Construction of New Genetic Tools as Alternatives for Protein Overexpression in Escherichia coli and Pseudomonas aeruginosa

Iranian Journal of Biotechnology
Chee Fah WongAbu Bakar Salleh

Abstract

Background:Pseudomonas protein expression in E. coli is known to be a setback due to significant genetic variation and absence of several genetic elements in E. coli for regulation and activation of Pseudomonas proteins. Modifications in promoter/repressor system and shuttle plasmid maintenance have made the expression of stable and active Pseudomonas protein possible in both Pseudomonas sp. and E. coli. Objectives: Construction of shuttle expression vectors for regulation and overexpression of Pseudomonas proteins in Pseudomonas sp. and E. coli. Materials and Methods:Pseudomonas-Escherichia shuttle expression vectors, pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo as well as E. coli expression vectors of pCon4 and pCon5 were constructed from pUCP19-, pSS213-, pSTBlue-1- and pPICZαA-based vectors. Protein overexpression was measured using elastase strain K as passenger enzyme in elastinolytic activity assay. Results: The integration of two series of IPTG inducible expression cassettes in pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo, each carrying an E. coli lac-operon based promoter, Plac, and a tightly regulated T7(A1/O4/O3) promoter/repressor system was performed to facilitate overexpression study of the organic solvent-tolerant elastase...Continue Reading

References

Jan 1, 1991·Methods in Enzymology·R K RothmelA Darzins
Oct 11, 1988·Nucleic Acids Research·S E West, B H Iglewski
Dec 1, 1988·Proceedings of the National Academy of Sciences of the United States of America·M Lanzer, H Bujard
Oct 18, 2001·Current Opinion in Biotechnology·H P Schweizer
Jul 6, 2004·Journal of Microbiological Methods·Sang-Jin SuhDennis E Ohman
Aug 24, 2010·Biotechnology and Applied Biochemistry·Chee Fah WongRaja Noor Zaliha Raja Abd Rahman

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