Abstract: A multifunctional polypeptide capable of hydrolyzing cellulosic materials, xylan, and mannan is disclosed. The polypeptide includes the catalytic core (cc) of Clostridium thermocellum Cthe_0797 (CelE), the cellulose-specific carbohydrate-binding module CBM3 of the cellulosome anchoring protein cohesion region (CipA) of Clostridium thermocellum (CBM3a), and a linker region interposed between the catalytic core and the cellulose-specific carbohydrate binding module. Methods of using the multifunctional polypeptide are also disclosed.

Abstract: Methods for the cell-free identification of polypeptide and polypeptide combinations with utility in biomass transformation, as well as specific novel polypeptides and cell-free systems containing polypeptide combinations discovered by such methods are disclosed.

Abstract: A multifunctional polypeptide capable of hydrolyzing cellulosic materials, xylan, and mannan is disclosed. The polypeptide includes the catalytic core (cc) of Clostridium thermocellum Cthe_0797 (CelE), the cellulose-specific carbohydrate-binding module CBM3 of the cellulosome anchoring protein cohesion region (CipA) of Clostridium thermocellum (CBM3a), and a linker region interposed between the catalytic core and the cellulose-specific carbohydrate binding module. Methods of using the multifunctional polypeptide are also disclosed.

Abstract: Compositions and methods are provided that are useful for predicting and controlling the stability of expressed polypeptides. The compositions and methods may be used to predict and as desired, increase or decrease the stability of proteins recombinantly expressed in mycobacteria, for example DesA3 expressed in Mycobacterium smegmatis. At the C terminus and the penultimate position, substitution to residues with charged side chains, large non-polar side chains, or no side chains can be used to reduce or inhibit the protein degradation. At the antepenultimate position from the C terminus, residues with no side chain or acidic side chains can increase the stability, i.e. reduce or inhibit the protein degradation. The combinational substitution of only the last three residues of polypeptides can make the polypeptides more stable during heterologous expression in mycobacterial hosts.

Abstract: Compositions and methods are provided that are useful for predicting and controlling the stability of expressed polypeptides. The compositions and methods may be used to predict and as desired, increase or decrease the stability of proteins recombinantly expressed in mycobacteria, for example DesA3 expressed in Mycobacterium smegmatis. At the C terminus and the penultimate position, substitution to residues with charged side chains, large non-polar side chains, or no side chains can be used to reduce or inhibit the protein degradation. At the antepenultimate position from the C terminus, residues with no side chain or acidic side chains can increase the stability, i.e. reduce or inhibit the protein degradation. The combinational substitution of only the last three residues of polypeptides can make the polypeptides more stable during heterologous expression in mycobacterial hosts.

Abstract: Expression systems and methods for the expression of functional membrane polypeptides such as human cytochrome b5 are provided. The systems include recombinant expression vectors capable of expressing soluble fusion proteins that include a solubilizing agent, a linker, and a membrane polypeptide, as well as one or more cleavers, e.g. proteases, capable of cleaving the linker to release the membrane polypeptide. When the fusion protein is expressed, the linker is cleaved by the cleaver to allow association of the membrane polypeptide with a membrane.

Abstract: Expression systems and methods for the expression of functional membrane polypeptides such as human cytochrome b5 are provided. The systems include recombinant expression vectors capable of expressing soluble fusion proteins that include a solubilizing agent, a linker, and a membrane polypeptide, as well as one or more cleavers, e.g. proteases, capable of cleaving the linker to release the membrane polypeptide. When the fusion protein is expressed, the linker is cleaved by the cleaver to allow association of the membrane polypeptide with a membrane.

Abstract: Methods for the cell-free identification of polypeptide and polypeptide combinations with utility in biomass transformation, as well as specific novel polypeptides and cell-free systems containing polypeptide combinations discovered by such methods are disclosed.

Abstract: Mycobacterium tuberculosis proteins and protein compositions that are components of a desaturase complex are provided. The Mycobacterium tuberculosis desaturase complex may include a desaturase and an oxidoreductase. The complex may include the rv3229c and rv3230c gene products of Mycobacterium tuberculosis. Vectors for expressing the desaturase and the oxidoreductase can be packaged together, including a label that indicates their use as a complex for analyzing desaturation of fatty acids. In addition, methods for screening target ligands specific for a desaturase complex are also provided.

