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High Throughput Protein Production by Novel E. coli Expression-Secretion System

Award Information

Agency:
Department of Health and Human Services
Branch:
N/A
Award ID:
79661
Program Year/Program:
2006 / SBIR
Agency Tracking Number:
GM079001
Solicitation Year:
N/A
Solicitation Topic Code:
N/A
Solicitation Number:
N/A
Small Business Information
MACCONNELL RESEARCH CORPORATION
9550 Waples Street Ste 120 SAN DIEGO, CA -
View profile »
Woman-Owned: No
Minority-Owned: No
HUBZone-Owned: No
 
Phase 1
Fiscal Year: 2006
Title: High Throughput Protein Production by Novel E. coli Expression-Secretion System
Agency: HHS
Contract: 1R43GM079001-01
Award Amount: $100,000.00
 

Abstract:

DESCRIPTION (provided by applicant): Protein production from cloned DNA sequences has become increasingly important in the post-genomic era. The availability of sequences from whole bacterial genomes, cDNAs, and tens of thousands of predicted mammalian genes have created the need to produce proteins for these genes for further studies. Of the many methods available, E. coli cell expression continues to be a viable choice as expression vector systems are well understood and the cost of E. coli growth and induction is minimal. E. coli expression, however, can present difficulties such as insolubility of synthesized protein, degradation, and cell toxicity; that detract from its utility as a universal expression protocol for production of a large numbers of proteins. In Phase I, we will develop a novel E. coli expression-secretion-purification system that will circumvent the difficulties associated with conventional E. coli expression. This system utilizes the already characterized E. coli hemolysin (HylA) protein secretion pathway that has been shown to allow efficient secretion of proteins when fused to the c-terminal portion of the HylA protein. Three other proteins found in E. coli, namely hemolysin B, D and TolC facilitate the secretion of hemolysin A by forming a multi-subunit channel that spans the inner and outer cell wall (1). The Hly B-D-TolC channel actively translocates the native HlyA or its c-terminal fusions through the cell wall of the E. coli, so that these proteins accumulate in the media after induction of expression. The HlyA fusion expression system has several advantages for protein production over conventional intracellular expression in E. coli. These advantages include: a) it avoids the formation of inclusion bodies, which require subsequent dissolution and renaturation, b) secretion circumvents toxicity problems, as the secreted protein does not accumulate in the bacterial cells, and c) the purification of fully secreted proteins is greatly simplified, as the protein can be concentrated and purified directly from the clarified growth media. Published work has shown that fusion of coding regions such as: scFv antibody chain, E. coli outer membrane porin (OmpF), chloramphenicol acetyltransferase (CAT), or L-asparagenase genes to the c-terminal 200 amino acid portions of the Hly A gene results in secretion of these fusion proteins into the growth medium. We will improve this published technology by introducing a proteolytic cleavage site between the protein to be expressed and the c-terminal HlyA domain (1,4). This proteolytic site will enable us to remove the HylA protein sequence from the protein of interest during its purification. Prior to Phase I, our company cloned the four key genes of the hemolysin secretion pathway from wild type E. coli, namely the: Hyl A, B, D and TolC proteins. The HylB, HylD, and TolC genes were inserted onto a plasmid designed to express these channel-forming proteins at low levels in the host DH5a E. coli cells. The c-terminal domain of the HylA gene was cloned into a pUC18 plasmid that was engineered with a 6His tag and the Hyl A c-terminus. This vector allowed us to demonstrate the expression and secretion of a green fluorescent protein fusion to HylA, a result that indicates that many different proteins can be secreted by this method. This system should allow for a standardized protocol to be applied to expression and purification of virtually any coding domain, as the expression in our test case did not require a specific induction protocol. In addition, affinity purification was greatly simplified by the fact that the secreted protein made up the majority of protein found in the growth medium. Our goal is to develop a standardized system for E. coli expression-secretion-purification that is applicable to a large number of diverse proteins. Phase I work will include: optimization of the growth/induction protocol, and introduction of a thrombin proteolytic recognition sequence between the HylA and inserted protein sequence; allowing removal of the hylA peptide from the secreted protein. A determination of the minimum portion of HylA that is needed for secretion will be made, and several protein coding regions will be tested with the system; including a toxic protein. Measurements of the enzyme activity of the secreted proteins will be carried out. The products that we will develop in the form of kits, vectors, and cells lines will be directly marketable to the 40,000 or more molecular biology and proteomics labs through academia, non-profit organizations, biotechnology, and pharmaceutical companies, including the many laboratories supported by the NIH. MacConnell Research Corp itself can directly market these products, as we have already established a customer base for our existing products. The products resulting from Phase II, if successful, will have a realistic potential of $10 - $20 million in sales per year. They will improve protein production and yield high quality protein. These products will be essentially ready for market at the end of Phase II.

Principal Investigator:

William P. Macconnell
8584522603
BMACCONNELL@MACCONNELL.COM

Business Contact:

William Macconnell
8584522603
bmacconnell@macconnell.com
Small Business Information at Submission:

MACCONNELL RESEARCH CORPORATION
6195 CORNERSTONE COURT EAST #114 SAN DIEGO, CA 92121

EIN/Tax ID: 330588488
DUNS: N/A
Number of Employees: N/A
Woman-Owned: No
Minority-Owned: No
HUBZone-Owned: No