2014•International Journal of Agriculture and BiologyRequires access
Lactose induced expression of Thermotoga petrophila α-amylase gene regulated by T7 promoter in E. coli codon plus (DE3).
Uzma Hameed, Ikram‐ul Haq, Mehmood Ali Khan
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Abstract
Current work is aimed to evaluate the lactose induced expression of Thermotoga petrophila alpha-amylase gene under T7 promoter in E. coli. Recombinant protein was expressed in nine different media (LB, ZB, 4xZB, ZBM, ZYB, 3xZYB, M9, ZYBM9 and 3xZYBM9) by using Isopropyl beta-D-1-thiogalactopyranoside (IPTG) and lactose as inducer. Lactose was found to be the better inducer in all the media with maximum expression in tryptone rich ZBM medium. The enzyme expression increased when host cells were given heat shock (42 degrees C) for 1 h immediately after induction with lactose. The parameters such as lactose concentration (200 mM), cell density at the time of induction (OD600 0.8), and incubation temperature (37 degrees C) were also optimized, that resulted in 1.5 fold increase in intracellular alpha-amylase activity. Thus, lactose as inducer is recommended to be used for the enhanced expression of recombinant alpha-amylase in tryptone rich medium. (C) 2014 Friends Science Publishers