The Effects Of Site-directed Mutation On Pegylated Arginine Deiminase

Objective:We conducted a site directed mutation modification of the wild type ADI(wADI),the E.coli recombinant expression system of mutant ADI(mADI)was constructed,high expression recombinant engineered bacteria was screened.Fermentation process and purification process of recombinant mADI were constructed.Comparative analysis of point mutation on enzyme activity.We modified the wild type and mutant ADI by PEG followed by purified.Analysis the differences on average number and activity of wADI and mADI modified by PEG.Methods :The design and construction of ADI mutations:(1)According to the wild-type ADI protein sequence of mycoplasma,the mutant ADI gene sequence of E.coli was designed(Considering its password preference)and its whole gene was synthesized.The whole gene synthesis mADI was inserted into p ET-3a to construct recombinant plasmid p ET-3a-mADI.(2)The recombinant plasmids p ET-3a-mADI were transformed into expression host strain E.coli BL21(DE3),the recombinant engineering bacteria was obtained by resistance screening method.(3)High expression strains were obtained from bottle activation culture and then transferred them to 30 L fermenter for enlarging cultivation.The research of purification process and enzyme activity of ADI:(1)The expression products were broken by high-pressure homogenizer?inclusion bodies were washed ?dissolved refolding.The product was purified with DEAE anion exchange chromatography and Butyl hydrophobic interaction chromatography column.Finally,the purified target protein was obtained.(2)The activity of the purified wADI and mADI were measured by UV method respectively followed by comparative analysis of point mutation on enzyme activity.ADI PEG-modified research:(1)Use different proportions of PEG-5k to modify the two enzymes.(2)The modified samples were purified by molecular sieve method,high purity modified samples were obtained.(3)The average degree of modification was determined by SEC-HPLC/UV-RI,The activity of modified samples was determined in vitro and comparative analysis of point mutation on PEG modified ADI.Conclution:The recombinant plasmid was identified by double enzyme digestion and gene sequencing,recombinant expression plasmid p ET-3a-mADI was successfully constructed.Target gene was expressed in E.coli as inclusion body and the expression amount was as high as 30%.Established and identified mADI's expression culture conditions in shake flask and amplification fermentor.Explored and established a stable purification process,the purity of SDS-PAGE and HPLC were higher than 95%.Comparative analysis found that mutations did not have a significant effect on the enzyme activity.PEG modification of wADI and mADI with different molar ratios was successfully achieved followed by purification.The differences and correlation of wADI and mADI of PEGylation was compared.It was proved that the mutation site could affect the binding of PEG on arginine deiminase,wADI corresponding to the site was very easy to be modified by PEG,mADI corresponding to the site,however,couldnt be modified by PEG.Under the same modification ratio,the average modification degree of mADI was 1 less than that of wADI.The same proportion of the modified products or the different modification proportion to obtain the products of same modification numbers,the enzyme activity of mADI was higher than that of wADI.This has laid a theoretical and practical basis for the development of low immunogenicity and high activity of arginine deiminase.