Full Length Research Paper

Enhanced production of subtilisin of Pyrococcus furiosus expressed in Escherichia coli using auto-inducing medium

Nadia Ikram¹, Shumaila Naz¹, M. Ibrahim Rajoka², Saima Sadaf^1,3 and M. Waheed Akhtar¹*

¹School of Biological Sciences, University of the Punjab, Lahore-54590, Pakistan.

²National Institute of Biotechnology and Genetic Engineering, Jhang Road, Faisalabad, Pakistan.

³Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore-54590, Pakistan.

*Corresponding author. E-mail: mwapu@brain.net.pk. Tel: +92-42-9230970.

Fax: +92-42-9230980.

Accepted 17 September, 2009

   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.

Key words: Subtilisin, Pyrococcus furiosus, auto-induction, enhanced production.