LUCIGEN YELLOW (LucY), A YELLOW FLUORESCENT PROTEIN

Abstract

Described herein are isolated polynucleotides that encode a fluorescent protein which is at least 80% sequence identical to a protein selected from the group consisting of SEQ. ID. NOS: 2, 4, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, and 74. Also described are expression constructs containing the polynucleotides, transformed host cells containing the expression constructions, the encoded fluorescent proteins themselves, and methods of using the nucleotides and encoded fluorescent proteins for bioanalytical research.

Description

BACKGROUND

Fluorescent proteins (FPs) make it possible to visualize biological processes through in vivo imaging and in vitro fluorescence labeling. Processes like protein expression, localization, degradation, and interaction can be observed through fusion of a protein of interest with a FP. Green Fluorescent Protein (GFP) from Aequorea victoria and its varied derivatives constitute a multi-colored toolbox ranging from blue to yellow, with red-shifted FPs (RFPs) originating mostly from the sea anemone Discosoma striata (Day and Davidson, 2009). Following the discovery and subsequent explosion of available FPs, came the development of their use in techniques like Fluorescence or Førster Resonance Energy Transfer (FRET), Bioluminescence Resonance Energy Transfer (BRET), and Bimolecular Fluorescence Complementation (BiFC), all of which capitalize on the variety of choices in excitation and emission maxima characteristic to each FP.

In GFP-like and RFP-like fluorophores, fluorescence emanates from a chromophore developed by the formation of an imidazolinone ring system between two centrally located residues. However, complete maturation of the chromophore requires oxidation of an adjacent tyrosine residue, making molecular oxygen a strict requirement for these systems (Tsien, 1998). For example, the chromophore of GFP itself is formed by the cyclization of the tripeptide Ser65-Tyr66-Gly67.

Bacterial MurB enzymes are a family of flavoproteins that non-covalently bind flavin adenine dinucleotide (FAD). The MurB enzyme family catalyzes a step in peptidoglycan biosynthesis. Because peptidoglycans are cell wall components, enzymes in this pathway have been targets for developing antimicrobial compounds. In a physiological setting, MurB flavoprotein enzymes catalyze a hydride transfer from NADPH to the substrate through FAD to produce the final reduced product, UDP-N-acetylmuramic acid (El Zoelby et al., 2003). A byproduct of FAD binding is fluorescence due to FAD's intrinsic fluorescent properties. Free FAD fluorescence is rather weak (Φ_F=0.032) due the quenching effects of the adenine moiety. However, sequestration within a protein environment can enhance its fluorescence (Munro and Noble, 1999).

SUMMARY OF THE INVENTION

Disclosed and claimed herein is a novel fluorescent protein (FP) initially identified in and isolated from a metagenomic library. The FP has been cloned and expressed in a variety of hosts. Nucleotides encoding the novel FP and variations thereof are also disclosed herein. The FP has been given the name “LucY” for Lucigen Yellow. LucY can be used in the same methods that employ conventional FPs. For example, the LucY protein and its derivatives are useful as fluorescent markers in the many ways that such markers are already in use by those of ordinary skill in the art. Such uses include determining subcellular localization and coupling the FP to antibodies, nucleic acids or other receptors for use in detection assays (for example immunoassays or hybridization assays). Further, LucY can be used to track the movement of proteins in cells by expressing the FP in an expression vector. For another example, the FP can be useful in systems to detect induction of transcription. The FP's described herein are also useful as a visualization tool to judge solubility of fusion proteins.

Additionally, LucY and its derivatives can be used in a novel method to facilitate positive identification of membrane protein crystals grown in lipidic cubic phase.

Additionally, LucY has been developed into a split-fluorescence system by which protein-protein interactions can be determined. Due to the non-covalent binding of the FAD molecule, LucY fluorescence is reversible, distinguishing it from other available FPs. This reversibility may allow the LucY split-fluorescent system disclosed herein to be used in drug-screening platforms when looking for inhibitors of protein-protein interaction. To evaluate the reversibility of the split-LucY system, the dissociation of split-LucY fragments can be experimentally controlled through a well characterized chemically reversible dimerization strategy. Inducible dimerization of the FK506 binding protein (FKBP) has been used to evaluate BiFC complex formation and signal development (Robida and Kerppola 2009). Typically FKBP and a truncated version of its binding partner, FRB, are used in a chemically induced dimerization strategy in which a ligand promotes dimerization (Chen et al., 1995). FKBP variants (F_M) have since been developed that form constitutive homodimers which dissociate upon addition of drug, thus providing a reversible protein interaction model (Rollins et al., 2000). Although ligand-reversible F_M dimerization has been used successfully, it is theoretically possible that FKBP specific ligands may interfere with LucY reassembly or fluorescence. If necessary an alternative drug-inducible dimerization/dissociation system may be used. The gyrase B N-terminal domain (GyrB NTD) contains binding sites for coumermycin and novobiocin (Gilbert et al., 1994). Coumermycin is a bivalent drug that binds simultaneously to two GyrB NTD monomers and promotes formation of parallel homodimers, while novobiocin is a monovalent analog that can displace coumermycin and thereby drive dimer dissociation. Fusions to the GyrB NTD have been used for demonstration of dimerization-dependent activation of Raf1 kinase activity (Farrar et al., 1996), and as a part of a dimerization-dependent transcriptional activation strategy for controlled gene expression (Zhao et al., 2003).

Nucleotides that encode LucY and its variants are also described herein. The FP may be introduced into a host cell by direct delivery or may be expressed by the host cell, e.g., by a vector. In addition, the FP expressed in bacterial, eukaryotic, insect, mammalian and in vitro systems can be used directly to monitor the interactions with fused partners in cell lysates, at the extracellular spaces, or in tissue samples. The FP's disclosed herein are very useful for high-throughput screening in drug discovery and identification procedures, and for new target validations of diseases.

Also disclosed herein are kits containing one or more compositions comprising the fluorescent proteins, which can be a portion of a fusion protein, or one or more polynucleotides that encode the fluorescent proteins. The kits may also can contain one or more recombinant nucleic acid molecules, which encode, in part, fluorescent proteins, which can be the same or different, and may further include, for example, an operatively linked second polynucleotide containing or encoding a restriction endonuclease recognition site or a recombinase recognition site, or any polypeptide of interest.

Thus, specifically disclosed herein is an isolated polynucleotide comprising a nucleotide sequence encoding a fluorescent protein which is at least 80%, 85%, 90%, 95%, and/or 97% sequence identical to a protein selected from the group consisting of SEQ. ID. NOS: 2, 4, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, and 74, as well as circular permutations thereof. Also disclosed are these same polynucleotides further comprising an in-frame subsequence encoding a poly-His sequence at a terminus of the encoded fluorescent protein. These His-tagged polynucleotides include polynucleotides selected from the group consisting of SEQ. ID NOS: 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, and 75, and polynucleotides having at least 80% 85%, 90%, 95%, and/or 97% sequence identity to them, as well as circular permutations thereof, exclusive of the poly-His subsequence.

Also disclosed are isolated polynucleotides as described in the immediately preceding paragraph, wherein the encoded fluorescent protein has absorbance maxima at about 274 nm, about 376 nm and about 460 nm, and an emission maxima at about 530 nm. Preferably, although not required, the isolated polynucleotide as described herein encodes between 279 and 347 amino acid residues. The polynucleotide sequence may optionally further encode at least one additional polypeptide of interest in-frame with the encoded fluorescent protein.

Also disclosed herein is an expression construct comprising any of the isolated polynucleotides as described herein. Also disclosed is a host cell comprising such an expression construct. Any and all suitable host cells are within the scope of the present disclosure. The host cell, for example, may be selected from the group consisting of unicellular prokaryote cells, unicellular eukaryote cells, insect cells, and mammalian cells. Also disclosed herein is a method for making a fluorescent protein comprising cultivating the transformed host cell.

Also encompassed by the present disclosure are novel fluorescent proteins having at least 80%, 85%, 90%, 95%, and/or 97% sequence identity to a protein selected from the group consisting of SEQ. ID. NOS: 2, 4, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, and 74, as well as circular permutations thereof. Also disclosed herein are proteins comprising a polypeptide of interest operationally linked to fluorescent protein which is at least 80%, 85%, 90%, 95%, and/or 97% sequence identity to a protein selected from the group consisting of SEQ. ID. NOS: 2, 4, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, and 74, or circular permutations thereof.

Also disclosed herein are methods for detecting molecular interactions. One such method comprises fragmenting a fluorescent protein which is at least 80% sequence identical to a protein selected from the group consisting of SEQ. ID. NOS: 2, 4, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, and 74, or a fluorescent circular permutation thereof, so that the fragmentation results in two or more non-fluorescent protein fragments and a reversible loss of protein fluorescence. The non-fluorescent protein fragments are separately fused or attached to other molecules. The non-fluorescent protein fragments are then re-associated through interactions of the molecules that are fused or attached to the non-fluorescent protein fragments. A resulting fluorescent signal is then detected. In the preferred version of this invention, the fluorescent protein is fragmented into two non-fluorescent protein fragments.

As noted previously, the fluorescent protein may optionally comprise a poly-His sequence at a terminus of the protein. The fluorescent protein may optionally comprise between 279 and 347 amino acid residues.

Another method for detecting molecular interactions disclosed herein comprises providing a first reagent comprising a first compound of interest linked to a first non-fluorescent protein fragment; and providing a second reagent comprising a second compound of interest linked to a second non-fluorescent protein fragment. Here, the first and second non-fluorescent protein fragments comprise complementary fragments of a fluorescent protein which is at least 80% sequence identical to a protein selected from the group consisting of SEQ. ID. NOS: 2, 4, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, and 74, or a fluorescent circular permutation thereof, and wherein the first and second non-fluorescent protein fragments generate a fluorescent detectable signal when associated. “Complementary” when used in this context means “serving to fill out or complete” or to be “complements” in the sense of “something that completes something else.” See “Merriam-Webster”® Online Dictionary (http://www.merriam-webster.com, © 2014, Merriam-Webster, Inc., Springfield, Mass., USA). The first and second non-fluorescent protein fragments are associated through interactions of the first and second compounds of interest, and any resulting fluorescent signal is detected. The first compound of interest, the second compound of interest, or both the first and second compounds of interest may comprise polypeptides.

Also disclosed herein are kits. A first kit comprises, in combination, a first non-fluorescent protein fragment in a first container; and a second non-fluorescent protein fragment in a second container. The first and second non-fluorescent protein fragments comprise complementary fragments of a fluorescent protein which is at least 80% sequence identical to a protein selected from the group consisting of SEQ. ID. NOS: 2, 4, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, and 74, or a fluorescent circular permutation thereof, and wherein the first and second non-fluorescent protein fragments generate a fluorescent detectable signal when associated.

Another kit disclosed herein comprises, in combination, a first isolated polynucleotide that encodes a first non-fluorescent protein fragment in a first container; and a second isolated polynucleotide that encodes a second non-fluorescent protein fragment in a second container; and wherein the first and second isolated polynucleotides encode complementary, non-fluorescent protein fragments of a fluorescent protein which is at least 80% sequence identical to a protein selected from the group consisting of SEQ. ID. NOS: 2, 4, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, and 74, or a fluorescent circular permutation thereof, and wherein the encoded first and second non-fluorescent protein fragments generate a fluorescent detectable signal when associated.

