This study was intended to construct 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 new RTX-tag. A 6.5 kb novel shuttle vector, designated as pSIT/RTX, was constructed from pCon2(3) as to improvise the expression of pCon2(3). pSIT/RTX was employed with tightly regulated promoter PT7(A1/O4/O3) for controlling gene expression, stabilizing fragment (SF) for replication and maintenance of plasmid in E. coli and P. aeruginosa, attB gene for genome integration, elastase strain K as passenger enzyme and RTX-tag which is located at C-terminal for 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 incooperation with 0.6 mM IPTG (Isopropyl β- d-1-thiogalactopyranoside). Elastase strain K-RTX fusion protein was purified using Ca2+ as novel ligand in immobilized-metal a.....

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