High Secretory Expression Of The Anti-VEGF Antibody Fragment Fab In Escherichia Coli

Currently,the antibody fragment Fab plays an important role in the treatment of a variety of human diseases,such as cancer,virus infection,and inflammation.Due to its lack of glycosylated Fc,the antibody fragment Fab does not need to be glycosylated.This structural feature allows it to be actively expressed in E.coli.The obvious advantage of E.coli as a host strain is its low cost,fast growth,easy control and high yield in a short time,and it has gradually become the backbone of producing antibodies.The main limiting factor in the production of antibody fragments Fab in E.coli is its low yield.The reason is that Fab is misfolded and aggregated in the cytoplasm,forming a non bioactive inclusion body.The host bacteria contain a variety of proteases,which have a certain degradation effect on Fab.Therefore,in order to improve the production of antibodies,we can study from several aspects,such as expression vectors,host bacteria,molecular chaperones and folding enzymes.In this study,the expression of Fab was optimized from three aspects:1)Based on the previous work,E.coli DH11 T7 was used as the host strain to improve the yield of Fab in E.coli by constructing different Fab expression vectors;2)Based on 1)work,by co-expressing multiple chaperones to further increase Fab yield in E.coli DH11;3)Based on the E.coli periplasmic protease-deficient strain DH11-ptr-degp(hereinafter abbreviated as 86D),a single amino acid mutation was carried out on the spr gene in its genome,and the host strain 86D-Mspr was obtained.At the same time,the dominant Fab expression vector and molecular chaperone were co-xpressed,and the yield of Fab in E.coli was raised by the fermentation experiment.For the construction of Fab expression vectors,the codons of the DsbA and STII sequences of the heavy chain signal peptides were optimized to change the intensity of the translation initiation region(TIR).The obtained 13 heavy chain fragments were respectively ligated with Pe1B-LC to the pET-3b vector backbone.The combination of DsbA8+HC+Pe1B+LC(pET-DH8PL),a Fab optimal expression vector,was obtained by ELISA analysis,and the total yield was about 14 mg/L.By further constructing a single molecular chaperone expression vector and multiple chaperone expression vectors,the secretion of Fab in E.coli can be improved to a certain extent,especially when combined with p15c-FkpA-SurA,the yield of Fab can reach 20 mg/L.When the host strain was changed from E.coli DH11 to E.coli periplasmic protease-deficient strain 86D,the yield of the antibody fragment Fab could be increased by about 20%.E.coli belongs to the prokaryotic expression system,and its fermentation process is relatively simple.The large-scale culture of the expression host is to obtain large-scale antibody proteins.In this study,a single amino acid mutation of E.coli 86D spr H145A was performed in a similar manner on the basis of the Red recombinant markless DNA deletion method constructed in the laboratory,and a genomic mutation strain 86D-Mspr was obtained.In the first step,the constructed donor plasmid p15AD2IC-Mspr-SacB and the helper plasmid pAAICDGBE were co-transfected into E.coli 86D,under the induction of L-arabinose,expressing the I-CreI endonuclease and the Red recombinase.The I-CreI enzyme cleaves the left and right homologous arms and the Cm resistance gene on the donor plasmid,performs homologous recombination under the action of the Red recombinase,and finally successfully integrates the Cm resistance gene on the E.coli 86D genome.In the second step,the helper plasmid SN3K was transformed into E.coli 86D containing the Cm resistance gene in the first step.Similarly,under the induction of L-arabinose,I-SceI endonuclease and Red recombinase were expressed.Under the action of I-SceI enzyme,the Is-Cm fragment on the genome was excised,and homologous recombination between the left and right homologous arms and the chromosome occurred under the action of the Red recombinase,thereby obtaining a genomic mutant strain 86D-Mspr.Finally,the preliminary fermentation process was studied.The yield of antibody fragment Fab in the 3 L fermentor reached 72 mg/L.This work laid the foundation for further increasing the production of antibody Fab.