Also disclosed herein is a method of increasing the aqueous solubility of a protein of interest. The method comprises fusing or attaching to the protein of interest a protein which is at least 80% sequence identical to a protein selected from the group consisting of SEQ. ID. NOS: 2, 4, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, and 74, or a circular permutation thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F. LucY expression. FIG. 1A depicts cell pellets (1 OD) from cultures expressing LucY from the rhaP_BAD promoter in response to rhamnose at the indicated concentrations. FIG. 1B is a graph depicting fluorescence response to rhamnose induction measured with a plate reader using excitation at 485 nm and emission at 528 nm. FIG. 1C is a photograph depicting induction of LucY expression in E. coli on a plate containing 0.2% rhamnose. FIG. 1D is a photograph depicting expression of LucY in the mammalian cells COS-7. FIG. 1E is a photograph depicting expression of LucY in the mammalian cells CHO-K1. FIG. 1F is a photograph depicting expression of LucY in the mammalian cells HeLa. All mammalian transfections were carried out with “TransIT-2020”-brand transfection-reagent (Mirus Bio LLC, Madison, Wis.) and imaged 24 h post-transfection with an inverted fluorescence microscope fitted with a GFP excitation/emission filter.

FIG. 2 is a photograph depicting the purification of LucY-His6 from E. coli. LucY was expressed with a carboxyl terminal His6 tag using Lucigen's Expresso™-brand T7 cloning and expression system. LucY-His6 was purified by metal affinity chromatography. Fractions from purification including whole cell pellet (lane 1), insoluble pellet after lysis (lane 2), soluble supernatant (lane 3), flowthrough (lane 4), wash (lane 5) and elution fractions (lanes 6-10) with imidazole were photographed under UV light (upper panel) and are aligned with corresponding gel lanes.

FIG. 3 is a graph depicting the superimposed excitation and emission spectra of LucY. The excitation scan (blue) was performed with emission at 528 nm and the emission scan (green) was performed with excitation at 465 nm.

FIG. 4A and FIG. 4B are photographs depicting E. coli cell pellets expressing candidate thermo-tolerant homologs of LucY and visualized under a hand-held UV lamp. In FIG. 4A: Sye=Sulfurihydrogenibium yellowstonense; Y4.1 MC1=Geobacillus sp. ((strain Y4.1MC1); Dtu=Dictyoglomus turgidum; Rma=Rhodothermus marinus; Aae=Aquifex aeolicus; Aac=Alicyclobacillus acidocaldarius; Ace1=Acidothermus cellulolyticus; Tma=Thermotoga maritima; Chy=Calsicellulosiruptor hydrothermalis; Tye=Thermodesulfovibrio yellowstonii. In FIG. 4B, Pma=Persephonella marina; Saz=Sulfurihydrogenibium azorense; Hth=Hydrogenobacter thermophilus; SspY=Sulfurihydrogenibium sp. (strain YO3AOP1); Tit=Thermoanaerobacter italicus; Tal=Thermocrinis albus; Hsp1=Hydrogenivirga sp. 128-5-R1-1.

FIG. 4C is a plot of fluorescence following 45 min incubation at temperatures ranging from 30° C. to 80° C. Fluorescence is expressed as a percentage of highest fluorescence recorded across temperature range. The temperature at which fluorescence diminished to 10% is listed. The abbreviations are the same as listed for FIGS. 4A and 4B.

FIGS. 5A and 5B are photographs depicting subcellular localization via LucY fluorescence. FIG. 5A is a photograph depicting E. coli cell pellets following illumination with UV light showing expression of LucY fused to the membrane GPCR protein, A1a, with growth at 25° C. and at 30° C. LSP=low-speed pellet; HSP=high-speed pellet. FIG. 5B depicts LucY fused to ATPb following induction with IPTG or arabinose.

FIG. 5C is a coomassie stained SDS-PAGE of fractions from E. coli expressing the LucY-ATPb fusion shown in FIG. 5B. GPCR-containing extracellular membranes separate with the high-speed pellet (HSP). ATPb separates with intracellular membranes in the low-speed pellet (LSP). HSS=high-speed supernatant; LSS=low-speed supernatant.

FIG. 6 is a photograph depicting expression of the COR-kinase domain of the LRRK2 enzyme as a C-terminal fusion with LucY from E. coli. Five different clones are shown. Each clone is yellow fluorescent, indicating positive fusion protein expression.

FIGS. 7A, 7B, 7C, and 7D are three-part photographs depicting expression of LucY as an intracellular loop fusion in mammalian cells. Each three-part series shows the fusion illuminated with a bright field (left), using a GFP filter (middle), and a merged bright field/GFP image (right). Each set of photos represents fusion at a different site within the intracellular loop 3 of a GPCR. FIG. 7A illustrates replacement of the GPCR intracellular loop positions 245-271 with LucY. FIG. 7B shows replacement of the GPCR intracellular loop positions 245-271 with LucY and GSG linker sequence. FIG. 7C illustrates replacement of the GPCR intracellular loop positions 245-274 with LucY and GSG linker sequence. FIG. 7D illustrates replacement of the GPCR intracellular loop positions 245-276 with LucY and GSG linker sequence. GSG linker sequence lies between LucY C-terminus and the GPCR.

FIG. 8 is a photograph depicting insect cells (“Hi 5”-brand; Life Technologies, Carlsbad, Calif., USA; generically BTI-TN-5B1-4 cells) expressing turkey β1-adrenergic receptor-LucY fusions. The far left tube is negative control, containing only unmodified “Hi 5”-brand cells. The other four tubes contain “Hi 5”-brand cells expressing LucY fused within the intracellular loop 3 of turkey β1-adrenergic receptor at four different junction points.

FIG. 9A is a photograph showing the utility of LucY as an indicator of detergent solubilization. ATPb was solubilized with 31 different detergents and the fluorescence was visualized by a UV light. FIG. 9B is a corresponding histogram of the fluorescence exhibited by the tubes in FIG. 9A as quantified with a Biotek Synergy 2 microplate fluorometer (BioTek Instruments, Inc., Winooski, Vt., USA).

FIGS. 10A, 10B, and 10C together demonstrate the utility of LucY as an indicator of protein expression and solubility. FIG. 10A is a photograph depicting Fisuc 1793-LucY expression on plates containing 0.2% rhamnose, with (right) or without (left) an amino-terminal SUMO tag. FIG. 10B is a photograph depicting cleared lysates from ˜5 ml cultures of cells expressing the indicated Fibrobacter genes as LucY fusions, with (right) or without (left) a SUMO tag. FIG. 10C is a series of Coomassie blue-stained polyacrylamide gels showing expression of LucY fusion proteins in total cell lysate (T), soluble fraction (S), and pellet fraction (P) after centrifugation at 12,000×G for 5 minutes. The asterisks indicate the induced fusion protein. No band was detected corresponding to the 1793-LucY protein without the SUMO tag.

FIGS. 11A, 11B, and 11C demonstrate insoluble expression of ABV-LucY and 4110-LucY as “solubility trap” fusion proteins. FIG. 11A is a photograph depicting weak fluorescence due to expression of SUMO-ABV-LucY fusion protein from rhaP_BAD. Plates contained no rhamnose (left), or 0.2% rhamnose (right). FIG. 11B depicts gel analysis of ABV-LucY protein expressed from rhaP_BAD, with or without an amino-terminal SUMO tag. FIG. 11C depicts gel analysis of 4110-LucY protein expressed from rhaP_BAD, with or without a SUMO solubility tag. The right and left portions of FIG. 11C are from the same gel. In FIGS. 11B and 11C, T represents total cell lysate; S represents the soluble fraction after centrifugation of the lysate; and P represents the insoluble pellet fraction after centrifugation.

FIGS. 12A and 12B are photos demonstrating the utility of LucY to select fluorescent colonies from a random fusion library in a “solubility trap” experiment. FIG. 12A is a photograph of a section of a primary selection plate containing 0.2% rhamnose. Several fluorescent colonies are indicated by arrows. FIG. 12B is a photograph of individual fluorescent colonies from the initial library screen depicted in FIG. 12A, re-streaked onto a plate containing 0.2% rhamnose.

FIGS. 13A and 13B demonstrate enhanced soluble expression of 4110-LucY fusion protein with amino-terminal fusion tags derived from shotgun library screens. FIG. 13A is a gel analysis of soluble expression of Geobacillus-4110-LucY library clone 11. The Control sample is 4110-LucY with no amino-terminal fusion. T represents total cell lysate; S represents the soluble fraction after centrifugation of the lysate; and P represents the insoluble pellet fraction after centrifugation. FIG. 13B is a photograph depicting increased partitioning of yellow fluorescence to the soluble fraction with Geobacillus library clone 11.

FIG. 14 is a topology diagram of LucY. LucY includes three domains as indicated by circles. Domains 1 and 2 are separated by a short loop; Domains 2 and 3 are connected by an approximately 20 amino-acid span.

FIGS. 15A, 15B, and 15C are schematic diagrams of various split LucY systems. FIG. 15A is a schematic depicting conventional bimolecular fluorescence complementation (BiFC). Here, a reporter protein is split into two sub-fragments, each of which is fused to a different protein. The two proteins (Protein X and Protein Y in the figure) have the potential to interact. Only after a positive interaction of Protein X and Protein Y is achieved will the two fragments combine and elicit a signal. FIG. 15B is a schematic diagram showing the three distinct domains (colored red, blue, and green) of LucY. A five-residue loop connects domains 1 and 2 and an approximately 18-residue loop connects domains 2 and 3. Five points within each of these loops were chosen as Split Points (SP), as indicated by underlined residues. FIG. 15C is a schematic diagram showing synthetic antiparallel leucine zippers (light green), which were used as protein interaction models to test reconstitution of split LucY fragments (right) and were compared to whole LucY fusions (left). LucY fused at the N-terminus of the leucine zipper is designated NZ; LucY fused at the C-terminus of the leucine zipper is designated CZ. Split LucY fragments fused to the N-terminus and C-terminus of the leucine zippers are referred to as SPNZ and SPCZ, respectively.

FIGS. 16A, 16B, 16C, and 16D are a series of histograms of fluorescence which demonstrate LucY reassembly. The image above each histogram shows whole cell pellets of the corresponding sample photographed under UV light. Fluorescence is represented as a percentage of CZ. Error bars represent standard deviation from the mean (n=3). FIG. 16A: Five split points between domains 1 and 2, referred to as SPa1-5, were made and their fusions with leucine zippers were coexpressed. FIG. 16B: The same conditions as in FIG. 16A, except the five split points were between domains 2 and 3 and are referred to as SPb1-5. FIG. 16C: SPbNZ5 paired with other split points to determine the pair with highest fluorescence. FIG. 16D: SPbNZ5 paired with either SPbCZ1 or SPbCZ5 was tested for fluorescence independently and without the leucine zipper (ΔZip).

FIGS. 17A, 17B, 17C, and 17D are a series of Coomassie-stained SDS-PAGE gels depicting various split point coexpressions of LucY. In each gel, the NZ and CZ bands are adjacent to the dot. FIG. 17A shows expression of NZ, CZ, and SPa1-5, corresponding to the data in FIG. 16A. FIG. 17B shows expression of NZ, CZ, and SPb1-5, corresponding to the data in FIG. 16B. FIG. 17C shows expression of SPbNZ5 and SPbCZ5 paired with a non-continuous CZ or NZ partner in whole cells, corresponding to the data in FIG. 16C. FIG. 17D shows expression of SPbNZ5, SPbCZ1 or 5 alone, in combination, or with their leucine zippers removed in whole cells, corresponding to data in FIG. 16D.

