Cloning and expression of thermostable alkaline protease 50a in E. coli BL21 (DE3) and TOP10

Abstract

Protease is one of the important enzymes utilize in numerous industrial application thus it requires high production to meet the market demand. Thermostable alkaline protease 50a (TAP50a) gene was previously isolated from Bacillus subtilis and was successfully cloned and expressed in E. coli BL21 (DE3) pLysS. The expression in this host was higher compared to the expression from the wild type strain. However it requires two types of antibiotics which contributed to higher production cost. This research aimed to express TAP50a gene in another potential host which may express high yield of enzyme with lower production cost. Two hosts, E. coli BL21 (DE3) and TOP10 were selected as they require only one antibiotic.TAP50a gene was extracted from the previous host, ligated into pGEX4T-1 and transformed into E. coli BL21 (DE3) and TOP10 competent cells. The validations of clones were carried out through restriction digestion analysis and PCR using specific primers incorporated with EcoR1 sites. Expression of TAP50a enzyme in both hosts were induced by IPTG (0.5 mM) at 30°C and incubated for 24 h. The expression of TAP50a enzyme in both hosts were occurred intra and extracellularly with higher level of expression observed in intracellular cell. In E. coli BL21 (DE3), the intra and extracellular expressions were 31.33 U/mL and 3.33 U/mL, respectively. Meanwhile, the intra and extracellular expressions of TAP50a in E. coli TOP10 were 12.67 U/mL and 7.67 U/mL, respectively. TAP50a enzyme was purified by heat treatment and showed a 2.54-fold increase in specific activity (819.58 U/mg) in E. coli BL21 (DE3) and a 2.61-fold increase in E. coli TOP10 with specific activity of 371.39 U/mg.