Reconstitution Of A Novel Human-like N-Glycosylation Pathway For Recombinant Proteins In Escherichia Coli

Escherichia coli is a favorite host for producing biotherapeutic proteins,which has the advantages of short production cycle and low cost compared with mammalian cell expression systems.However,natural E.coli lacks post-translational processing,such as the ability to glycosylate proteins,which limits its application.In the past decade,the functional application of oligosaccharide transferase(pglB)from Campylobacter jejuni to E.coli has enabled the expression of N-glycosylated recombinant proteins in the periplasm of E.coli with many N-oligosaccharides,such as the heptose of Campylobacter jejuni and human core oligosaccharide(Man3GlcNAc2).However,the structures of these oligosaccharides differ greatly from those of human glycoproteins,and their modification efficiencies are generally not high.Furthermore,N-glycosylation modified with the terminal sialylation can significantly improve the physicochemical properties of recombinant proteins.It remains a challenge to produce terminally sialylated N-glycoproteins economically and efficiently in E.coli,since non-pathogenic E.coli does not naturally produce sialic acids.This study developed a novel pathway for producing human-like N-glycosylated proteins with terminally sialylation in the periplasm of E.coli,aiming to achieve a high yield with low cost.The main work of the thesis was listed as follows:(1)A model of receptor protein for studying N-glycosylation pathway was established in E.coli.The flexible chains were designed to link oligosaccharides to different regions of FN3 proteins.Receptor protein expression vectors containing different glycosylation sequons in different sites and the vector pACYCpgl containing N-glycosylated gene cluster from were transformed into the periplasm of E.coli CLM3 7.A series of proteins were N-glycosylated via the glycosylation machinary from Campylobacter jejuni(with GalNAc-?-1,4-GalNAc-?-1,4-[Glc-?-1,3-]GalNAc-?-1,4-GalNAc-?-1,4-GalNAc?-1,3-Bac-).The efficiency of N-glycosylated FN3 was optimized systematically by different induction methods and culture conditions.It was found that auto-induction method could greatly improve the glycosylation efficiency;the glycosylation efficiency of FN3-Gly1(also named as FN3-Gly,"Gly" means carrying a glycosylation sequon)could be increased to 100%by using the optimized glycosylation sequon DQNAT constructed in the C-terminal flexible region of FN3 proteins.(2)The human-like N-glycosylation pathway with terminal sialylation was established in the periplasm of E.coli,and the efficiency of N-glycosylation was optimized.1)Constructing a human-like N-glycosylation pathway:The vector p15-Ara-lsg was constructed by synthesizing the genes of O-antigen synthase WecA from E.coli,pglB transferase,flippase pglK from C jejuni and the lsgCDEF locus from Haemophilus influenzae.Thus,the oligosaccharide chain of Gal-?-1,4-GlcNAc-?-1,3-Gal-?-1,3-GlcNAc(Gal-?-1,4-GlcNAc-is human-like)was thesised.Then the efficiencies of synthesizing human-like oligosaccharide chains and N-glycosylation were optimized by combination of different types of promoters,different induction methods and culture conditions.It was found that the glycosylation efficiency could reach 100%by the inducible Ara promoter under optimized auto-induction conditions.2)Constructing a pathway to produce terminally sialylated N-glycoproteins in the periplasm of E.coli,mimicking the sialylated moiety(Neu5Ac-?-2,6-Gal-?-1,4-GlcNAc-)of human glycan.The vector pIG6-Sia-FN3-Gly-P2-plst6 was reconstituted,and Neu5Ac?-2,6-Gal-?-1,4-GlcNAc-?-1,3-Gal-?-1,3-GlcNAc-was synthesized through the coexpressed glycosyltransferases LsgCDEF from Haemophilus influenzae,Campylobacter jejuni NeuBCA enzymes and Photobacterium leiognathi ?-2,6-sialyltransferase in an engineered E.coli strain which produces CMP-Neu5Ac.And the C.jejuni oligosaccharyltransferase pglB was used to transfer the terminally sialylated glycan onto a glycosylation sequon in the FN3-Gly.Sialylation of the target protein was 60-70%.(3)An extracellular secretory expression system for enlarging production of humanlike N-glycosylated recombinant proteins has been successfully established.1)Establishing a secretory expression system:The lipoprotein lpp gene in E.coli outer membrane was knockout to constructe the leakage strain.The N-glycosylation recombinant protein FN3-Gly(14.2 kDa),Enhanced Green Fluorescent protein(eGFP-Gly,29.4 kDa)and anti-VEGFR3 scFv(scFv-vEGFR3-Gly,39.8 kDa)were further explored.It was found that the leakage fractions into the medium decreased gradually with the increasing molecular weight of recombinant N-glycosylated proteins.The highest yield of FN3-Gly was 106±7.4 mg/L.2)Applications of the secretory expression system:An anti-VEGFR2 monobody FN3vEGFR2-Gly(also named as C7+-Gly)expression vector was constructed.Through systematic optimization of secretory expression conditions,the N-glycosylated antiVEGFR2 monobody FN3VEGFR2-lsg(with Gal-?-1,4-GlcNAc-?-1,3-Gal-?-1,3-GlcNAc-)was successfully produced in CLM37?lpp,with glycosylation efficiency reaching 100%and a high yield(50±3.7 mg/L).3)A secretory system of expressing sialylatd human-like N-glycosylated recombinant proteins was established.Firstly,leakage strain CLM37?lpp?nanKETA was constructed,and pIG6-Sia-FN3vEGFR2-Gly-P2-plst6 vector(carrying Campylobacter jejuni NeuBCA locus,FN3VEGFR2-DQNAT sequon,P2 promoter and Photobacterium leiognathi ?-2,6sialyltransferase),together with p15-Ara-lsg vector capable of synthesizing human-like oligosaccharides,was applied for secreting monobody with sialylated human-like oligosaccharide chain.A large number of sialylated human-like N-glycosylated FN3VEGFR2Sia can be produced by using the optimized conditions,and the sialylated efficiency reached 70%,and the yield reached 40±4.3 mg/L.(4)The physicochemical properties and biological activity changes of sialylated human-like N-glycosylated monobody were investigated.It was found that human-like Nglycosylation of FN3VEGFR2-Sia could improve the solubility,chemical stability,thermal stability and serum stability,but had no significant effects on the biological activity and affinity.In conclusion,this study aims to establish a novel human-like N-Glycosylation pathway for producing sialylated human-like N-glycosylated proteins in E.coli.The yield and N-glycosylation efficiency of recombinant glycoproteins are greatly improved through the combined application of using the leakage strain and auto-induction.This study is of great significance for large-scale producing recombinant glycoproteins with high efficiency and low cost.It also opens a new way for the production of glycosylated protein drugs in E.coli.