FIGS. 18A and 18B are Coomassie-stained SDS-PAGE gels depicting split point solubility and expression of LucY. FIG. 18A shows insoluble (P) and soluble (S) fractions of SPbNZ5 and SPbCZ4, alone, coexpressed, with leucine zippers, and without leucine zippers. FIG. 18B shows expression of the C-terminal fragments of LucY without leucine zipper, uninduced (UI), and induced (I).

FIG. 19A is a schematic diagram illustrating circular permutation of LucY. LucY was circularly permutated such that domains 1 and 3 were connected with a small linker (dashed line) and new N and C-termini were created between domains (red loops). Break points (BP) were introduced between domains 1 and 2 (BPs1-5) or domains 2 and 3 (BPs6-10). FIG. 19B is a histogram comparing the fluorescence of the circularized permutations to wild-type LucY. FIG. 19C is a histogram showing fluorescence of LucY permutations in which split points were made from a circular permuted LucY such that domains 3 and 1 make up one half and domain 2 makes up the other. In FIGS. 19B and 19C, fluorescence is represented as a percentage of wild-type LucY and CZ, respectively. The image above each histogram is a photograph of the whole cell pellets of the corresponding sample under UV light. Error bars represent standard deviation from the mean (n=3).

FIGS. 20A and 20B are Commassie-stained SDS-PAGE gels showing expression of circular permutations of LucY and splits made from them. FIG. 20A is a gel showing expression of each circular permutation, designated BP-1 through BP-10. The gel shown in FIG. 20A corresponds to the data presented in FIG. 19B. FIG. 20B is a gel showing split point trials derived from circularly permuted LucY. The gel shown in FIG. 20B corresponds to the data in FIG. 19C.

FIGS. 21A-21J make up a series of photographs showing split LucY systems expressed in HEK 293T cells. FIG. 21A is a photograph of NZ-transformed cells viewed with a band pass filter. FIG. 21B is a photograph of CZ-transformed cells viewed with a band pass filter. FIG. 21C is a photograph of SPbNZ5-transformed cells viewed with a band pass filter. FIG. 21D is a photograph of SPbCZ4-transformed cells viewed with a band pass filter. FIG. 21E is a photograph of SPbNZ5+SPbCZ4-transformed cells viewed with a band pass filter. FIG. 21F is a photograph of NZ-transformed cells viewed with bright field and band pass filters merged. FIG. 21G is a photograph of CZ-transformed cells viewed with bright field and band pass filters merged. FIG. 21H is a photograph of SPbNZ5-transformed cells viewed with bright field and band pass filters merged. FIG. 21I is a photograph of SPbCZ4-transformed cells viewed with bright field and band pass filters merged. FIG. 21J is a photograph of SPbNZ5+SPbCZ4-transformed cells viewed with bright field and band pass filters merged. FIG. 21K is a photograph of an immunoblot gel using anti-HA to detect HA-tagged leucine zipper/LucY fusions either whole, NZ and CZ, or splits SPbNZ5 and SPbCZ4.

FIGS. 22A-22E are a series of fluorescent photomicrographs demonstrating the use of LucY to visualize protein crystals. FIG. 22A is a photomicrograph of LucY crystals visualized under UV light. FIG. 22B, FIG. 22C, FIG. 22D, and FIG. 22E are photomicrographs of putative crystals of a β1-AR-IL3-LucY fusion proteins in lipidic cubic phase (LCP) visualized under UV light.

FIG. 23A is a gel depicting enhanced soluble expression of TEV protease as a fusion to C-terminal LucY. E. coli cultures harboring plasmids encoding TEV protease with a C-terminal His6 fusion or a C-terminal LucY-His6 fusion were grown at 37° C. and induced with 0.2% rhamnose. The induced cells were harvested and lysed by sonication, and the lysates were separated into soluble and insoluble fractions by centrifugation. Samples of the total lysate (T), soluble (S), and insoluble (I) fractions were run on SDS PAGE gel (4-20%) and proteins were stained with Coomassie blue stain. FIG. 23B depicts soluble and insoluble fractions of TEV-LucY-His6 lysate photographed under long-wavelength UV light. FIG. 23C is a gel depicting sequence-specific protease activity of the TEV-LucY-His6 fusion protein.

DETAILED DESCRIPTION

Abbreviations and Definitions

ABV=Acidianus bottle-shaped virus. ATPb=ATP synthase (part of the Fo complex; subunit a). β1-AR-IL3=cardiac β1-adrenergic receptor+interleukin 3 fusion. BiFC=bimolecular fluorescence complementation. DNAP=DNA polymerase. FP=fluorescent protein. GFP=green fluorescent protein. GPCR=G protein-coupled receptor. HA tag=human influenza hemagglutinin tag. IPTG=isopropyl β-D-1-thiogalactopyranoside. LRRK2=leucine-rich repeat kinase 2. Ni-NTA=nickel-nitrilotriacetic acid resin. PCR=polymerase chain reaction (see U.S. Pat. Nos. 4,683,195 and 4,683,202). RFP=red-shifted fluorescent protein. SDS-PAGE=sodium dodecyl sulfate polyacrylamide gel electrophoresis. SUMO=small ubiquitin-like modifier protein.

Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in biochemistry and genetic engineering. For purposes of the present disclosure, the following terms are specifically defined.

The term “nucleic acid molecule” or “polynucleotide” refers to a deoxyribonucleotide or ribonucleotide polymer in either single-stranded or double-stranded form, and, unless specifically indicated otherwise, encompasses polynucleotides containing known analogs of naturally occurring nucleotides that can function in a similar manner as naturally occurring nucleotides. For example, this term can refer to single and double stranded forms of DNA and/or RNA.

The term “recombinant nucleic acid molecule” refers to a non-naturally occurring polynucleotide containing two or more linked polynucleotide sequences. A recombinant nucleic acid molecule can be produced by recombination methods, particularly genetic engineering techniques, or can be produced by a chemical synthesis method. A recombinant nucleic acid molecule can encode a fusion protein, for example, a fluorescent protein as disclosed herein linked to a polypeptide of interest. The term “recombinant host cell” refers to a cell that contains or can express a recombinant nucleic acid molecule.

The term “encoding” when referring to a polypeptide or protein (the terms are used synonymously herein) refers to the transcription of a corresponding polynucleotide and translation of the mRNA produced therefrom to yield the polypeptide. The encoding polynucleotide is considered to include both the coding strand, whose nucleotide sequence can be identical to an mRNA, as well as its complementary strand. Encoding polynucleotides explicitly include degenerate codons which encode the same amino acid residues or functionally equivalent amino acid residues. Nucleotide sequences encoding a polypeptide or protein can include polynucleotides containing introns and exons.

The term “expression construct” refers to a polynucleotide molecule containing at least one sub-sequence encoding a protein of interest which is operationally linked to one or more regulatory sub-sequences which drive expression of the encoded protein when the construct is transformed into a suitable host cell. Such constructs may also contain sub-sequences encoding proteins for selecting host cells transformed to contain the construct, such as sub-sequences which confer antibiotic resistance or dietary limitations to transformed cells. An expression construct may also include one or more of the FP's disclosed herein.

The terms “control sequences (or sub-sequences),” “regulatory sequences (or sub-sequences),” and the like refer to polynucleotide sequences that are necessary to effect the expression of coding and non-coding sequences in a host cell. Such control sequences can include promoters, ribosomal binding sites, transcription termination sequences, and the like. These terms are used synonymously herein and include, at a minimum, components whose presence can influence expression and also include additional components whose presence is advantageous, such as leader sequences. Fusion partner sequences may sometimes also be control sequences.

The term “operationally linked” when referring to joined polynucleotide sequences denotes that the sequences are in the same reading frame and upstream regulatory sequences will perform as such in relation to downstream structural sequences. Polynucleotide sequences which are operationally linked are not necessarily physically linked directly to one another but may be separated by intervening nucleotides which do not interfere with the operational relationship of the linked sequences. Similarly, when referring to joined polypeptide sequences, operationally linked means that the functionality of the individual joined segments are substantially identical as compared to their functionality prior to being operationally linked. For example, a fluorescent protein can be fused to a polypeptide of interest and in the fused state retain its fluorescence, while the fused polypeptide of interest also retains its original biological activity.

As used herein, the term “brightness,” with reference to a fluorescent protein, is measured as the product of the extinction coefficient (c) at a given wavelength and the fluorescence quantum yield (Φ_F).

The term “probe” refers to a substance that specifically binds to another substance (a “target”). Probes include, for example, antibodies, polynucleotides, receptors and their ligands, and may (or may not) be labeled so as to provide a means to identify or isolate a molecule to which the probe has specifically bound.

The term “label” refers to a composition that is detectable with or without instrumentation, for example, by visual inspection, spectroscopy, or a photochemical, biochemical, immunochemical or chemical reaction. Exemplary labels (non-limiting) include ³²P, fluorescent dyes and proteins, electron-dense reagents, enzymes (such as those commonly used in an ELISA), and binding labels or tags, such as biotin, digoxigenin, or other haptens or peptides for an antiserum or antibody. For example, a label can generate a measurable signal such as fluorescent light in a sample.

The terms “polypeptide” and “protein” refer to a polymer of two or more amino acid residues. For purposes of this disclosure, the two terms are synonymous. “Polypeptides” and “proteins” are polymers of amino acid residues that are connected through amide bonds. As defined herein, the term “amino acid” includes natural α-amino acids and unnatural α-amino acids (e.g. beta-alanine, phenylglycine, homoarginine, N-alkyl α-amino acids and the like). All optical isomers are included within the definition of “amino acid.”

The term “isolated” or “purified” refers to a material that is substantially or essentially free from components that normally accompany the material in its native state in nature. Purity generally can be determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis, high performance liquid chromatography, and the like. A polynucleotide or a polypeptide is considered to be isolated when it is the most predominant species present in a preparation.

The term “naturally occurring” refers to a protein, nucleic acid molecule, cell, or other material that exists in nature. A naturally occurring material can be in its “native” form, that is as it exists in nature. Naturally-occurring materials may also be modified by human intervention so that they are in an isolated or purified form.

Two or more amino acid sequences or two or more nucleotide sequences are considered to be “substantially identical” or “substantially similar” if the amino acid sequences or the nucleotide sequences share at least 80% sequence identity with one another, or with a reference sequence over a given comparison window. Thus, substantially similar sequences include those having, for example, at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity. The terms “sequence identity” or “sequence identical” are defined to mean sequence identity as measured using the cluster database at high identity with tolerance method (i.e., CD-HIT). In simplified terms, terminal gaps are ignored and identity is calculated from the remaining aligned columns. An “identity” or “match” is a column having the same amino acid residues or nucleotide bases; a “mismatch” is a column with two different amino acid residues or nucleotide bases. An “indel” is a consecutive series of gaps in one sequence. Percent identity is then calculated by dividing the number of matches by the length of the shorter of the two sequences being compared. CD-HIT is well known in the field and will not be discussed in any detail herein. For a full description, see “Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences,” Weizhong Li & Adam Godzik Bioinformatics, (2006) 22:1658-9; and Limin Fu, Beifang Niu, Zhengwei Zhu, Sitao Wu and Weizhong Li, “CD-HIT: accelerated for clustering the next generation sequencing data,” Bioinformatics, (2012), 28(23):3150-3152.

