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

• Published in: Iranian journal of biotechnology Vol. 15; no. 3; pp. 194 - 200
• Main Authors: Wong, Chee Fah, Rahman, Raja Noor Zaliha Raja Abd, Basri, Mahiran, Salleh, Abu Bakar
• Format: Journal Article
• Language: English
• Published: Iran National Institute of Genetic Engineering and Biotechnology 27.09.2017
• Subjects: T7(A1/O4/O3); LacIq; Regulation; Overexpression; Elastase strain K
• ISSN: 1728-3043; 2322-2921
• DOI: 10.15171/ijb.1524

protein expression in is known to be a setback due to significant genetic variation and absence of several genetic elements in for regulation and activation of proteins. Modifications in promoter/repressor system and shuttle plasmid maintenance have made the expression of stable and active protein possible in both sp. and . Construction of shuttle expression vectors for regulation and overexpression of proteins in sp. and . shuttle expression vectors, pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo as well as 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. The integration of two series of IPTG inducible expression cassettes in pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo, each carrying an lac-operon based promoter, Plac, and a tightly regulated T7 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 P promoter, which conferred tight regulation and repression of basal expression due to existence of respective double operator sites, O3 and O4, and lacI . The constructs offered remarkable assistance for overexpression of heterogeneous genes in sp. and for downstream applications such as in industries and structural biology study.