Abstract: The present invention provides methods of systems and methods of site specific methylation.

Abstract: The present invention provides an in vitro directed evolution selection system to create modified methyltransferases which improve methyltransferase specificity and use it to optimize and provide fusion proteins comprising a zinc finger methyltransferase derived from M.SssI. The resulting fusion proteins show increased target methylation specificity and greatly decreased non-target methylation compared to wild-type enzyme activity. Methods of use of such fusion proteins in both prokaryotic and eukaryotic cells are also provided.

Abstract: The present invention features a novel protein engineering strategy by combining the domains of two independent proteins into a molecular switch. The invention features polypeptides comprising a prodrug activating enzyme and a protein that binds a cancer specific marker, polynucleotides encoding the polypeptides, and molecular switches for converting a prodrug into a toxin, comprising the polypeptides. The invention also features methods for converting a prodrug into a toxin, methods for treating cancer, and methods for making the molecular switches, as well as kits.

Abstract: The invention provides molecular switches which couple external signals to functionality and to methods of making and using the same. The switches according to the invention can be used, for example, to regulate gene transcription, target drug delivery to specific cells, transport drugs intracellularly, control drug release, provide conditionally active proteins, perform metabolic engineering, and modulate cell signaling pathways. Libraries comprising the switches and expression vectors and host cells for expressing the switches are also provided.

Abstract: The present invention features a novel protein engineering strategy by combining the domains of two independent proteins into a molecular switch. The invention features polypeptides comprising a prodrug activating enzyme and a protein that binds a cancer specific marker, polynucleotides encoding the polypeptides, and molecular switches for converting a prodrug into a toxin, comprising the polypeptides. The invention also features methods for converting a prodrug into a toxin, methods for treating cancer, and methods for making the molecular switches, as well as kits.

Abstract: A strategy to improve protein stability by domain insertion. TEM 1 beta-lactamase (BLA) and exo-inulinase, as model target enzymes, are inserted into a hyperthermophilic maltose binding protein from Pyrococcus furiosus (PfMBP). Unlike conventional protein stabilization methods that employ mutations and recombinations, the inventive approach does not require any modification on a target protein except for its connection with a hyperthermophilic protein scaffold. For that reason, target protein substrate specificity was largely maintained, which is often modified through conventional protein stabilization methods. The insertion was achieved through gene fusion by recombinant DNA techniques.

Abstract: The invention provides molecular switches which couple external signals to functionality and to methods of making and using the same. The switches according to the invention can be used, for example, to regulate gene transcription, target drug delivery to specific cells, transport drugs intracellularly, control drug release, provide conditionally active proteins, perform metabolic engineering, and modulate cell signaling pathways. Libraries comprising the switches and expression vectors and host cells for expressing the switches are also provided.

Abstract: The invention provides molecular switches which couple external signals to functionality and to methods of making and using the same. The switches according to the invention can be used, for example, to regulate gene transcription, target drug delivery to specific cells, transport drugs intracellularly, control drug release, provide conditionally active proteins, perform metabolic engineering, and modulate cell signaling pathways. Libraries comprising the switches and expression vectors and host cells for expressing the switches are also provided.

Abstract: The invention provides molecular switches which couple external signals to functionality, and combinatorial methods of making and using the same involving circular permutation of nucleic acid and amino acid sequences. The switches according to the invention can be used, for example, to regulate gene transcription, target drug delivery to specific cells, transport drugs intracellularly, control drug release, provide conditionally active proteins, perform metabolic engineering, and modulate cell signaling pathways. Libraries comprising the switches, expression vectors and host cells for expressing the switches are also provided.

Abstract: A series of methods that utilize the incremental truncation of nucleic acids are described to create a plurality of modified nucleic acids and hybrid polypeptides. A plurality of substantially all possible single base-pair deletions of a given nucleic acid sequence is created. A method of making shuffled incremental truncated nucleic acids, which is independent of nucleic acid sequence homology, is also described. These methods can be used in protein engineering, protein folding, protein evolution, and the chemical synthesis of novel hybrid proteins and polypeptides.

Abstract: A strategy to improve protein stability by domain insertion. TEM 1 beta-lactamase (BLA) and exo-inulinase, as model target enzymes, are inserted into a hyperthermophilic maltose binding protein from Pyrococcus furiosus (PfMBP). Unlike conventional protein stabilization methods that employ mutations and recombinations, the inventive approach does not require any modification on a target protein except for its connection with a hyperthermophilic protein scaffold. For that reason, target protein substrate specificity was largely maintained, which is often modified through conventional protein stabilization methods.

Abstract: The invention provides molecular switches which couple external signals to functionality, and combinatorial methods of making and using the same involving circular permutation of nucleic acid and amino acid sequences. The switches according to the invention can be used, for example, to regulate gene transcription, target drug delivery to specific cells, transport drugs intracellularly, control drug release, provide conditionally active proteins, perform metabolic engineering, and modulate cell signaling pathways. Libraries comprising the switches, expression vectors and host cells for expressing the switches are also provided.

Abstract: A series of methods that utilize the incremental truncation of nucleic acids are described to create a plurality of modified nucleic acids and hybrid polypeptides. A plurality of substantially all possible single base-pair deletions of a given nucleic acid sequence is created. A method of making shuffled incremental truncated nucleic acids, which is independent of nucleic acid sequence homology, is also described. These methods can be used in protein engineering, protein folding, protein evolution, and the chemical synthesis of novel hybrid proteins and polypeptides.

Abstract: A series of methods that utilize the incremental truncation of nucleic acids are described to create a plurality of modified nucleic acids and hybrid polypeptides. A plurality of substantially all possible single base-pair deletions of a given nucleic acid sequence is created. A method of making shuffled incremental truncated nucleic acids, which is independent of nucleic acid sequence homology, is also described. These methods can be used in protein engineering, protein folding, protein evolution, and the chemical synthesis of novel hybrid proteins and polypeptides.

Abstract: A series of methods that utilize the incremental truncation of nucleic acids are described to create a plurality of modified nucleic acids and hybrid polypeptides. A plurality of substantially all possible single base-pair deletions of a given nucleic acid sequence is created. A method of making shuffled incremental truncated nucleic acids, which is independent of nucleic acid sequence homology, is also described. These methods can be used in protein engineering, protein folding, protein evolution, and the chemical synthesis of novel hybrid proteins and polypeptides.

Abstract: Disclosed are methods of producing eukaryotic disulfide bond-containing polypeptides in bacterial hosts, and compositions resulting therefrom. Co-expression of a eukaryotic foldase and a disulfide bond-containing polypeptide in a bacterial host cell is demonstrated. In particular embodiments, the methods have been used to produce mammalian pancreatic trypsin inhibitor and tissue plasminogen activator (tPA) in soluble, biologically-active forms, which are isolatable from the bacterial periplasm. Also disclosed are expression systems, recombinant vectors, and transformed host cells.