Additionally, two or more amino acid sequences or two or more nucleotide sequences are considered to be “substantially identical” or “substantially similar” if the amino acid sequences or the nucleotide sequences are circular permutations of each other. Not all sequence-based algorithms for determining percent sequence identity will detect circular permutations. However, several well known programs that require an input consisting only of a linear sequence of amino acids or nucleotides will detect circular permutations of the protein (or encoded protein in the case of nucleotides). Such programs include SHEBA (Jung, J.; Lee, B. (2001). “Circularly permuted proteins in the protein structure database,” Protein Science 10(9):1881-1886); Multiprot (Shatsky, M.; Nussinov, R.; Wolfson, H. J. (2004). “A method for simultaneous alignment of multiple protein structures,” Proteins: Structure, Function, and Bioinformatics 56(1):143-156); RASPODOM (Weiner, J.; Thomas, G.; Bornberg-Bauer, E. (2005). “Rapid motif-based prediction of circular permutations in multi-domain proteins,” Bioinformatics 21(7):932-937); CPSARST (Lo, W. C.; Lyu, P. C. (2008). “CPSARST: An efficient circular permutation search tool applied to the detection of novel protein structural relationships,” Genome Biology 9(1):R11); GANGSTA+(Schmidt-Goenner, T.; Guerler, A.; Kolbeck, B.; Knapp, E. W. (2010). “Circular permuted proteins in the universe of protein folds,” Proteins: Structure, Function, and Bioinformatics 78(7):1618-1630.); SANA (Wang, L.; Wu, L. Y.; Wang, Y.; Zhang, X. S.; Chen, L. (2010). “SANA: An algorithm for sequential and non-sequential protein structure alignment,” Amino Acids 39(2):417-425); and CE-CP (Prlic, A.; Bliven, S.; Rose, P. W.; Bluhm, W. F.; Bizon, C.; Godzik, A.; Bourne, P. E. (2010). “Pre-calculated protein structure alignments at the RCSB PDB website,” Bioinformatics 26(23):2983-2985).

The term “fluorescent properties” refers to the molar extinction coefficient at an appropriate excitation wavelength, the fluorescence quantum efficiency, the shape of the excitation spectrum or emission spectrum, the excitation wavelength maximum and emission wavelength maximum, the ratio of excitation amplitudes at two different wavelengths, the ratio of emission amplitudes at two different wavelengths, the excited state lifetime, or the fluorescence anisotropy.

The term “fluorescent protein” refers to any protein capable of light emission when excited with an appropriate electromagnetic energy. Fluorescent proteins include proteins having amino acid sequences that are either natural or engineered, such as the fluorescent proteins derived from Aequorea victoria fluorescent proteins.

The term “mutant” or “variant” also is used herein in reference to a fluorescent protein that contains a mutation with respect to a corresponding wild-type fluorescent protein. In addition, reference is made herein to a “spectral variant” or “spectral mutant” of a fluorescent protein to indicate a mutant fluorescent protein that has a different fluorescence characteristic with respect to the corresponding wild-type fluorescent protein. Similarly, thermo-tolerant mutants or variants are fluorescent protein that display fluorescent characteristics at elevated temperatures as compared to the corresponding wild-type.

The recombinant polynucleotides described herein are incorporated into a suitable host cell. The host cell may be any host cell now known or developed in the future which is amenable to transformation, including, but not limited to prokaryotic and eukaryotic unicellular host cells, e.g., bacteria, yeast, and the like, such as unicellular microbes of the genera Saccharomyces, Bacillus, Aspergillus, Pichia, Kluyveromyces, Escherichia and the like, or isolated, high-order cells from multi-cellular organisms, such as insect and mammalian host cells.

Many of the steps noted below for the manipulation of polynucleotides and proteins, including digesting with restriction endonucleases, amplifying by PCR, hybridizing, ligating, separating and isolating by gel electrophoresis, transforming cells with heterologous DNA, selecting successful transformants, and the like, are well known and widely practiced by those skilled in the art and are not extensively elaborated upon herein. Unless otherwise noted, the standard protocols utilized herein are described extensively in Michael R. Green & Joseph Sambrook, “Molecular Cloning: A Laboratory Manual (Fourth Edition),” © 2012, Cold Spring Harbor Laboratory Press: New York, N.Y., ISBN 978-1-936113-42-2.

Numerical ranges as used herein are intended to include every number and subset of numbers contained within that range, whether specifically disclosed or not. Further, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 2 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All references to singular characteristics or limitations described herein shall include the corresponding plural characteristic or limitation, and vice-versa, unless otherwise specified or clearly implied to the contrary by the context in which the reference is made.

All combinations of method or process steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.

The methods, compositions, and kits described herein can comprise, consist of, or consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, preparatory steps, subsequence steps, or limitations described herein or otherwise useful or desired.

Overview:

Disclosed herein are nucleic acid sequences and amino acid sequences of a novel yellow fluorescent protein designated LucY. The nucleic acid sequences and amino acid sequences encoding the FP are useful for monitoring and visualizing physiological processes, such as protein localization, expression of genes, solubility of protein, etc. The LucY protein, its sequence variants, and homologues, have broad applicability in characterizing cells and organisms and in detecting or measuring various cellular parameters, notably in thermo-tolerant organisms.

LucY, its sequence variants, and homologs may also be used to identify protein binding peptide partners (such as protein-protein or protein-peptide interactions) at the cellular level. More particularly, the split-protein approach described below can be used to determine peptide partners, even if previously unknown, in bacteria and eukaryotes and can be used to visualize cellular and sub-cellular protein localization in multicellular organisms. The FP also can be used to monitor signaling processes and molecular interactions in conjunction with other fluorescent entities such as other fluorescent proteins via FRET. LucY, its sequence variants, and homologues can be used in these protocols, as well as in other conventional protocols that use fluorescent markers.

Thus, LucY, its sequence variants, and homologues can be used for coupling fluorescent protein variants to antibodies, polynucleotides or other receptors for use in detection assays such as immunoassays or hybridization assays, or to track the movement of proteins in cells. For intracellular tracking studies, a first polynucleotide encoding LucY, a LucY sequence variant, or a LucY homologue is fused to at least a second polynucleotide encoding a protein of interest. The construct, if desired, can be inserted into an expression vector. Upon expression inside a heterologous host cell, the protein of interest can be localized based on fluorescence.

LucY, its sequence variants, and its homologs are also useful in systems to detect induction of transcription. For example, a nucleotide sequence encoding a non-fluorescent protein can be fused to nucleotide sequence encoding LucY, and further linked to a promoter or other expression control sequence of interest, which can be contained in an expression vector. The construct is then transfected into a host cell. Induction of the promoter (or other regulatory element) is then measured by detecting the presence or amount of fluorescence, thereby enabling the responsiveness of a signaling pathway to be evaluated. These and other methods of using the LucY protein and its corresponding polynucleotide are described in greater detail below.

Kits:

A kit for use in transfecting host cells may be assembled using the nucleic acid molecules encoding the FPs, or for labeling target polypeptides with the FP. Host cell transfection kits may comprise at least one container containing one or more of the nucleic acid molecules encoding a FP (or a composition comprising one or more of the nucleic acid molecules or plasmids described herein), which nucleic acid molecule preferably comprises plasmid. These kits optionally may comprise at least one additional container that may contain, for example, a reagent for delivering the FP nucleic acid molecule into a host cell.

Further, kits may contain chemical reagents (e.g., polypeptides or polynucleotides) as well as other components. For example, kits may include apparatus and reagents for sample collection and/or purification, apparatus and reagents for product collection and/or purification, reagents for host cell transformation, reagents for eukaryotic cell transfection, previously transformed or transfected host cells, sample tubes, holders, trays, racks, dishes, plates, instructions to the kit user, solutions, buffers or other chemical reagents, suitable samples to be used for standardization, normalization, and/or control samples. Kits may also be packaged for convenient storage and safe shipping. In some versions, the kits might include a FP as disclosed herein, a polynucleotide vector (e.g., a plasmid) encoding a FP as disclosed herein, bacterial cell strains suitable for propagating the vector, reagents for purifying the expressed fusion proteins, and the like. The FPs and kits using such proteins and/or their corresponding nucleotides may be configured or optimized to carry out one or more of the analytical methods described herein.

Samples Useful with LucY:

The samples that can be assayed or analyzed using LucY, its sequence variants, and its homologs include biological samples, environmental samples, or any other samples for which it is desired to determine whether a particular molecule is present therein. With some embodiments, the sample can include a cell or a cell extract from any source, without limitation (prokaryotic, eukaryotic, single-celled, multi-celled, etc.).

Further, the cells may be obtained from a culture of such cells, for example, a cell line, tissue line, or can be isolated from an organism. As such, the cell can be contained in a tissue sample, which can be obtained from an organism by any means commonly used to obtain a tissue sample, for example, by biopsy of a human or other organism. Where the method is performed using an intact living cell or a freshly isolated tissue or organ sample, the presence of a molecule of interest in living cells can be identified, thus providing a means to determine, for example, the intracellular compartmentalization of the molecule.

Measuring Fluorescence:

Methods for detecting the FP or of a cell expressing a FP may comprise, for example, illuminating the FP or cell expressing the FP with an illumination source such that the FP or cell expressing the FP emits radiation. Such detection methods may use an illumination source such as an incandescent light source, a fluorescent light source, a halogen light source, a laser light source, sunlight, and other equivalent sources. When illuminated by such an illumination source, the FP will emit fluorescent light that may be detected by unaided observation or by other qualitative or quantitative methods. Suitable methods for measuring fluorescence of samples are known and understood by those with ordinary skill in the art. Alternatively, the fluorescence signal and absorbance may be measured directly from the FP. The native LucY protein has strong absorbance maxima at 247, 376, and 460 nm. Thus the absorption at any of these wavelengths may be used to detect the FP via absorption spectroscopy. Further, the native LucY protein may be detected directly from a fluorescence emission at 530 nm.

Suitable methods for measuring fluorescence of samples are known and understood by those with ordinary skill in the art. They will not be described in any detail herein. Representative known methods of performing assays on fluorescent materials are described in, e.g., Lakowicz, J. R., Principles of Fluorescence Spectroscopy, (Plenum Press 1983); Herman, B., Resonance Energy Transfer Microscopy, Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, vol. 30, pp. 219-243 (ed. Taylor, D. L. & Wang, Y.-L., Academic Press 1989); Turro, N.J., Modern Molecular Photochemistry, pp. 296-361 (Benjamin/Cummings Publishing, Inc. 1978). There are numerous commercial suppliers of suitable fluorimeter, fluorescence spectroscopy, fluorescence microscopy, and confocal laser scanning microscopy equipment, such as Agilent Technologies (Santa Clara, Calif., USA; maker of Cary Eclipse-branded instruments) and PerkinElmer, Inc. (Waltham, Mass., USA).

