Abstract: In order to investigate thoroughly the enzymatic properties of 4-coumarate:coenzyme A ligase(4CL1) from Populus tomentosa,the prokaryotic expression vector of 4CL1 gene pQE31-4CL1 was constructed and transformed into E.coli M15(pREP4).The recombinant 4CL1 fused protein was expressed successfully in M15(pREP4) after M15(pREP4) was induced by IPTG for 4 h.SDS-PAGE revealed that the mass of the induced protein was about 60 kD consistent with the predicted value.The expression system of the 4CL1 gene in E.coli was optimised,showing that the most effective concentration of IPTG was 0.4 mmol/L,the OD₆₀₀ of bacteria density of E.coli ranged from about 0.3 to 0.5 and the best expression time was 2 h.The 4CL1 fused proteins existed almost in the form of inclusion bodies expressed at 37℃.The soluble 4CL1 protein which was expressed at 28℃ was active to several substrates.Agarose coupled with Ni²⁺-NTA was the filling of the metal affinity column chromatography;the electrophoresis purity 4CL1 protein was acquired after one-step purification.The enzymatic activity ratio of the electrophoresis purity 4CL1 to the five substrates,respectively,as follows:3 949.0 pkat/mg to 4-coumarate,2 214.0 pkat/mg to caffeic acid,715.0 pkat/mg to ferulate,84.9 pkat/mg to cinnamate,no activity to sinapate.