Construction of New Genetic Tools as Alternatives for Protein Overexpression in and .

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.