Abstract
| Grant Number: | 5R44GM068404-03 |
| Project Title: | SUMO Fusions to Enhance Expression and Secretion of Protiens |
Abstract:
DESCRIPTION (provided by applicant): As more proteins are identified from the genome-sequencing projects, there is an increasing need for reliable and cost efficient means for expression and purification of proteins of academic and clinical importance. LifeSensors has developed a novel SUMO-fusion system to enhance expression, and solubility of a wide variety of proteins in E. coli, and to facilitate the downstream purification of the protein via an affinity tag attached to SUMO. An important feature of the system is the protease that cleaves the SUMO-fusion from expressed proteins. The removal of the fusion leaves no N-terminal overhang, so that any desired amino acid can be cloned at the N-terminus of the expressed protein. Several research or therapeutically important proteins either are not expressed well in bacteria or are inefficiently secreted. Recent data from the Protein Structure Initiative suggest that production of correctly folded protein is a bottleneck for success in our quest for Structural Genomics. In phase I, we proposed to exploit the chaperoning properties of SUMO to enhance expression and secretion of proteins in E. coli. Recognizing the well-known protein secretion properties of Bacillus subtilis and Lactococcus lactis we also proposed to test the role of N-terminal of SUMO in secretion of protein in these gram-positive organisms. Lactococcus lactis is well known for its secretory properties and widely used in the food industry, and yet this bacteria has not been well exploited for protein secretion in the biopharmaceutical industry. Our phase I data suggest that while E. coli is good for intracellular expression, it is not the best host for protein secretion. Lactococus lactis has emerged as the most suitable host for secretion and production of large quantities of proteins. In this phase II, LifeSensors proposes a plan to build on its success with L. lactis to test a wide variety of difficult to express protein and further validate the usefulness and role of SUMO and L. lactis as a host for protein secretion. Development of an efficient secretory system in L. lactis that allows production of correctly folded, difficult to express protein will open the bottleneck for Structural Genomics. It will also create an inexpensive platform for endotoxin free protein production that will help the biopharmaceutical industry in the post-genomic era.
Thesaurus Terms:
binding protein, gene expression, method development, protein purification, secretion, ubiquitin
chimeric protein, endopeptidase, functional /structural genomics, genetic library, proteolysis
Bacillus subtilis, Escherichia coli, Streptococcus lactis, affinity chromatography, bioengineering /biomedical engineering, protein quantitation /detection, transfection /expression vector
| Institution: | LIFESENSORS, INC. |
| 271 GREAT VALLEY PKY |
| MALVERN, PA 19355 |
| Fiscal Year: | 2007 |
| Department: | |
| Project Start: | 01-MAR-2006 |
| Project End: | 28-FEB-2008 |
| ICD: | NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES |
| IRG: | ZRG1 |
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