Prokaryotic Expression, Purification And Identification Of Human Lysozyme N-terminal Fragment-Exendin-4 Chimeric Peptide

Objectives:Exendin-4 is a polypeptide of 39 amino acids which has recently been clinically used and demonstrated to have good therapeutic efficacy for type 2 diabetes. Human lysozyme N-terminal polypeptide has been shown to be able to bind AGEs and to increase the clearance of AGEs from body. Thus, this peptide may delay or prevent the development of diabetic complications. In this study, our aim was to link these two polypeptide genes by using a linker sequence which encodes for an amino acid sequence recognized by thrombin and dipeptidyl peptidase IV, so that the chimeric peptide can be cleaved into lysozyme N-terminal fragment and exendin-4 by enzymes in the body, and both of these products may play different roles in the treatment of diabetes. The expression, purification and identification of this chimeric peptide in E. coli will provide an experimental foundation for development of a new type of dual-targeting protein drugs.Methods:The gene sequence for human lysozyme N-terminal fragment(1-74aa) was prepared by RT-PCR from human leucocytes. This sequence was then linked to exendin-4 gene sequence by using recombinant PCR. The linker sequence between lysozyme fragment and exendin-4 contained a site recognized by thrombin and another site by dipeptidyl peptidase IV. HLYZ(N74)-exendin-4 gene was cloned into a prokaryotic expression vector, pET-32a(+), and then transformed into E. coli BL21(DE3). After induced with IPTG, the recombinant protein was identified by SDS-PAGE and Western blotting. After optimization of the expression condition, a high expression level of the recombinant protein were achieved. The polypeptide of interest was purified by Ni2+ affinity chromatography under denaturing conditions, followed by renaturation of the recombinant protein by dialysis, and digestion with enterokinase to release the chimeric peptide. After cleavage of the recombinant protein by thrombin, the hydrolisis fragments were finally separated by HPLC and identified by mass spectrometry.Results:DNA sequence analysis showed that the recombinant plasmid pET-32a/hLYZ(N74)-Ex4 was constructed correctly. The recombinant protein was highly expressed, and reached a maximum level of about 30% of total somatic proteins after induction with 0.6mmol/L IPTG at 37℃for 4h. SDS-PAGE analysis showed that the molecular weight of the recombinant protein was about 30kD. Western blotting analysis suggested that the recombinant protein had a strong reaction with anti-His-tag antibody, exhibiting as a single clear band. The recombinant protein was expressed mainly in the form of inclusion bodies and could be highly purified by Ni2+ affinity chromatography. The recombinant protein was renatured by dialysis, and the refolding conditions was found to be 50μg/mL of recombinant proteins,4℃, and with addition of L-Arg and GSH/GSSG in the dialysate, which contributed to a higher level of yield, at about 23.8%. Our results also showed that the recombinant protein was cleaved into two fragments by enterokinase, the Trx-His tag and the chimeric peptide hLYZ(N74)-exendin-4. After cleavage of the recombinant protein by thrombin, the exendin-4 fragment was detected at the molecular weight of 4341.0 by mass spectrometry.Conclusion:The prokaryotic expression vector for hLYZ(N74)-exendin-4 fusion gene was successfully constructed, and the recombinant protein was highly expressed in E. coli. This recombinant protein can produce the chimeric peptide, hLYZ(N74)-exendin-4, after digestion with enterokinase, which contains a thrombin hydrolytic site between the human lysozyme N-terminal fragment and exendin-4.