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

Document Type: Research Paper

Authors

1 Department of Biology, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris, 35900 Tanjong Malim, Perak, Malaysia.

2 Enzyme and Microbial Technology Research Centre, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

3 Enzyme and Microbial Technology Research Centre, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.

4 Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.

5 Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

Abstract

Background: Pseudomonas protein expression in E. coli is known to be a setback due to signifi cant 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.

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