Journal of Biotechnology & Biomaterials
Open Access

Abstract

A gene encoding a thermostable, methanol-stable and enantioselective lipase YLIP9 of Yarrowia lipolytica MSR80 was cloned and expressed in pEZZ10-HB101 vector-host system. pEZZ18 vector has a 14 kDa ZZ-tag and the purified protein of 60 kDa was obtained. In this system, constitutive extracellular expression of ZZ-YLIP9 was observed, after 48 hours incubation at 37�º C/300 rpm with expression level of 0.25�±0.15 U/ml. As the titres were low, this gene was sub-cloned and expressed in vectors with different tags-pET22b (C-terminal His-tag), pET51b (N-terminal Strep and C-terminal His-tag), pET22b-SUMO (N-terminal His, 10 kDa SUMO-tag and C-terminal His-tag) and pGEX-4t1 (N-terminal 26 kDa GST-tag) with protein of 45, 45, 55 and 70 kDa, respectively. Periplasmic expression in pET22b and pET22b-SUMO and intracellular expression in pET51b and pGEX-4t1 was observed 3 h after IPTG induction. In pET22b the expressed protein was inactive while expression of 3.25�±0.13, 3.5�±0.09 and 1.75�±0.21 U/ml was obtained with pET51b, pET22b-SUMO and pGEX-4t1, respectively. Around 14- fold enhancement was observed with pET22b-SUMO. However, the expression was not substantial so the usage of codons in E. coli was compared to that of Y. lipolytica using the codon usage database. About 80% of the codons were changed and the gene was synthesized, re-sequenced followed by its sub cloning and expression in pET22b, pET51b, pET22b-SUMO and pEZZ18 vector-E. coli host systems. As compared to earlier observation this time the protein that was expressed in pET22b was active with an expression level of 2.98�±0.18 U/ml and a further 2-3 fold enhancement was observed for pET51b and pET22b-SUMO, whereas, a significant 40-fold enhancement was observed in pEZZ18-HB101 system. Hence, host-vector combinations, tags and codon usage have a significant effect on protein expression.

Biography

Email: Poonam.88.30@gmail.com