Abstract
A subtilisin gene identified in the reported genome sequence of Pyrococcus furiosus was amplified and inserted in pET-22b(+) vector to produce the recombinant plasmid pET-SB. Escherichia coli BL-21 (DE3) CodonPlus was transformed with this plasmid and the enzyme was expressed up to 30% of the total cell protein on induction with IPTG. The expressed protein appeared at a position corresponding to ~20 kDa on SDS-PAGE as compared to theoretical molecular mass of 17.6 kDa. This aberrant electrophoresis mobility could be due to specific amino acid composition of the protein. Auto-induction with lactose also produced a similar level of expression but the total amount of the enzyme produced was 2.4 fold greater than that when produced with IPTG induction. This was due to a higher cell density obtainable in the auto-inducing medium. The enzyme expressed in the insoluble state could be partially refolded after denaturation with urea at high pH. This study reports for the first time high-level expression of subtilisin of P. furiosus in E. coli using an auto-inducing medium.
Highlights
Studies have continued to produce commercially important enzymes like proteases with better thermostability and other favorable properties for applications
Escherichia coli BL-21 (DE3) CodonPlus was transformed with this plasmid and the enzyme was expressed up to 30% of the total cell protein on induction with IPTG
Auto-induction with lactose produced a similar level of expression but the total amount of the enzyme produced was 2.4 fold greater than that when produced with IPTG induction
Summary
Studies have continued to produce commercially important enzymes like proteases with better thermostability and other favorable properties for applications. Escherichia coli BL-21 (DE3) CodonPlus was transformed with this plasmid and the enzyme was expressed up to 30% of the total cell protein on induction with IPTG. Auto-induction with lactose produced a similar level of expression but the total amount of the enzyme produced was 2.4 fold greater than that when produced with IPTG induction.
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