Study On The Effect Of EndoH On The Half-life Of The HSA Fusion Protein Expressed In Pichia Pastoris

With the rapid development of biopharmaceutical technology,protein drugs has become the important ingredient of the new biotechnology drugs,such as,steroids,cytokines drugs,soluble receptors and receptor antagonists,Thrombolytic drugs and other recombinant protein drugs.Most of them have as the character of higher physiological activity,low immunogenicity and minor side effects.But they are also easy to be removed by the kidney and liver and other organs or proteolysis.Most protein and peptide drugs show short plasma half-life.It commonly used long acting mechanisms of fusion proteins,PEG-modified biotechnology to development of long acting new protein and polypeptide drugs.Presently,an increasing number of recombinant proteins have been produced by HSA fusion protein technology,such as GLP1/HSA,IFNa/HSA,GM-CSF/HSA,etc.P.pastoris expression system is the new generation of protein production platform.But the protein expressed in P.pastoris with varying degrees of glycosylation,which can be cleaned by receptor in liver or lymphocytes.Steric produced by HSA fusion also affect the activity of the fusion protein.The study carried out for these two problems:?As a model to GM-CSF/HSA,research the influence on pharmacokinetic of this class protein that deglycosylated by Endo H.? As a model to IFNa2b/HSA,study the impact of the fusion protein in vitro biological activity which linked with Hinge region.Firstly,this research used Endo H to hydrolyze of high-mannose type oligosaccharide of GM-CSF/HSA which expressed in P.pastoris GS115,and analyze the internal half-life of GM-CSF/HSA-Endo H in mice.In this study,we expressed Endo H in P.pastoris JC308(ura3-,his4-,arg4-).One complete gene was synthesized on the basis of the cDNA sequence encoding Streptomyces plicatus reported in GeneBank.The gene was cloned into the expression vector pPIC9.The expressionvector pPIC9-EndoH was transformed into P.pastoris(JC308).The expression products were induced by methanol,purified by two-step of chromatography,used to analyze the glycan structures of RNaseB by the DSA-FACE(DNA sequencer assisted fluorophore-assisted carbohydrate electrophoresis)methods,and finally compared with PNGaseF.Endo H can retrieve the shortcoming of PNGaseF in the location and functional analysis of N-glycosylation.And then,the high-mannose type oligosaccharide of GM-CSF/HSA which expressed in GS115 digested with Endo H.Then it compared with GM-CSF/HSA use MTT colorimetric method.The result show we that the activity of GM-CSF/HSA is 39.99IU/?l at 0.25 h.and it decrease of about four-fold at 0.75h.The activity of HSA/GM-CSF-Endo H is 319.98IU/?l at 0.25 h,and it almost no activity is lost at 0.75h.Half value is at 6h,after a slow decreased showed metabolism.So the half-life of GM-CSF/HSA/Endo H is significantly prolonged in mice.Secondly,exposure of active binding sites of the protein may affect after the HSA fusion protein.This causes the issue of degrade of biological activity.We added a linker between HSA and the fusion protein for increase spatial distance between them,Look forward to obtain a Novel fusion protein with longer half-life and higher biological activity.This research locus on the preparation of IFNa2b/Hinge/HSA expressed in pichia pastoris,Hinge and pHIL-D2/HSA/IFN is used for template,fragment of Hinge and HSA is amplification with PCR,and then the gene was cloned into the expression vector pPICZaA.IFNa2b was cloned into the recombination of pPICZaA/Hinge/HSA.The expression vector pPICZaA/IFNa2b/Hinge/HSA was transformed into P.pastoris.The expression products induced with methanol and purified by chromatography.Use the method of cytopathic inhibition assay to analyze activity of IFNa2b/Hinge/HSA and IFNa2b/GGGGS/HSA.The result showed that activity of IFN/Hinge/HSA is 2.8 times then IFNa2b/GGGGS/HSA.And then GLP-1/Hinge/HSA is expressed in GS115.To solve the problem of GLP-1 which is easily identified and degradation by DPP-IV,Ala8 is insteaded of Glu8 and Gly8 using mutation.Three amino acids GLP-1 was cloned into the expression vector pPICZaA/Hinge/HSA.The recombination expression vector pPICZaA/GLP-1/Hinge/HSA was transformed into GS115.The expression products induced with methanol and purified by two steps of chromatography.The results of the biological activity of GLP-1 required follow-up experiments.Summary,This study was established techniques of EndoH efficient preparation in Yeast.Found HSA fusion protein glycosylated with the enzyme has a longer half-life in vivo.We design and preparation of methods for improving HSA fusion proteins in vitro biological activity by added a joint area.