Document Type: Research Paper
Department of Biology, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris, 35900 Tanjong Malim, Perak, Malaysia.
Enzyme and Microbial Technology Research Centre, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
Enzyme and Microbial Technology Research Centre, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
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.