One method for measuring fluorescence in samples is through the use of fluorimeters. Radiation is passed through the sample under controlled conditions. As the radiation passes through the sample at an excitation wavelength, the FP in the sample emits radiation at distinct wavelength(s), which then are captured as data by the fluorimeter. Both excitation and emission spectra are taken to determine the excitation and emission maxima for optimal fluorescence signals under any given set of conditions. The data is saved on a computer and or it can be further analyzed by computer. The scanned data is typically compared to control samples, such as calibration samples, negative and positive controls, and the like. The analyte concentration may be determined by extrapolating the fluorescence of the sample with a calibration curve.

LucY Identification and Isolation:

A 10 g sample of corn stalks collected from a field outside Middleton, Wis. in the fall of 2007 was added to YTP-2 medium containing (per liter) 2.0 g yeast extract, 2.0 g tryptone, 2.0 g sodium pyruvate, 1.0 g KCl, 2.0 g KNO₃, 2.0 g Na₂HPO₄.7H₂O, 0.1 g MgSO₄, 0.03 g CaCl₂, and 2.0 ml clarified tomato juice. (See Gao et al. (2011) Biotechnol Biofuels 4:5.) The sample was grown at 55° C. and 200 rpm in a 2 L flask containing 1 L of medium. After 2 weeks the media was filtered through a Miracloth-brand filter (EMD Millipore, Philadelphia, Pa.; pore size: 22-25 μm) and the remaining material was centrifuged to pellet the microbial cells. The cells from the enrichment culture were resuspended in lysis buffer and high molecular weight genomic DNA was purified using the Qiagen (Valencia, Calif.) Genomic-tip kit. The DNA was randomly fragmented to 3-6 kb using a Hydroshear Apparatus (Digilab, Marlborough, Mass.). The ends of the DNA were made blunt using the DNATerminator kit (Lucigen, Middleton, Wis.). The sheared, end-repaired DNA was gel purified and ligated to the pEZSeq vector (Lucigen, Middleton, Wis.). The ligation reaction was transformed into E. coli 10 G cells (Lucigen, Middleton, Wis.). The cells were plated on LB media containing 30 m/ml kanamycin and grown overnight at 37° C. The plates containing several hundred colonies each were moved to a dark room and checked for fluorescence using a 365 nm long wavelength hand-held UV lamp (UVP, Upland, Calif.). A single colony out of approximately 1000 was a fluorescent yellow color under the UV lamp. The single yellow colony was grown overnight in terrific broth. The plasmid DNA was purified using standard procedures and the nucleotide sequence of the entire 3684 bp recombinant insert was determined by Sanger sequencing biochemistry on an Applied Biosystems 3100 Genetic Analyzer (Foster City, Calif.).

The DNA sequence of the yellow fluorescent recombinant insert was compared to the GenBank database using BLASTN (Altschul, et al., 1990) and close homology (83% sequence identity) was found to a segment of the Bacillus licheniformis ATCC 14580 genome. Analysis of the cloned region revealed that it contains homologues of bacterial murG and murB genes and portions of the spoVE and divIB genes. The murB gene encodes UDP-N-acetylenolpyruvoylglucosamine reductase, a flavoprotein that functions in the synthesis of the peptidoglycan cell wall (El Zoelby et al., 2003). The novel metagenomic clone encodes a MurB homologue with 95% amino acid sequence identity to the uncharacterized Bacillus licheniformis MurB protein. A lower level of homology to the well-studied MurB proteins from Staphylococcus aureus (39% identity; 63% similarity) and Escherichia coli (26% identity; 43% similarity) was noted. Previous studies have reported that the purified MurB protein from E. coli exhibits a yellow color (Benson et al., 1993). Quenching of the fluorescence of the tightly bound FAD cofactor has been used as the basis for evaluating binding of compounds to S. aureus MurB protein (Yang et al., 2006).

To confirm that the novel MurB protein is responsible for the observed fluorescence, its coding region (SEQ. ID. NO. 1) was cloned into a bacterial expression plasmid under the control of a bacteriophage T7 promoter. When this plasmid was introduced into BL21(DE3) cells expressing bacteriophage T7 RNA polymerase, colonies exhibited bright yellow fluorescence. Accordingly, the name LucY (Lucigen Yellow) was chosen for the novel MurB homologue. The amino acid sequence of the wild-type LucY protein is given in SEQ. ID. NO. 2.

Expression in Different Hosts:

E. coli: LucY has been expressed in bacterial cells under the control of the IPTG-inducible T7 promoter and the rhamnose-inducible promoter. Strong fluorescence was observed for both, as evidenced by FIGS. 1A, 1B, and 1C, and FIG. 2. FIG. 1A depicts cell pellets (1 OD) from cultures expressing LucY from the rhaP_BAD promoter in response to rhamnose at the indicated concentrations. FIG. 1B is a graph that maps fluorescence response to rhamnose induction measured with a plate reader using excitation at 485 nm and emission at 528 nm. FIG. 1C is a photograph of LucY-transformed, fluorescent E. coli colonies on a plate containing 0.2% rhamnose.

Referring to the tubes and corresponding gel lanes in FIG. 2, LucY was expressed with a carboxyl terminal His6 tag using Lucigen's Expresso™-brand T7 cloning and expression system. LucY-His6 was purified by metal affinity chromatography. Fractions from purification including whole cell pellet (lane 1), insoluble pellet after lysis (lane 2), soluble supernatant (lane 3), flow through (lane 4), wash (lane 5) and elution fractions (lanes 6-10) with imidazole were photographed under UV light (upper panel) and are aligned with the corresponding gel lanes.

HI-Control BL21(DE3) cells harboring the LucY gene under the control of the T7 promoter in Lucigen's pETite C-His vector were induced with 1 mM IPTG for 4 hours. For expression under the control of the rhamnose-inducible promoter, LucY was cloned into pRham (Lucigen), transformed into 10 G cells and expression was induced with varying percentages of rhamnose. Again see FIGS. 1A and 1B.

Mammalian cell culture: LucY was cloned into Lucigen's mammalian expression plasmid, pME, which contains the constitutively active cytomegalovirus (CMV) promoter. Various mammalian cell lines have been transfected with this construct, including CHO-K1, COS-7, and HeLa cell lines. Cells exhibiting strong yellow fluorescence were observed when visualized with Nikon Eclipse TE2000-S epifluorescence microscope, fitted with a Diagnostic Instruments 11.2 color camera and long pass GFP filter cube. See FIGS. 1D (expression of LucY in the mammalian cells COS-7), 1E (expression of LucY in the mammalian cells CHO-K1), and 1F (expression of LucY in the mammalian cells HeLa).

LucY Purification:

LucY was expressed in HI-Control BL21(DE3) E. coli cells with a C-terminal 6× histidine tag and purified by nickel affinity chromatography. See FIG. 2 (described previously). Clarified lysate was loaded onto an equilibrated Ni-NTA column and then washed with 30 column volumes wash buffer final pH 8.0; 10 mM Imidazole, 1×PBS pH 7.4 (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na phosphate dibasic, heptahydrate, and 1.9 mM K phosphate, monobasic) and eluted in 5 mL fractions with 5 column volumes of elution buffer final pH 8.0; 500 mM imidazole, 1×PBS pH 7.4. Fluorescent elution fractions visualized under UV light were pooled and dialyzed overnight in 2 liters 150 mM NaCl, 50 mM Tris pH 7.5 at 4° C. LucY contained minimal aggregates after dialysis and was spun for 10 minutes at 10,000 rpm (12,062×g) to clarify prior to storage at −80° C. Final protein concentration was determined to be 8 mg/mL by Bradford assay.

LucY Fluorescence Properties:

The fluorescence characteristics of LucY and related MurB proteins from S. aureus, E. coli, and T. thermophilus were determined through measurements on a Tecan Infinite M1000 monochromator-based plate reader (Tecan Group AG, Männedorf, Switzerland). See FIG. 3 and Table 1. Excitation scans from 230 nm to 540 nm show maximal excitation at three wavelengths, 274 nm, 376 nm, and 460 nm.

TABLE 1
Fluorescence characteristics of LucY and other MurB homologs.
	Excitation	Emission		ΦF	ΦF	Brightness
	Max	Max	ε	based on	based on	(% of
	(nm)	(nm)	(M−1cm−1)	FAD	FMN	LucY)
FAD	270/375/452	522	11,900	—	0.032	9
FMN	268/376/448	525	12,500	0.273	—	82
LucY	276/377/460	528	11,662	0.356	0.351	100
S. aureus	271/377/460	527	13,511	0.224	0.221	73
E. coli	276/369/460	527	12,008	0.109	0.108	32
T. thermophilus	276/375/460	522	13,745	0.051	0.051	17
ε, extinction coefficient
ΦF, quantum yield
Published ΦF for FAD and FMN are 0.032 and 0.27, respectively.

Quantum yield estimates for LucY and other MurB family members were ascertained via the comparative method in relation to the well characterized FAD and flavin mononucleotide (FMN) cofactors. The quantum yield (Φ_F) of LucY was determined to be 0.349 and 0.357 using FAD or FMN as a reference standard, respectively. Brightness values were also ascertained by taking the product of the extinction coefficient and quantum yields for each fluorophore and are expressed as a percentage of LucY. Brightness values show that LucY enhances FAD brightness by 10-fold and that it is the brightest of the MurB homologs.

LucY Thermo-Tolerant Homologs:

Because of their extreme growth conditions, few fluorescent biological reporters are viable in thermophilic organisms. Thus, what are otherwise routine techniques using GFP are not possible in many thermophilic organisms. Enhanced thermo-tolerant fluorescent proteins also have great potential in the thermo-stabilization of GPCRs suitable for crystallization. Thus, a search was conducted for thermo-tolerant LucY homologs using the BLAST function of UniProt (www.uniprot.org). Candidate homologs that originated from thermophilic organisms were selected for further investigation. Seventeen of these genes (Table 2) were synthesized de novo with their coding sequences optimized for expression in E. coli (SEQ. ID. NOS. 5-76) and cloned under the control of an inducible promoter (DNA2.0-brand; DNA 2.0, Inc., Menlo Park, Calif., USA). Cultures were grown at 37° C. in LB medium and expression was induced with IPTG. Induced cells were harvested by centrifugation and cell pellets were visualized with a hand-held UV lamp. See FIG. 4A. Proteins whose expression resulted in substantial fluorescence were purified by nickel affinity chromatography. The effect of temperature on fluorescence output was evaluated by incubating known quantities of protein at temperatures ranging from 30° C. to 80° C. and recording fluorescence every 15 minutes for 45 minutes in a Biotek Synergy 2 microplate fluorometer. LucY homologs from Y4.1MC1 and Thermoanaerobacter italicus (Thit) were the most thermostable LucY homologs in terms of fluorescence, with fluorescence diminishing to approximately 10% at 75.2° C. and 79.0° C. respectively. See FIG. 4B. Because Thit and Pma (Persephonella marina) were visually the brightest appearing homologs, quantum yield measurements were taken as stated above and are approximately, 0.485 and 0.406, respectively. FIG. 4C is a plot of percent fluorescence versus temperature following the 45 min incubation at temperatures ranging from 30° C. to 80° C. Fluorescence is expressed as a percentage of highest fluorescence recorded across temperature range.

