Abstract
| Grant Number: | 1R43GM075731-01 |
| Project Title: | Split SUMO for protein expression and purification |
Abstract:
DESCRIPTION (provided by applicant): The "bottleneck" for structural and functional Genomics and Proteomics is efficient expression and purification of the proteins of interest. Advances in fusion systems have played a major role in enhancing expression of proteins. However, in many cases, using a fusion tag does not increase yield of expressed proteins because the fusion is cleaved by the host enzymes and/or the tag does not have sufficient chaperoning effects. Although intact SUMO fusion system can significantly improve production of recombinant proteins in E. coli, it is not applicable to eukaryotes because the fusion proteins are cleaved in vivo by the host proteases. We propose to develop a novel fusion technology, termed "Split SUMO", for enhancing expression and purification of proteins of interest. The Split SUMO fusions are not cleaved in eukaryotes. We hypothesize that the Split SUMO will have similar or greater advantages than the intact SUMO. Using insect cells rather than E. coli to express the Split SUMO fusions would also improve the yield and preserve biological activities of the proteins. The key features of this novel technology are: 1) Attachment of the C-terminal half of SUMO (CTHS) to the N-terminus of a target protein, enhancing the latter's expression in cells; 2) Introduction of N-terminal half of SUMO (NTHS) as affinity tag, allowing rapid purification of proteins; 3) Combination of the two halves of SUMO in vitro to reconstitute SUMO structure, allowing cleavage of fusion protein by SUMO proteases to generate desired amino termini.
Thesaurus Terms:
chimeric protein, posttranslational modification, protein engineering, protein purification, technology /technique development, ubiquitin
functional /structural genomics, protein binding, protein structure, proteomics, resin
bioengineering /biomedical engineering, biotechnology
| Institution: | LIFESENSORS, INC. |
| 271 GREAT VALLEY PKY |
| MALVERN, PA 19355 |
| Fiscal Year: | 2006 |
| Department: | |
| Project Start: | 01-APR-2006 |
| Project End: | 31-MAR-2008 |
| ICD: | NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES |
| IRG: | ZRG1 |
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