High-level expression of 4-coumarate:coenzyme A ligase gene Pt4CL1 of Populus tomentosa in E. coli

Abstract

In order to investigate the enzymatic properties of the 4CL1 of Populus tomentosa, the recombinant expression vector pQE31-4CL1 was constructed. The recombinant was identified by three restriction endonucleases, then the vector pQE31-4CL1 was transformed into expression host M15 (pREP4) and induced by isopropyl-α-D-thiogalactoside (IPTG) to express 60 kD fused protein Pt4CL1. The biologically active Pt4CL1, expressed as soluble protein, was achieved with 0.6 mmol·L−1 IPTG induction as the expression temperature declined from 37 to 28°C. The 6×His tag facilitates affinity binding to Ni2+-nitrolotriacetic acid (NTA) and enables one-step purification to acquire the molecular SDS-PAGE electrophoresis purity of the active 4CL1 protein by agarose coupled with Ni2+-NTA affinity chromatography. The optimal substrate for Pt4CL1 was 4-coumarate.