Expressione Of Human Type ? Collagen In Pichia Pastoris

Background and Objectives:Type ? collagen has widespread applications in cosmetics,food,and healthcare industries.It also has promising application prospect for the treatment of wound healing,controlled-drug release,and has been used as scaffold material to construct organs in foreign.But the problem limiting the use of collagen is raw material.Currently,there are two main sources of collagen,one is purified from animal skins and tendons.They are used for cosmetics and health products.Because of their immunogenicity and potential pathogens contamination,While medical collagen is mostly recombinant protein,which is obtained by microbial fermentation.The difficulty of this method lies in how to increase collagen production and form active triple helix structure.This study aims to overcome these two difficulties.Collagens are highly hydroxylated proteins,The hydroxylation of proline which is catalyzed by 4-proline hydroxylase is pivotal for collagen biosynthesis.Eukaryotic cell lines including HEK-293 and CHO,have been explored for the recombinant expression of collagens.But due to the high cost and low yield,it's hard for large scale production.Pichia pastoris is an eukaryotic microbe generally regarded as safe(GRAS),it has plethora of advantages for heterologous expression,including uncomplicated nutrient demand for culture,rapid proliferation rate,applicable for large-scale production,high yield,suitable for secretive protein expression,and easy genetic manipulation.However,Pichia pastoris cells do not express 4-proline hydroxylase,so the genes encoding the two subunits of 4-proline hydroxylase need to be introduced into cells before the expression of type ? collagen.In this study,a novel balanced expression of two subunits of 4-proline hydroxylase was achieved by using self-cleaving 2A peptide.Type ? collagen expression strain was obtained,and the production of type ? collagen was greatly increased.Methods:In this study,the Pichia pastoris expression system was selected to explore the expression of human type ? collagen.The main experimental methods include:1.Due to the lack of commercial Pichia expression vector with Arg4 complementary function,we self-designed and constructed a Pichia expression vector with Arg4 gene element according to the principle of homologous recombination.PCRs were carried out to amplify necessary elements from p Bluescript SK,p PICZ?A,and Pichia pastoris genomic DNA,overlapping PCR and Gibson assembly techniques were used to construct Arg4 complementary expression vector p Arg4.2.The coding sequences of human type ? collagen alpha chain(COL3A1)and two subunits(P4H? and P4H?)of 4-proline hydroxylase were codon-optimized for Pichia expression.The optimized sequences were synthesized and cloned into p PICZ?A,p ARG4?p PIC9 K vector respectively.The designed sequence will be used for gene synthesis after codon optimized.Molecular cloning techniques including overlapping PCR and Gibson assembly were applied to construct polycistronic expression plasmid in which the self-cleaving polypeptide 2A was in-frame fused between P4H? and P4H? and was cloned into expression vector p PIC9 K.3.Linearized p ARG4/P4H? and p PIC9K/P4H? plasmid were transformed into GS200 strain,which is renamed as GS200/P4H?+?;The linearized p PIC9K/P4H?-2AP4H? was transformed into GS115,which is renamed as GS115/P4H?+?.Genomic DNA PCR and Western blotting were applied to assess whether the ? and ? subunits were successfully inserted into the chromosome and also expressed.4.By electroporation,linearized p PICZ?A/COL3A1 was transformed into GS200/P4H?+? or GS115/P4H?+?.Methanol was used to induce the recombinant proteins to express.The extracellular protein and intracellular protein were extracted and assessed for the expression of type ? collagen by Western blotting technique.Results:1.The Pichia expression vector p ARG4 was successfully constructed and the sequence was confirmed with restriction enzymes digestion and DNA sequencing.2.The three sets of optimized gene sequences were cloned into the expression vector,and finally four recombinant plasmids,p ARG4/P4H?,p PIC9K/P4H?,p PIC9K/P4H?-2A-P4H? and p PICZ?A/COL3A1 were obtained.3.PCR identification showed that p ARG4/P4H? and p PIC9K/P4H? were successfully transformed into GS200;p PIC9K/P4H?-2A-P4H? was successfully transformed into GS115.The engineered strains were induced to express recombinant proteins,and Western blotting showed that P4H? and P4H? were successfully expressed in Pichia pastoris.GS200/P4H?+? and GS115/P4H?+? engineered strains were obtained.4.PICZ?A/COL3A1 was transformed into GS200/P4H?+? or GS115/P4H?+?strain.Zeocin-resistant clones were induced with methanol to express recombinant proteins.and select a single colony to induce expression.The results of Western blotting technique showed that type ? collagen was successfully expressed in Pichia pastoris.Conclusion:P4H? and P4H? genes were recombinantly introduced into Pichia genome and successfully expressed to obtain functional 4-proline hydroxylase,which can promote collagen to form a correct triple helix structure.On this basis,the COL3A1 gene was introduced,and the expression was induced by adding 1% methanol,and finally human type ? collagen was successfully obtained.