TABLE 2
Thermo-tolerant LucY homologs
UniProt	Organism	Abbreviation
A0LRK5	Acidothermus cellulolyticus	Acel
F8IH59	Alicyclobacillus acidocaldarius	Aac
O66805	Aquifex aeolicus	Aae
E4Q8N4	Caldicellulosiruptor hydrothermalis	Chy
B8E323	Dictyoglomus turgidum	Dtu
E3IC15	Geobacillus strain Y4.1MC1	Y4.1MC1
G2SID5	SG0.5JP17-172	Rma
C4FHX3	Sulfurihydrogenibium yellowstonense SS-5	Sye
B5YFT2	Thermodesulfovibrio yellowstonii strain	Tye
	ATCC 51303
Q9X239	Thermotoga maritima strain ATCC 43589	Tma
B2V7Y9	Sulfurihydrogenibium sp. (strain YO3AOP1)	SspY
C1DVM7	Sulfurihydrogenibium azorense	Saz
C0QUP5	Persephonella marina	Pma
A8UZI2	Hydrogenivirga sp. 128-5-R1-1	Hsp1
D3DK91	Hydrogenobacter thermophilus	Hth
D3SPD6	Thermocrinis albus	Tal
D3T3U7	Thermoanaerobacter italicus	Thit
B4U6R2	Hydrogenobaculum sp. (strain Y04AAS1)	HspY

LucY as a Fluorescent Protein Fusion Partner and as an Indicator of Protein Expression:

LucY has been used successfully as a reporter of protein expression. Fusing the LucY nucleotide sequence (and its variants and homologs) to various proteins of interest at different junction points have resulted in bright fluorescence.

• • Expression in bacterial cells: C-terminal fusions to G-protein coupled receptor (GPCR), specifically A1a, have been visualized by bright yellow fluorescence at both 25° C. and 30° C. See FIG. 5A. In the figure, LSP=low-speed pellet; HSP=high-speed pellet. FIG. 5B corresponds to FIG. 5A and depicts LucY fused to ATPb following induction with IPTG or arabinose. FIG. 5C is a coomassie stained SDS-PAGE of fractions from E. coli expressing the LucY-ATPb fusion shown in FIG. 5B. GPCR-containing extracellular membranes separate with the high-speed pellet (HSP). ATPb separates with intracellular membranes in the low-speed pellet (LSP). HSS=high-speed supernatant; LSS=low-speed supernatant. Similar data have been obtained when LucY was fused to the COR-kinase domain of the Parkinson's disease related protein LRRK2. See FIG. 6, which is a photograph under UV light of the E. coli host cells transformed to contain and express the COR-kinase-LucY fusion protein.
  • Expression in mammalian cells: Turkey β1-adrenergic receptor fused to LucY in the 3^rd intracellular loop has been expressed in mammalian cells (human embryonic kidney cells; HEK-293T) under the control of the CMV promoter. The recombinant receptor is expressed as indicated by visible yellow fluorescence. Varying the junction points of insertion did not alter the fluorescence of the fusion protein. See FIGS. 7A, 7B, 7C, and 7D. Each figure is three-part photo series depicting expression of LucY as an intracellular loop fusion in mammalian cells. Each three-part series shows the fusion illuminated with a bright field (left), using a GFP filter (middle), and a merged bright field/GFP image (right). Each set of photos represents fusion at a different site within the intracellular loop 3 of a GPCR. FIG. 7A illustrates fusion at positions 244-272; FIG. 7B at positions 244-GSG-272; FIG. 7C at positions 244-GSG-275; and FIG. 7D at positions 244-GSG-278. GSG is the linker sequence between LucY C-terminus and the GPCR.
  • Expression in insect cells: The same intracellular loop constructs used for expression in HEK-293T were also used to express LucY as a fusion protein in insect cells. When expressed in baculovirus-infected insect cells (“Hi 5”-brand cells), the cells exhibited positive protein expression as evidenced by the yellow fluorescence in the cell pellets. See FIG. 8, which is a photograph of the Hi-5 host cells transformed to express the LucY fusion protein.

LucY as a Label to Determine Subcellular Localization:

A genetically encoded fluorescent marker is extremely useful for determining cellular localization. LucY is a soluble, highly expressed protein amenable to fusion to a variety of proteins. Its fluorescence can be used to track the location of its fusion partner. The GPCR A1a protein preferentially fractionates with extracellular membranes, which can be separated by centrifugation at a low speed and visualized by fusion to LucY. See FIG. 5A, discussed earlier. Alternatively, the single-pass transmembrane protein ATPb fractionates with internal membranes following centrifugation at high speeds and can likewise be visualized by fusion to LucY. See FIGS. 5B and 5C, described previously.

LucY as an Indicator of Detergent Solubilization:

Tracking the fluorescence in the soluble fraction of a detergent solubilized sample for integral membrane proteins offers an easy and quick solution for high throughput screening of different solubilization combinations. ATPb (a single-pass membrane protein) was used as a typical example of a membrane protein to demonstrate the effectiveness of different screening conditions. Thirty-one (31) different detergents belonging to various classes were tested. Solubilization was performed over night at 4° C. in an end-over rotor. The solubilized fraction was clarified with a high-speed centrifugation step and fluorescence was measured (FIG. 9A) as well as visualized (FIG. 9B) under UV light.

LucY as an Indicator of Protein Expression and Solubility.

Fluorescence enables straightforward visual evaluation of the expression and solubility of proteins fused to LucY. This visual readout can be exploited to identify expression conditions that improve protein expression, and to screen a variety of fusion partners that may promote soluble expression.

A large-scale effort to identify hydrolytic enzymes from Fibrobacter succinogenes identified several enzymes that were initially poorly expressed or insoluble in E. coli using a T7 bacteriophage polymerase expression system. Several of these recalcitrant proteins were cloned under control of the rhaP_BAD promoter with carboxyl terminal fusions to LucY, with or without an amino terminal SUMO solubility tag. Expression of the fusion proteins was induced by inclusion of 0.2% rhamnose in plates or in liquid media. Fluorescence was monitored in colonies, in cell pellets from liquid cultures, and in soluble and insoluble fractions of induced cell lysates.

Results are shown in FIGS. 10A, 10B, and 10C for three different proteins exhibiting different expression levels. In the case of Fisuc_1793, no yellow fluorescence was observed in the absence of SUMO (FIG. 10A, left), and fusion to an amino-terminal SUMO tag led to a dramatic increase in yellow fluorescence (FIG. 10A, right). See also the top pair of tubes in FIG. 10B. A second example, Fisuc_2201, produced moderate yellow fluorescence in the absence of SUMO, but increased fluorescence with the SUMO tag (FIG. 10B, middle pair of tubes). A third case, Fisuc_2442, produced strong fluorescence that was not significantly enhanced by the SUMO tag (FIG. 10B, bottom pair of tubes).

Lysates from the induced cells were centrifuged to separate soluble and insoluble fractions, and fluorescence, when present, was found primarily in the supernatant (soluble) fraction in each case. Gel analysis of fusion protein expression by SDS-PAGE correlated well with the fluorescence results. See FIG. 10C (top panel is for 1793; middle panel for 2201; bottom panel for 2442). No detectable Fisuc_1793-LucY fusion protein was present in either the soluble or insoluble fraction when expressed without the SUMO tag, while the SUMO tagged counterpart was expressed well and in a mostly soluble form. Results with Fisuc_2201 and Fisuc_2442 similarly recapitulated the fluorescence data, with the SUMO tag enhancing the relatively weak expression of Fisucc_2201, but not significantly increasing the already strong expression of Fisuc_2442.

A potential limitation of whole cell fluorescence as an indicator of soluble expression is the possibility of false positives. Fluorescence may be observed, for example, if the proper folding of LucY is allowed despite formation of insoluble aggregates via a fusion partner. Importantly, the stable fluorescence of LucY allows the preparation and fractionation of lysates to evaluate the partitioning of fluorescence between soluble and insoluble fractions. Fluorescent fusion proteins that are found exclusively in the insoluble fraction can be eliminated from further consideration, potentially saving the time and expense of analysis by gel electrophoresis. Alternatively, the fluorescence can be used to screen for conditions that allow solubilization of the protein. For example, the use of nonionic detergents, or different buffer conditions (salt concentrations or pH) may allow dispersal of fluorescent aggregates and recovery of soluble protein.

Fluorescence can also be used as the basis for a solubility “trap” screen, in which an insoluble protein fused to LucY is used to identify for novel fusion partners that impart greater levels soluble expression. Two different insoluble proteins were used to evaluate this screening strategy. In both cases LucY was fused to the C terminus of an insoluble DNA polymerase. The DNA polymerase (DNAP) genes were derived from Acidianus bottle-shaped virus (ABV DNAP), or from a screen for novel thermostable DNAPs (“4110” DNAP). These DNAP-LucY fusions were cloned under the rhaP_BAD promoter. Fluorescence development and expression were then monitored both on plates and in liquid media with and without 0.2% rhamnose. The effect of an amino-terminal SUMO fusion on the fluorescence development and solubility of each DNAP-LucY fusion protein was also tested. The ABV-LucY fusion protein was poorly soluble, and its solubility was not enhanced by the amino-terminal SUMO tag. The low-level solubility of the SUMO-ABV-LucY fusion resulted in weak yellow fluorescence on plates containing 0.2% rhamnose. See FIG. 11A. The 4110-LucY fusion was also largely insoluble, but fusion to SUMO partially rescued solubility. Both ABV-LucY and 4110-LucY were exploited as “solubility traps” in a screen for solubility-enhancing tags. FIG. 11B depicts the gel analysis of ABV-LucY protein expressed from rhaP_BAD, with or without an amino-terminal SUMO tag. FIG. 11C depicts the gel analysis of 4110-LucY protein expressed from rhaP_BAD, with or without a SUMO solubility tag. In FIGS. 11B and 11C, T represents total cell lysate; S represents the soluble fraction after centrifugation of the lysate; and P represents the insoluble pellet fraction after centrifugation.

For each DNAP-LucY fusion, separate libraries were constructed using genomic fragments from several sources including E. coli, bacteriophage lambda, and a thermophilic Geobacillus species. In each case, genomic DNA was physically sheared by nebulization to generate random fragments ranging in size from ˜100-700 bp. The DNA fragment ends were made blunt and phosphorylated using Lucigen's DNA terminator kit, and the fragments were cloned into the rhaP_BAD expression vector between the ATG start codon and the second residue of the ABV-LucY or 4110-LucY solubility trap fusion. Library transformants were plated on media containing kanamycin and 0.2% rhamnose. In these small-scale test screens, approximately 5,000 to 10,000 clones were plated on each of 6 to 10 large-diameter (13 cm) plates. Plates were observed under illumination from a hand-held long-wavelength UV lamp, and colonies with varying degrees of fluorescence were detected. See FIG. 12A for an example of a primary screening plate and FIG. 12B for an example a secondary screen plate with re-streaked candidates. The 4110-LucY fusion was screened with bacteriophage λ, and Geobacillus genomic inserts, and the ABV-LucY solubility trap was screened with E. coli genomic inserts.

The ABV-LucY fusion was first screened with genomic inserts from E. coli. In this screen, bright fluorescent colonies were observed at a frequency of <0.1%. Twenty-two (22) of these bright fluorescent colonies were chosen for further analysis. All 22 were found to have significant deletions removing large portions of the ABV sequence, re-creating an in-frame fusion that presumably resulted in expression of an ABV-LucY fragment with increased solubility. While these results illustrate the utility of LucY fusions to map soluble domains of poorly-soluble proteins, the objective of the screen is to identify novel solubility tags. These deletion clones were not analyzed further.

