Expression And Purification Of HSA ScFv By Fiusion SUMO Tag And Structure Analysis

Human serum albumin HSA is a protein that is present in human plasma and is abundant in content.It is often used as a carrier protein for binding and transport.HSA has a long half-life in the human body and can be used to fuse polypeptide drug proteins to extend the half-life of drugs.However,HSA fusion polypeptides have large molecular weight,difficult to express,and difficult to penetrate through the blood vessel walls.Therefore,the subject has studied new single-stranded anti-HSA chains.Compared with intact antibody molecules,the antibody and scFv have the characteristics of small molecular weight,easy passage through the blood vessel wall,and no aglycosylation in the structure.So they are suitable for expression and purification in E.coli.Compared to mammalian cell expression,E.coli has a short life cycle and low cost of training.However,there are also some problems in the expression of scFv in E.coli,such as the low expression level and difficulty in soluble expression.Hybridoma cells were prepared by immunizing mice with HSA,and sequences of anti-HSA antibodies were obtained by sequencing.The SUMO tag was used for the expression of scFv,the expressed Ulp1 was used for excising SUMO-tag,and the soluble scFv was successfully obtained.The structural analysis of the scFv was performed.The main research results are as follows:?1?Hybridoma cells were prepared by immunizing mice with HSA,mouse ascitic fluid was aspirated,and antibodies were purified for affinity analysis.It was determined that the antibodies produced by hybridoma cells of 1H11 strain had the strongest affinity.Total RNA of hybridoma cell was extracted and reverse transcribed into cDNA.The anti-HSA antibody sequences was obtainded by PCR and sequencing.The sequences of the heavy chain variable region and the light chain variable region of the antibody were ligated with a short linker ?G₄S?₃ to obtain a single chain antibody?sc Fv?.Expression of scFv using psvT7 as an expression vector showed low amount in the form of inclusion bodies in E.coli.Therefore,the SUMO tag was used for the expression of scFv and a soluble SUMO-scFv fusion protein was obtained.Its optimal expression conditions were determined by optimizing the expression conditions.IPTG was added 7 h after seeding and the final concentration of IPTG was 0.05 mmol.L^-1.?2?In order to further obtain the scFv without a tag,it is necessary to use the SUMO protease to excise the SUMO tag.First,the expression and purification of SUMO protease Ulp1 was performed:The gene ulp1with a polyhistidine tag?His6?at the N-terminus and C-terminus was synthesized by the gene engineering technology.The psvT7 was used as the expression vector,and high-throughput screening technique was used to quickly determine the optimal expression.Under the condition that BL21?DE3?was used as the expression host and IPTG was added 7 h after transfer,the final concentration of IPTG was 0.1 mmol·L^-1,the induction time was 16 h,and the final protein expression accounted for 34.5%of total bacterial protein.The expression level of recombinant protein Ulp1 was 190 mg·L^-1.Ulp1with a purity of 95%or more was obtained by one-step purification of Ni-NTA.The enzyme activity was 5.19 U·?L^-1 and the specific enzyme activity was 5.23×10⁴ U·mg^-1,which was1.87 times that of the previous reported enzyme activity.The apparent activity of Ulp1 was analyzed by mechanical analysis of enzyme activity.The Km value of the constant is 0.859g·L^-1,and Vm is 5.10?g·?mL·min?^-1.The purified fusion protein SUMO-scFv was digested and purified using the expressed Ulp1.At last,the scFv was obtained with a purity of more than 90%and no N-terminal excess amino acids.?3?In order to further improve the affinity of the antibody,the modeling analysis of the antibody was performed.Homologous modeling was constructed in the form of Fab,except that the similarity of the CDR3 of the variable region of the heavy chain and the known crystal structure was 53.8%,and the consistency of the rest was higher than 75%.The results of the Lagrangian plot showed that the homology was modeled.The result is reliable.The molecular docking study of Fab modeling and known crystal structures of HSA,indicates that the binding site of antibody and antigen HSA is mainly in domain III of HSAIn summary,SUMO protease Ulp1 was expressed and purified using molecular biology techniques,and successfully used for the expression and purification of scFv,providing raw materials for the development of scFv drugs with independent intellectual property rights.Through antibody modeling analysis,preliminary determination of antibody antigen binding sites provides direction for further verification of docking accuracy,and a basis for future virtual amino acid mutations to improve antibody affinity and antibody humanization.