Construction of New Genetic Tools for Protein Overexpression in E. coli-Pseudomonas host systems
Keywords:
pSIT/RTX, overexpression, elastase strain K, rapid purification, RTX-tagAbstract
This study reported the construction of a new Escherichia coli-Pseudomonas shuttle vector for overexpression of elastase strain K in both E. coli and Pseudomonas as well as for rapid purification using the new RTX-tag. A 6.5 kb novel shuttle vector, designated as pSIT/RTX, was constructed from pCon2(3) in order to improvise the expression of pCon2(3). pSIT/RTX was harbour a tightly regulated promoter PT7(A1/O4/O3), for controlling gene expression; stabilising fragment (SF) for replication and the maintenance of plasmid in E. coli and P. aeruginosa; attB gene for genome integration; elastase strain K as the passenger enzyme and RTX-tag rapid purification. E. coli TOP10/pSIT/RTX was chosen to proceed with purification as the highest amount of proteolytic activity was detected at 12 h after incorporation with 0.6 mM IPTG (Isopropyl β- d-1-thiogalactopyranoside); the pSIT/RTX showed a clear difference between culture in Luria-Bertani broth without ampicillin (5.4 X103 CFU) and 3.6 X103 CFU for culture in ampicillin.
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