Screening of the 4110-LucY solubility trap yielded several candidate solubility-enhancing tags. Small-scale screens of the 4110-LucY construct were conducted with two different genomic insert libraries derived from bacteriophage λ, and from a thermophilic Geobacillus species. Partial deletions of the 4110 DNAP coding region were obtained far less frequently than with the ABV-LucY trap construct. For the λ, insert library, 24 fluorescent clones were selected for analysis. Sequences were obtained for 23 of these clones, and all 23 were found to contain inserts of λ, genomic DNA. Twenty-two (22) clones were found to have fusions that restored the correct frame between the ATG initiation codon and the 4110-LucY coding region. A single clone contained a fragment that included a promoter and the amino-terminal portion of a protein coding region, fused in-frame to 4110-LucY. The genomic inserts ranged in size from 93 to 669 base-pairs, and encoded peptides of 31 to 223 residues. Interestingly, two different polypeptide regions were each represented twice by non-identical clones. The repeated isolation of these regions among only 23 clones of 100-700 base-pairs from the 48 kb lambda genome strongly suggests non-randomness in the screen results. Clones were grown in liquid LB media and induced with 0.2% rhamnose for preparation of lysates to evaluate solubility. An example of a library fusion showing a significant increase in solubility of the 4110-LucY protein is presented in FIGS. 13A and 13B. These two figures demonstrate the enhanced soluble expression of 4110-LucY fusion protein with amino-terminal fusion tags derived from the shotgun library screens. FIG. 13A is a gel analysis of soluble expression of Geobacillus-4110-LucY library clone 11. The Control sample is 4110-LucY with no amino-terminal fusion. T represents total cell lysate; S represents the soluble fraction after centrifugation of the lysate; and P represents the insoluble pellet fraction after centrifugation. FIG. 13B is a photograph depicting increased partitioning of yellow fluorescence to the soluble fraction with Geobacillus library clone 11. Again, S represents the soluble fraction after centrifugation of the lysate and P represents the insoluble pellet fraction after centrifugation.

The Split-LucY System for Protein-Protein Interaction Studies:

The ability of a monomeric protein to be split and reassembled was first demonstrated with ubiquitin (Johnsson and Varshavasky, 1994) and has since been adopted for use with reporter proteins like GFP (Ghosh et al., 2000) and luciferase (Paulmurugan and Gambhir, 2003). LucY is a 32.7 kDa protein made up of three discrete domains, suggesting candidate split points lie between domains. FIG. 14 presents a schematic diagram of the three domains of the native LucY. In practice, a reporter protein is split in half and each half is expressed as a translational fusion to two proteins of interest for which protein-protein interaction is a possibility and their interaction is assayed in living cells. The reporter protein provides an output signal, like fluorescence, only if its two halves are brought together in a complementary manner due to the interaction of the fusions. This is illustrated schematically in FIG. 15A. The yellow fluorescence emitted by the FAD-binding capacity of LucY is an ideal reporter of protein-protein interaction.

Five points were tested between the short loop between domains 1 and 2 (residues 84 to 88) and within the long loop between domains 2 and 3 (residues 217-234) of LucY. See FIG. 15B, which depicts these split points schematically. Antiparallel leucine zippers were used as an idealized model for interacting protein partners (Ghosh et al., 2000). Because these leucine zippers interact in an antiparallel fashion, one fragment of LucY was fused at the amino-terminus of a leucine zipper (Split Points SPaNZ1-5 and SPbNZ1-5) while the complementary fragment was fused to the carboxy-terminus of the partner leucine zipper (SPaCZ1-5 and SPbCZ1-5). This is depicted schematically in FIG. 15C. The letters a and b and numbers 1 through 5 indicate the location of the split; see Table 3. Matching split point numbers indicates that the CZ split point immediately follows the residue at the carboxy end of the NZ split point. Fusion pairs containing complementary LucY fragments (e.g. SPaNZ1+SPaCZ1; SPbNZ4+SPbCZ4, etc.) were tested for protein expression and for fluorescence complementation. Fluorescence produced by coexpressed fragment pairs fused to leucine zippers was compared to fluorescence of fully intact LucY fused to either the N- or C-terminus of one of the zippers (NZ and CZ). Fluorescence was determined visually in whole cell pellets and quantitatively by a fluorometer.

TABLE 3
Start and end residues for each split point (SP). NZ constructs
start with a 6x-His tag. CZ constructs end with the linker sequence
SLSTPPTPSTPPT, followed by an Avi-tag. Constructs destined
for expression in mammalian cells instead contain an HA-tag. Split
Pairs c and d were constructed from a circular permutation construct.
	Start	End	includes domains:
	SPaNZ1	D2	G84	1
	SPaNZ2	D2	A85	1
	SPaNZ3	D2	G86	1
	SPaNZ4	D2	L87	1
	SPaNZ5	D2	D88	1
	SPaCZ1	A85	R303	2&3
	SPaCZ2	G86	R303	2&3
	SPaCZ3	L87	R303	2&3
	SPaCZ4	D88	R303	2&3
	SPaCZ5	H89	R303	2&3
	SPbNZ1	D2	P217	1&2
	SPbNZ2	D2	P221	1&2
	SPbNZ3	D2	S225	1&2
	SPbNZ4	D2	N229	1&2
	SPbNZ5	D2	H234	1&2
	SPbCZ1	V218	R303	3
	SPbCZ2	C222	R303	3
	SPbCZ3	I226	R303	3
	SPbCZ4	P230	R303	3
	SPbCZ5	A235	R303	3
	SPcNZ1	L87	P217	2
	SPcCZ1	V218	G86	3&1
	SPcNZ2	L87	H234	2
	SPcCZ2	A235	G86	3&1
	SPdNZ1	P217	G86	3&1
	SPdCZ1	L87	Q216	2

Fragmenting LucY at any of the five residues between domains 1 and 2 did not result in fluorescence when fused to leucine pairs and coexpressed (FIG. 16A). A contributing factor may have been the low expression of the CZ half of each of the split points in comparison to the complementary NZ half. See the gel shown in FIG. 17A. However, bright fluorescence was seen in four out of the five pairs split between domains 2 and 3, with highest fluorescence seen with the SPbNZ5 (ending at His 234) and SPbCZ5 partners (beginning at A1a 235). See FIG. 16B. All fusions expressed well and expression did not correlate with fluorescence. See the gel shown in FIG. 17B. Interestingly, the one split point combination between domains 2 and 3 that did not show fluorescence was at Ser 225, a residue previously shown to be important for catalysis in other MurB proteins (Benson et al., 1995).

Because the SPbNZ5 and SPbCZ5 when reconstituted showed the highest fluorescence, each was tested with other split points between domains 2 and 3 to determine if overlap, or alternatively gaps, in the amino acid sequence would affect fluorescence. Pairing SPbNZ5 with SPbCZ4 was the most successful reconstituted pair and contains a four-residue overlap with SPbNZ5 ending at residue 234 and SPbCZ4 beginning at residue 230. Interestingly, the opposite pair (SPbNZ4 and SPbCZ5) did not show substantial fluorescence. See FIG. 16C. All pairs showed some level of expression. See FIG. 17C.

To verify that neither LucY fragment was fluorescent on its own and that the leucine zippers were driving the interaction, SPbNZ5 and SPbCZ4 were tested independently and without their leucine zipper fusions. Neither SPbNZ5 nor SPbCZ4 were fluorescent on their own. However, when expressed on its own, SPbNZ5 was less soluble than when coexpressed with SPbCZ4. Removal of the leucine zipper from SPbNZ5 did not affect expression, while removal of the leucine zipper from SPbCZ4 caused a loss of expression. See FIG. 18A. Because the zipperless SPbC4 was not expressed, it was not possible to fully obtain the background fluorescence level of the SPb:NZ5+CZ4 pair. Therefore, the fluorescence of the remaining SPbCZ fragments that exhibited fluorescence were used as a measure when coexpressed with SPbNZ5. Only SPbCZ1 and SPbCZ5 expressed when their zippers were removed. See FIG. 18B. Therefore these were chosen for further study. It was found that the SPb:NZ5+CZ5 pair possessed the highest level of fluorescence (33% of CZ control) with the least background (8% of CZ control). See FIG. 16D, and the corresponding gel showing expression in FIG. 17D.

Because LucY fluorescence is dependent on the binding of a non-covalently attached small molecule, fluorescence emission will dissipate following separation of the interacting pairs and release of the small molecule. Thus, the split-LucY system is reversible making it possible to test for inhibitors of protein interaction.

Circular Permutation of LucY:

Reorganization of a polypeptide chain, called circular permutation, has been used as a means to introduce change into a protein scaffold without amino acid substitutions (Yu and Lutz, 2011) and has been successfully investigated with GFP (Baird et al., 1999). A welcome consequence to domain reorganization of LucY would be an increase in fluorescence due to increased binding affinity to FAD. Novel termini also offer variation in points of attachment to fusion partners, as well as new possibilities in split points. The wild-type LucY amino- and carboxy-termini are 16.7 Å apart and thus require a greater than four-residue linker to span the intervening space. To build a circularly permuted LucY, two tandem repeats were constructed with a 6-residue linker connecting the C-terminus of one copy to the N-terminus of the second. Deletions within this construct were performed such that new N and C termini were introduced between either domains 1 and 2 or between domains 2 and 3. This is shown schematically in FIG. 19A. The same residues used as split points above were used as breakpoints (BPs) here. Ten (10) circularly permuted LucY proteins were made. BP1-5 comprised a 2-3-1 domain arrangement, while BP6-10 comprised a 3-1-2 domain arrangement. See Table 4.

TABLE 4
N- and C-terminal residues of LucY circular permutations,
with order of domain occurrence listed. Amino acid
numbering is that of wild-type.
	N-term	C-term	Domain arrangement
	BP1	G84	L83	2-3-1
	BP2	A85	G84	2-3-1
	BP3	G86	A85	2-3-1
	BP4	L87	G86	2-3-1
	BP5	D88	L87	2-3-1
	BP6	P217	Q216	3-1-2
	BP7	P221	N220	3-1-2
	BP8	S225	G224	3-1-2
	BP9	N229	R228	3-1-2
	BP10	H234	D233	3-1-2

Circularly permuted LucY proteins all showed some degree of fluorescence. See the histogram of FIG. 19B. However, none of the circularly permuted LucY variations were any brighter than the wild-type. The best candidate was BP6, which has a 3-1-2 domain arrangement beginning at the long loop connecting domains 2 and 3 in wild-type. Four out of the 10 circular permutation trials (BP4, BP7, BP9 and BP10) resulted in poorly expressed variants. See the gel of FIG. 20A. New split points became available by reorganizing the domain architecture of LucY, such that domains 3 and 1 could make up one LucY fragment and domain 2 could make up the other. To determine if a split-LucY system of this type was more viable then previous trials, three (3) additional split pairs were made: SPcNZ1/CZ1, SPcNZ2/CZ2, and SPdNZ1/CZ1. SPc pairs comprise domain 2 as the SPNZ fragment and domains 3 and 1 as the SPCZ fragment, with the numbers indicating different start and end locations. SPd comprises the opposing pairing, with domains 3 and 1 as the SPNZ fragment and domain 2 as the SPCZ fragment. See Table 3. These new split pairings did not exhibit fluorescence when reconstituted despite high levels of expression. See FIG. 19C and FIG. 20B, which further show that a domain 1+2/domain 3 pair (as in SPb fusions) offers the best split-LucY system.

