KITS FOR PRODUCING RECOMBINANT POLYPEPTIDES VIA CYSTEINE PROTEASE AUTOPROCESSING OF FUSION PROTEINS

Research output: Patent

Abstract

Recombinant Protein Production and Purification without Affinity Tags NU 2008-069 Inventor Karla Satchell Abstract A Northwestern researcher has developed a new method for researchers to produce and purify recombinant proteins with a patented technology that auto-cleaves extraneous sequences leaving the native protein sequence behind. The technology can be easily packaged as a single kit, including the package vector, cloning enzymes and affinity purification reagents. Currently, recombinant proteins are produced in model organisms such as E. coli and are important tools for industrial processes, antigens, and research. While these proteins are most commonly cloned and produced with short peptide tags that facilitate easy one-step affinity purification, the tags can often interfere and impact protein characteristics such as enzyme activity, biophysical studies, structure, and antibody production. For these reasons, cumbersome peptide removal is sometimes required and involves the use of exogenous enzymes such as thrombin or TEV protease. Northwestern's newly patented technology offers a novel process by which recombinant proteins can be purified and subsequently treated with an inexpensive small molecule that initiates auto-processing removal of extraneous sequences beyond the recombinant protein. The technology involves fusing the recombinant protein to auto-processing cysteine protease (CPD) under the control of the T7 promoter. Upon the simple addition of inositol hexakisphosphate (phytic acid), CPD auto-cleaves at a leucine residue. The inventor has designed two vectors-- one with a ligation-independent cloning (LIC) site featuring blunt end restriction sites and a second without a LIC but is compatible with BamHI or recombination-based insertional methods for DNA and PCR products. The former vector leaves four residues upon auto-processing, and the latter leaves a single leucine remaining on the recombinant protein. Application o Production and purification of recombinant protein for: o Research o Antibody Production o Peptide Synthesis o Industrial Processing Advantages o Purification and tag removal accomplished in one step o Preservation of protein characteristics related to enzyme activity, biophysical studies, structure, antibody production o High throughput o Easy scalability o Use of inexpensive small molecule that initiates auto-removal of extraneous tag IP Status Issued US Patent Nos. 8,257,946,B2 and 8,383,400,B2 Marketing Contact Michael Moore, PhD Invention Manager (e) michaelmoore@northwestern.edu (p) 847.491.4645
Original languageEnglish
Patent number8383400
Publication statusPublished - Nov 22 2012

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