Construction of new genetic tools as alternatives for protein overexpression in escherichia coli and pseudomonas aeruginosa

Wong Chee Fah, Raja Abd. Rahman Raja Noor Zaliha, Basri Mahiran, Salleh Abu Bakar,

QR Code Link :
Type :	article
Subject :	Q Science (General)
ISSN :	1728-3043
Main Author :	Wong Chee Fah, Raja Abd. Rahman Raja Noor Zaliha, Basri Mahiran, Salleh Abu Bakar,
Title :	Construction of new genetic tools as alternatives for protein overexpression in escherichia coli and pseudomonas aeruginosa

Year of Publication :

2017

Full Text :

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. Modifi cations 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 pPICZαAbased 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 strain K. These constructs have demonstrated an elastinolytic fold of as high as 1464.4 % in comparison to other published constructs. pCon4 and pCon5, on the other hand, are series of pCon2(3)-derived vectors harboring expression cassettes controlled by PT7(A1/O4/O3) promoter,which conferred tight regulation and repression of basal expression due to existence of respective double operator sites, O3 and O4, and lacIq. Conclusions: The constructs off ered remarkable assistance for overexpression of heterogeneous genes in Pseudomonas sp. and E. coli for downstream applications such as in industries and structural biology study

References

1. Schweizer HP. Vectors to express foreign genes and techniques to monitor gene expression of Pseudomonads. Curr Opin Biotechnol. 2001;12:439-445. DOI: 10.1016/S0958-1669 (00)00242-1 2. Rothmel RK, Chakrabarty AM, Berry A, Darzins A. Genetic systems in Pseudomonas. Methods Enzymol. 1991;204:485-515. DOI: 10.1016/0076-6879(91)04025-J 3. Pasloske BL, Drummond DS, Frost LS, Paranchych W. The activity of the Pseudomonas aeruginosa pilin promoter is enhanced by an upstream regulatory site. Gene. 1989;81:25-34.DOI: 10.1016/0076-6879(91)04025-J 4. West SEH, Iglewski BH. Codon usage in Pseudomonas aeruginosa. Nucleic Acids Res. 1988;16: 9323-9335. DOI: 10.1093/nar/16.19.9323 5. Choi K, Trunck LA, Kumar A, Mima T, Karkhoff -Schweizer RR, Schweizer HP. Pseudomonas Genomics and Molecular Biology. Norfolk: Caister Academic Press; 2008. 6. Schumann W. Plasmids Current Research and Future Trends.Norfolk: Caister Academic Press; 2008. DOI: 10.1086/603490 7. Schweizer HP. Escherichia-Pseudomonas shuttle vectors derived from pUC18/19. Gene. 1991; 97:109-112. DOI: 10.1016/0378-1119(91)90016-5 8. Suh S, Silo-Suh LA, Ohman DE. Development of tools for the genetic manipulation of Pseudomonas aeruginosa. J Microbiol Methods. 2004; 58:203-212. DOI: 10.1016/j. mimet.2004.03.018 9. Wong CF, Salleh AB, Basri M, Rahman RNZRA. Organic solvent stability of elastase strain K overexpressed in an Escherichia-Pseudomonas expression system. Biotechnol Appl Biochem. 2010;57:1-7. DOI: 10.1042/BA20100224 10. Baharum SN, Salleh AB, Razak CNA, Basri M, Rahman MBA, Rahman RNZRA. Organic solvent tolerant lipase by Pseudomonas sp. strain S5: stability of enzyme in organic solvent and physical factors aff ecting its production. Ann Microbiol. 2003; 53:75-83. 11. Choi K, Kumar A, Schweiser H. A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells:Application for DNA fragment transfer between chromosomes and plasmid transformation. J Microbiol Methods. 2006;64:391-397. DOI: 10.1016/j.mimet.2005.06.001 12. Ohman DE, Cryz SJ, Iglewski BH. Isolation and characterization of a Pseudomonas aerugionsa PAO mutant that produces altered elastase. J Bacteriol. 1980; 142: 836-842. 13. Lanzer M, Bujard H. Promoters largely determine the effi ciency of repressor action. Proc Natl Acad Sci USA. 1988; 85: 8973-8977.

This material may be protected under Copyright Act which governs the making of photocopies or reproductions of copyrighted materials.
You may use the digitized material for private study, scholarship, or research.

Back to previous page