Gene Editing On Bacterial Genome Associated With Improvement Of Disulfide Bonding And Glycosylation Implantation And Its Application On Expression Of Full-Length Engineering Huamn Antibody

In recent years,genetically engineered antibodies have been widely used in scientific research and clinical practice,and has a very broad market prospects.How to express active antibodies efficiently and economically becomes a growing problem.At present,the Chinese hamster ovary(CHO)cell expression system is already quite mature and has been widely used in antibody expression.Nevertheless,this system is very time consuming,complicated to operate,easy to pollute and high cost of goods relative to prokaryotic expression system.E.coli prokaryotic expression system is the most attractive expression system,it has many advantages such as clear genetic background,fast growth and reproduction,high yields,ease of use,relatively low cost and so on.The full-length antibodies need to rely on disulfide bonds to form a complete structure,and need be modified by glycosylation to function.However,the cytoplasm of E.coli is the highly reduced state and can not form a stable disulfide bond.In addition,E.coli lacks an effective glycosylation system and can not glycosylate the antibody.Thus,antibodies are misfolded,poorly expressed,and has low activity when expressed in the cytoplasm of E.coli.Therefore,this is of great significance to constructing a novel expression system which can promote the correct pairing of disulfide bonds and be able to glycosylate the exogenous protein based on E.coli prokaryotic expression system.The Red mediated recombination requires the participation of three different homologous recombinases of Exo,Beta,Gam and linear DNA targeting fragments containing short homologous arms,this system can be used to delete or insert DNA sequences on the E.coli genome accurately.The high frequency of Targeted point,simple design and operation,various forms,high specificity and efficiency of CRISPR-Cas9 make it be the first selected technology for gene editing at any position of prokaryotic and eukaryotic cell genome in recent years.The aim of this study is to genetically edit the E.coli genome to obtain a protein expression strain that could form disulfide bonds in the cytoplasm.Full-length antibodies were expressed by the strain,their propertie were determined and compared to other protein expression strains.Further more,an initial attempt for construction of glycosylation system in E.coli was made on the base of above researchs.Firstly,the trxB gene encoding the thioredoxin reductase and the gor gene encoding glutathione reductase were knocked out by Red homologous recombination system.The CRSPR-Cas9 gene editing system was used to integarte the coding region of DsbC containing the T7 regulatory element into the E.coli genome to overexpress the disulfide isomerase DsbC with chaperone activity in the cytoplasm.We obtained an novel E.coli protein expression strain named TGD,whose cytoplasm was highly oxidized and it could form stable disulfide bonds.Secondly,we constructed two kinds of light chain and heavy chain co-expression plasmid of hepatitis B virus surface antibody 23E7 and HUE6F6,the pET-duet containing double multiple cloning sites was used as the vector.Antibodies expression was performed using the TGD strain constructed in this study,the SHuffle strain saled by NEB company which can correctly folding disulfide bonded proteins in its cytoplasm,and the recipient ER2566 strains respectively.The cell morphology and the growth status of the three strains was similar.The.purity of expressed antibodies was highly purified after purified by affinity chromatography,The expression level of antibody 23E7 in TGD strain was 6.4 times that of SHuffle and ER2566 strains,the expression level of antibody HUE6F6 in TGD strain was 4.1 times higher than that of SHuffle and ER2566 strain,the expression of two antibodies in TGD strain was significantly improved.Thirdly,the properties of antibodies expressed by three strains were tested and compared by SDS-PAGE,Western blot,molecular exclusion chromatography,analytical ultracentrifugation,antigen-binding activity evaluation and virus neutralization activity.Compared with SHuffle and ER2566 strains,The antibodies expressed by the TGD strain can form tetramer,components are simple,with high purity,antigen binding activity and neutralizing activity were stronger than those expressed in SHuffle and ER2566 strains.The properties of antibodies expressed by TGD strains had obvious advantages.Finally,the waaL gene encoding O-antigen ligase in E.coli was knocked out using the Red homologous recombination system.The pglB gene encoding oligosaccharide transferase in Campylobacter jejuni was constructed into the pTO-T7 vector,and the plasmid was transformed into E.coli for expression.Antibodies expressed by glycosylated strain can detect the obvious oligosaccharide signal,may contain a preliminary glycosylation modification,which provides a reference for the establishment of an N-linked glycosylation system in E.coli.In conclusion,the E.coli strain whose cytoplasm is oxidized and promotes the formation of disulfide bonds constructed in this study can express the full-length antibody efficiently,which lays the foundation for the study of the expression of full-length antibody by E.coli.The introduction of the N-linked glycosylation system of Campylobacter jejuni is a reference for the establishment of glycosylation system in E.coli,and it is expected to construct a novel prokaryotic expression system which can promote the correct pairing of disulfide bonds and glycosylate subsequently.