Protein complementation assays like BiFC are often used in higher order systems, such as mammalian cells. Therefore, the best leucine zipper split-LucY pair from the above testing (SPbNZ5+SPbCZ4) was tested in HEK 293-T cells (a mammalian host cell). Only when each fragment was present did fluorescence occur. The results are depicted in the photographic series of FIGS. 21A through 21J. FIGS. 21A, 21B, 21C, 21D, and 21E are band pass filter photos of NZ-transformed cells, CZ-transformed cells, SPbNZ5-transformed cells, SPbCZ4-transformed cells, and SPbNZ5+SPbCZ4-transformed cells, respectively. FIGS. 21F, 21G, 21H, 21I, and 21J are merged band pass and bright field filter photos of NZ-transformed cells, CZ-transformed cells, SPbNZ5-transformed cells, SPbCZ4-transformed cells, and SPbNZ5+SPbCZ4-transformed cells, respectively. FIG. 21K is a photograph of an immunoblot gel using anti-HA to detect HA-tagged leucine zipper/LucY fusions either whole, NZ and CZ, or splits SPbNZ5 and SPbCZ4.

LucY as a Visualization Tool for Crystallization:

Typically protein crystals are visualized using a standard light microscope. When LucY was crystallized for the first time, bright fluorescent yellow crystals were visualized. Observation of the fluorescent crystals opens up a new avenue for the application of LucY in which crystal detection is problematic due to small crystal size, murky mother liquor, and/or excessive precipitation. One such challenging application is growth of membrane crystals in Lipidic Cubic Phase (LCP) (Caffrey, 2009), which mimics the lipid environment in which membrane proteins are most stable. Fusion of LucY to a membrane protein of interest provides a fluorescent beacon for successful crystallization. LucY crystals themselves form within 24 hours in a variety of conditions using a conventional hanging drop method and were easily visualized using a UV light source and light microscope. FIG. 22A is an exemplary photograph. LucY was fused to the GPCR, β1-adrenergic receptor and the resulting fusion protein crystallized using LCP. Screening for crystals within a LCP matrix was visualized with Nikon Eclipse TE2000-S epifluorescence microscope, fitted with a Diagnostic Instruments 11.2 color camera and long pass GFP filter cube. Exemplary photographs of the fusion protein (LucY-β1-adrenergic receptor) in a LCP matrix are shown in FIGS. 22B, 22C, 22D, and 22E.

LucY as a Solubility-Enhancing Fusion Partner:

Solubility-enhancing fusion partners such as maltose binding protein (MBP), thioredoxin (TRX), small ubiquitin-like modifier (SUMO), glutathione S-transferase (GST), NusA, and others are most frequently employed as fusions to the amino terminus of the protein of interest. In this context, fusion of LucY to the carboxyl terminus of the target protein provides a useful visualization tag to evaluate solubility of the fusion protein. Examples presented above illustrate this application of LucY.

In addition to functioning as a visual indicator of soluble expression, it has been found that fusion to LucY at the carboxyl terminus of a target protein can also enhance the soluble expression of the protein, regardless of fusion to an amino-terminal partner. FIGS. 23A, 23B, and 23C present an example in which the Tobacco Etch Virus (TEV) protease was expressed in E. coli from the rhamnose-inducible rhaP_BAD promoter. Consistent with previous studies (Kapust and Waugh 1999; van den Berg et al. 2006), TEV protease expressed with a C-terminal 6×His tag was found almost exclusively in the insoluble (pellet) fraction after centrifugation of a cell lysate. In contrast, expression of TEV protease with LucY fused to its C-terminus resulted in expression of a large proportion of the TEV-LucY fusion protein in a soluble form. See FIGS. 23A and 23B. The proportion of TEV-LucY fusion protein found in the soluble fraction is comparable to that reported with an amino-terminal MBP-TEV fusion protein, and greater than that observed with amino-terminal GST or TRX (Kapust and Waugh 1999). Thus, LucY effectively promotes the soluble expression of TEV protease when fused to the protease as a C-terminal partner.

The TEV-LucY-His6 fusion protein was purified by nickel-affinity chromatography and assayed for sequence-specific protease activity using a purified substrate consisting of two similar-sized fluorescent proteins joined by a linker containing the TEV protease recognition sequence, ENLYFQ/G. FIG. 23C shows that incubation of the TEV-LucY-His6 fusion protein with the substrate protein results in generation of products. Thus the TEV-LucY-His6 fusion protein exhibits the sequence-specific proteolytic activity of TEV protease.

REFERENCES CITED

The following documents are incorporated herein by reference.

• Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215:403-410 (1990).
• Baird, G. S., Zacharias, D. A. & Tsien, R. Y. Circular permutation and receptor insertion within green fluorescent proteins. Proc Natl Acad Sci USA 96, 11241-11246 (1999).
• Benson, T. E., Filman, D. J., Walsh, C. T. & Hogle, J. M. An enzyme-substrate complex involved in bacterial cell wall biosynthesis. Nat Struct Biol 2, 644-653 (1995).
• Caffrey M. Crystallizing membrane proteins for structure determination: use of lipidic mesophases. Annu Rev Biophys 38, 29-51 (June 2009).
• Chen J, Zheng X F, Brown E J, Schreiber S L (1995) Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. Proc Natl Acad Sci USA. 92:4947-4951.
• Day, R. N. & Davidson, M. W. The fluorescent protein palette: tools for cellular imaging. Chem Soc Rev 38, 2887-2921 (2009).
• El Zoeiby, A., Sanschagrin, F. & Levesque, R. C. Structure and function of the Mur enzymes: development of novel inhibitors. Mol Microbiol 47, 1-12 (2003).
• Farrar M A, Alberol-Ila J, Perlmutter R M (1996) Activation of the Raf-1 kinase cascade by coumermycin-induced dimerization. Nature. 383:178-181.
• Ghosh, I., Hamilton, A. & Regan, L. Antiparallel leucine zipper-directed protein reassembly: Application to the green fluorescent protein. J. Am. Chem. Soc. 122, 5658-5659 (2000).
• Gilbert E J, Maxwell A (1994) The 24 kDa N-terminal sub-domain of the DNA gyrase B protein binds coumarin drugs. Mol Microbiol. 12:365-373.
• Johnsson, N. & Varshaysky, A. Split ubiquitin as a sensor of protein interactions in vivo. Proc Natl Acad Sci USA 91, 10340-10344 (1994).
• Munro, A. W. & Noble, M. A. Fluorescence analysis of flavoproteins. Methods Mol Biol 131, 25-48 (1999).
• Paulmurugan, R. & Gambhir, S. S. Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation. Anal Chem 75, 1584-1589 (2003).
• Robida A M, Kerppola T K (2009) Bimolecular fluorescence complementation analysis of inducible protein interactions: effects of factors affecting protein folding on fluorescent protein fragment association. J Mol Biol. 394:391-409.
• Rollins C T, Rivera V M, Woolfson D N, Keenan T, Hatada M, Adams S E, Andrade L J, Yaeger D, van Schravendijk M R, Holt D A, Gilman M, Clackson T (2000) A ligand-reversible dimerization system for controlling protein-protein interactions. Proc Natl Acad Sci USA. 97:7096-8101.
• Tsien, R. Y. The green fluorescent protein. Annu Rev Biochem 67, 509-544 (1998).
• Yu, Y. & Lutz, S. Circular permutation: a different way to engineer enzyme structure and function. Trends Biotechnol 29, 18-25 (2011).
• Zhao H F, Boyd J, Jolicoeur N, Shen S H (2003) A coumermycin/novobiocin-regulated gene expression system. Hum Gene Ther. 14:1619-1629.

Claims

1. A recombinant polynucleotide comprising a nucleotide sequence encoding a fluorescent protein which is at least 90% sequence identical to a protein having a sequence selected from the group consisting of SEQ. ID. NOS: 22, 26, 34, 46, 50, 54, 66, 70, and 74.

2. The recombinant polynucleotide of claim 1, wherein the nucleotide sequence further encodes at least one additional polypeptide of interest in-frame with the encoded protein.

3. The recombinant polynucleotide of claim 2, wherein the at least one additional polypeptide of interest comprises a tag.

4. The recombinant polynucleotide of claim 3, wherein the tag comprises a poly-His tag.

5. The recombinant polynucleotide of claim 1, wherein the encoded protein is thermostable.

6. The recombinant polynucleotide of claim 1, wherein fluorescence of the encoded protein at about 50° C. is greater than about 10% of fluorescence of the encoded protein at about 30° C.

7. The recombinant polynucleotide of claim 1, wherein fluorescence of the encoded protein at about 60° C. is greater than about 10% of fluorescence of the encoded protein at about 30° C.

8. The recombinant polynucleotide of claim 1, wherein fluorescence of the encoded protein at about 70° C. is greater than about 10% of fluorescence of the encoded protein at about 30° C.

9. The recombinant polynucleotide of claim 1, wherein fluorescence of the encoded protein at about 75° C. is greater than about 10% of fluorescence of the encoded protein at about 30° C.

10. The recombinant polynucleotide of claim 1, wherein the nucleotide sequence is optimized to express the fluorescent protein in a heterologous host.

11. The recombinant polynucleotide of claim 1, wherein the nucleotide sequence encodes a fluorescent protein which is at least 95% sequence identical to a protein having a sequence selected from the group consisting of SEQ. ID. NOS: 22, 26, 34, 46, 50, 54, 66, 70, and 74.

12. An expression construct comprising a recombinant polynucleotide as recited in claim 1.

13. A host cell comprising an expression construct as recited in claim 12.

14. A method for making a fluorescent protein comprising cultivating the host cell of claim 13.

15. A fluorescent protein made by cultivating a host cell, wherein the host cell comprises an expression construct, the expression construct comprises a recombinant polynucleotide, and the recombinant polynucleotide comprises a nucleotide sequence encoding a fluorescent protein which is at least 90% sequence identical to a protein having a sequence selected from the group consisting of SEQ. ID. NOS: 22, 26, 34, 46, 50, 54, 66, 70, and 74.

16. A fusion protein comprising a polypeptide of interest operationally linked to a fluorescent protein which is at least 90% sequence identical to a protein having a sequence selected from the group consisting of SEQ. ID. NOS: 22, 26, 34, 46, 50, 54, 66, 70, and 74.

17. A method of increasing the aqueous solubility of a protein of interest comprising fusing or attaching the protein of interest to a protein which is at least 90% sequence identical to a protein having a sequence selected from the group consisting of SEQ. ID. NOS: 22, 26, 34, 46, 50, 54, 66, 70, and 74.

18. A method of assaying gene expression comprising:

cultivating a host cell comprising an expression construct, wherein the expression construct comprises a recombinant polynucleotide and the recombinant polynucleotide comprises a nucleotide sequence encoding a fluorescent protein which is at least 90% sequence identical to a protein having a sequence selected from the group consisting of SEQ. ID. NOS: 22, 26, 34, 46, 50, 54, 66, 70, and 74; and

detecting fluorescence in the cell or an extract thereof.