Prokaryotic Expression And Purification Of Recombinant Rat Lysozyme Fragment And Its Biological Activity

Objectives:To highly express the recombinant rat lysozyme C-terminal fragment in prokaryotes and to investigate the biological activity of the recombinant fragment LY77 in vitro.Methods:①Total RNA was extracted from leucocytes of Sprague-Dawley rats, and the cDNA sequences of rat lysozyme C-terminal fragment were prepared using RT-PCR.②The sequence,encoding for the C-terminal fragment of rat lysozyme, was then inserted into an expression vector,pET-30α(+),to construct the recombinant expression vector,pLY70,which was identified by double restriction endonuclease digestion and sequencing.③The recombinant expression vector pLY70 was transfected into E.coli Rosetta(DE3) for expression of the recombinant rat lysozyme C-terminal fragment(LY70) induced by IPTG..Optimization of the inducing conditions including temperature,IPTG concentration and induction time was done to obtain the maximum yield of the recombinant fragment.④The expression products were purified by using Ni~+-NTA agarose chromatography,and the prepared rat lysozyme fragment LY70 was identified by using SDS-PAGE and Western blotting.⑤Advanced glycation end products(BSA-AGEs) were prepared in vitro,for study of the recombinant peptide LY70's binding activity with AGEs.⑥Cellular experiments were done to study whether the recombinant peptide LY77(the N-terminal fragment of rat lysozyme) can facilitate the clearance of AGEs by cells.Results:1) The recombinant vector was successfully constructed in the study:①The total RNA isolated gave three typical bands in agarose electrophoresis,28s,18s and 5s,respectively.The ratio of these band densities was determined to be 5:3:1,in well consistence with the levels of the three RNAs.The product fragment of 210bp was amplified through RT-PCR,and it was showed to be the right length as designed.②The recombinant expression vector pLY70 was finally identified by sequencing.2) High expression of the recombinant rat lysozyme C-terminal fragment in prokaryotes:①The recombinant expression vector pLY70 in E.coli Rosetta(DE3) was highly expressed with induction of IPTG..The expression product LY70 was identified by using SDS-PAGE and Western blotting.It was demonstrated that the recombinant peptide LY70 was highly soluble in water with a molecular weight of about 10.5KD.②The Optimal inducing conditions were found to be:37℃for induction temperature,0.2mmol/L for IPTG concentration,and 5 hours for induction time.Expression of LY70 was found to be satisfactory(90mg/L) in E.coli Rosetta(DE3).The expression product was determined to be about 60%of the total soluble proteins in the expression system.3) By using Ni~+-NTA agarose chromatography,the expression product LY70 was obtained at a high level of purity,and advanced glycation end products(BSA-AGEs) were successfully prepared in vitro through Maillard reaction.4) ELISA analysis showed that LY70 had no binding activity with the prepared albumin-AGEs in vitro.5) A recombinant peptide,LY77(the N-terminal fragment of rat lysozyme), prepared in our previous study,was found to be able to bind with AGEs and to down regulate the expression of AGE's receptor-RAGE,in our cellular experiments.Conclusion:1) The recombinant expression vector pLY70 was highly expressed as a fusion protein,LY70,in E.coli Rosetta(DE3).2) LY70 showed no AGEs-binding activity in vitro.3) LY77,with AGEs-binding activity and ability to down regulate the expression of AGE's receptor RAGE,promotes clearance of AGEs by cells.