Abstract: The invention discloses a method of determining protease activity, in real time, using fluorescence polarization technology. In particular, the invention provides vectors and a method for their use, which expresses uncharacterized proteins conjugated to a fluorescence tag, which binds specifically to a fluorescent ligand. Cleavage of the recombinant protein results in a fragment of the expressed peptide and results in a change in fluorescence polarization of the fluorophore. The rate of change in fluorescence polarization can be measured in real time and is equivalent to the rate of protease cleavage.

Abstract: Methods for refining the compositions of bacterial growth media to improve heterologous expression of desired recombinant target genes are provided. Also provided are compositions and culture media obtained using the above methods.

Abstract: Mycobacterium tuberculosis proteins and protein compositions that are components of a desaturase complex are provided. The Mycobacterium tuberculosis desaturase complex may include a desaturase and an oxidoreductase. The complex may include the rv3229c and rv3230c gene products of Mycobacterium tuberculosis. Vectors for expressing the desaturase and the oxidoreductase can be packaged together, including a label that indicates their use as a complex for analyzing desaturation of fatty acids. In addition, methods for screening target ligands specific for a desaturase complex are also provided.

Abstract: Expression system for components of a desaturase complex is provided. The system includes expression of a desaturase and an oxidoreductase. The system may be used for expression of mycobacterial desaturases or for expression of mammalian desaturases. The system may further include cell-free expression of other components of the desaturase complex. The expression system may include expression of stearoyl-CoA. The expression system may further include expression of cytochrome b5. The expression system may also include expression of cytochrome b5 reductase. The expression system may also include expression of Rv3230c. In addition, methods for assaying the activity of a stearoyl-CoA desaturase in vitro are provided.

Abstract: Disclosed are apo-, holo-, and acylated-acyl carrier proteins modified to have a non-radioactive label covalently bonded to a modified tyrosine residue within the acyl carrier protein. Also disclosed are methods of using the labeled acyl carrier proteins to investigate reaction involving or mediated by acyl carrier proteins. Also disclosed are kits containing the non-radioactively-labeled acyl carrier proteins.

Abstract: A method is disclosed for degradation of a halogenated hydrocarbon compound such as trichloroethylene (TCE) which utilizes a soluble methane monooxygenase or a bacterium comprising the monooxygenase. Methylosinus trichosporium OB3b is a soluble methane monooxygenase-producing bacterium which when cultivated by continuous culturing comprising exposing the bacterium to a continuous-flow gas mixture of air and methane in a ratio of about 25:1-1:20, respectively. Methylosinus trichosporium OB3b is capable of degrading TCE at rates from about 500-10,000 micromoles per hour per gram cells. The present method is useful to degrade halogenated hydrocarbon compounds such as TCE at initial concentrations up to 10,000 micromoles/l.

Abstract: A purified hydroxylase component of the soluble methane monooxygenase enzyme present in the bacterium Methylosinus trichosporium OB3b is found capable of oxidizing hydrocarbons under aerobic conditions in the presence of suitable reducing agents. The hydroxylase can be reduced by commercial reducing agents, such as sodium dithionite and photo- and electrochemical means when in the presence of electron transport components, such as methyl viologen and proflavin. The hydroxylase component can be obtained with high final specific activity when ferrous iron compounds and cysteine are included in the purification buffers used to extract the hydroxylase from bacterial cells.

Abstract: A method of incorporating at least one fiber tube into a plastic pipe, such that the pipe becomes useable for carrying optical signals along the fiber optic tube as well for carrying a fluid such as water. The fiber optic tube is affixed to the wall of the plastic pipe during extrusion of the latter and in a first embodiment, the fiber optic tube is extruded with the pipe. In a second embodiment, the fiber optic tube is secured to the pipe when the pipe is in a heated state after extrusion.

Abstract: An anti-scald apparatus for a tub and shower single control faucet which incorporates a novel cam and cam follower arrangement on the hot and cold water valve means which permits substantially more than 180 degrees of rotation of the control handle. Adjustable temperature stops provide both a comfort stop for normal use and a safety stop which can be reached only by activation of a release button carried by the faucet handle by means of which the comfort stop may be overridden by continuing the rotation of the handle.

Abstract: An anti-scald apparatus for a tub and shower single control faucet which incorporates a novel cam and cam follower arrangement on the hot and cold water valve means which permits substantially more than 180 degrees of rotation of the control handle. Adjustable temperature stops provide both a comfort stop for normal use and a safety stop which can be reached only by activation of a release button carried by the faucet handle by means of which the comfort stop may be overridden by continuing the rotation of the handle.