Expression,Purification And Characterization Of HTGF-β3 In Pichia Pastoris

Objective:Transforming growth factor beta 3(TGF-β3),a member of the TGF-βfamily,has a wide range of cellular biological activities,such as promoting cell growth,differentiation,migration,apoptosis,extracellular matrix production and immunity.But unlike other family members TGF-β1 and TGF-β2,TGF-β3 is mainly expressed in embryonic development and plays an important role in embryonic development.Fibrosis of liver,lung,heart and other major organs and tissues seriously threatens the life and health of patients in clinical practice.Excessive scar formation during wound healing on the body surface also seriously affects the appearance of the face and normal functioning,and causes psychological burden on patients.TGF-β1 can promote the transformation of fibroblasts into myofibroblasts,stimulate collagen secretion,and play an important role in tissue fibrosis and scar formation,while TGF-β3 can promote scarless wound healing and is used in clinical and daily medical beauty.has important application value.Therefore,establishing a green,cheap and efficient preparation process is of great significance for the development of TGF-β3 in clinical applications.The three family members of TGF-β have high structural similarity and sequence homology,and the mature proteins are all homodimers.The precursor of TGF-β3 has a total of 412 amino acids and consists of three segments: a signal peptide composed of 23 amino acids,a latency-associated peptide(LAP)of 277 amino acids,and a mature peptide of 112 amino acids.Amino acids 74,135,142 and 293 are glycosylation sites.The mature TGF-β3protein structure is a homodimer composed of two identical monomeric peptide chains connected by a pair of interchain disulfide bonds.The mature peptide monomer of TGF-β3 has 9 highly conserved cysteines,of which 8cysteines form 4 pairs of intra-monomer disulfide bonds,and the remaining cysteines form a connection to another monomer of inter-monomer disulfide bonds.Due to the complex structure of TGF-β3,it is difficult to efficiently prepare TGF-β3 protein by means of genetic engineering.At present,TGF-β3protein has been expressed in prokaryotic and eukaryotic expression systems.However,because prokaryotic host cells lack a perfect post-translational processing mechanism,the expression products often exist in the form of inclusion bodies.Due to the complex structure,the in vitro renaturation of TGF-β3 inclusion body protein is difficult,and it is difficult to obtain active hTGF-β3 protein.The most common eukaryotic expression vector for TGF-β3is the CHO(Chinese hamster ovary cell)expression system,but the product expressed in the precursor form exists as a mature dimer and a fusion protein with LAP,which affects the downstream separation.Purification process efficiency,reagent grade TGF-β3 preparations all exist as a mixture of the two structural forms.The Pichia pastoris expression system is a mature eukaryotic expression system with relatively complete post-translational processing and modification functions.It can achieve high-density fermentation through a simple inorganic salt medium,and can efficiently secrete and express foreign proteins.It is ideal for TGF-β3.Industrial production host systems.Some people have used the Pichia pastoris methanol-inducible expression system to construct a mutant secretion expression system of another family member TGF-β1.The mutant is a cysteine substitution mutation involved in the formation of interchain disulfide bonds,and the expression product is a monomer.But it still has biological activity with application value.In this study,a green,efficient and inexpensive production process of human TGF-β3(human TGF-β3,hTGF-β3)was established by using the methanol-inducible Pichia pastoris constitutive secretory expression system,laying the foundation for further research and development of hTGF-β3.Base.Research content and methods:1.Expression system construction strategy(1)Construction of engineering bacteria expressing products in three structural formsOne is direct secretion and expression of hTGF-β3 mature peptide pulled by Saccharomyces cerevisiae alpha factor signal peptide(MFα),and the other is to use the auxiliary folding and transport functions of LAP to construct MFα-pulled LAP and hTGF-β3 fusion protein for secretion and expression.In order to improve the recovery efficiency of the mature peptide and obtain the natural hTGF-β3 at the N-terminus,an enterokinase recognition site(EK)was set between the LAP and the mature peptide,and the secreted product was in the form of LAP-EK-TGF-β3.Considering that there is a glycosylation site in the natural LAP sequence,and the potential site at position 293 is close to the enterokinase recognition site,the polysaccharide chain may mask the recognition sequence of the protease,so set a between LAP and EK by A flexible Lingker composed of(Gly4Ser)3,whose structural form is LAP-Linker-EK-TGF-β3.(2)Construction of a methanol-inducible green production system using a constitutive promoterAt present,most Pichia pastoris expression systems are methanol-inducible.This type of inducible relies on toxic methanol as a carbon source and inducer,which is likely to cause methanol pollution to the environment and products.Glyceraldehyde-3-phosphate dehydrogenase(GAP)is a relatively powerful constitutive promoter in the Pichia pastoris system,which can mediate high-level expression of target genes,and its expression capacity is higher than that of methanol.Regulated alcohol oxidase 1(AOX1)promoter.In addition,the promoter uses non-toxic substances such as glycerol or glucose as the sole carbon source during fermentative expression,does not require methanol induction,avoids the pollution and danger caused by the use of a large amount of methanol in the production process,and has high expression capacity and simple fermentation process.,more suitable for mass production.(3)using pure natural N-terminal designThe target gene is cloned downstream of the α-factor signal peptide of the p GAPzαA vector,and the final fusion protein expression product includes the signal peptide,protease recognition sequence,restriction enzyme cleavage site and the target protein,so usually the extra amino acids introduced at the N-terminus of the target protein in the secretion product will To a certain extent,it affects the activity of recombinant protein.Therefore,the construct that directly secretes and expresses hTGF-β3 utilizes the Xho I and Xba I sites within the signal peptide to clone the fusion gene.The expression of fusion with LAP utilizes the enterokinase recognition site to obtain the native N-terminal sequence.2.Construction and screening of engineering bacteria(1)Construction of expression vector: All target genes were optimized online by Pichia pastoris,and then synthesized by the company,and cloned into p GAPzαA vector using Xho I-Xba I double digestion.In addition,in order to facilitate screening of engineered bacteria and identification of expression products,a His6 tag sequence was introduced at the C-terminus of all constructs.(2)Transformation of Pichia pastoris: First,the expression vector was linearized with restriction enzyme Bln I,and then the vector was transferred into the host cell Pichia X33 by electroporation,and then the transformants were screened by bleomycin resistance screening method.(3)Screening of high expression recombinants : Anti-his6 antibody was used as the antibody for the detection of expression products,and high-expressing recombinants were screened by in situ dot immunoblotting,and the recombinants with strong immune signals were selected for seed preservation.These recombinants were then cultured according to the conventional culture method,and the supernatants were collected by centrifugation,and the high-expressing recombinants were screened again by dot blotting.(4)Screening of highly expressed recombinants by SDS-PAGE and Western blot: Shake the screened recombinants by conventional Pichia culturing method for three days,draw the supernatant to prepare samples,and perform SDS-PAGE and Western blot expression analysis respectively,so as to facilitate more Accurately screen out the highly expressed recombinants.3.Isolation and purification of expression productsA weak cation CM-sepharose FF chromatographic column and a weak anion DEAE-sepharose FF column,which are easy for industrial scale-up,were used to establish a purification process for secreted expression products.4.Characterization of expression products(1)hTGF-β3: The expression products were identified by Western Blot,and the structural forms of the expressed products were identified by reducing and non-reducing SDS-PAGE electrophoresis,respectively.(2)LAP-EK-hTGF-β3 、 LAP-linker-EK-hTGF-β3: The size of the expressed recombinant protein was analyzed by SDS-PAGE and Western Blot,and then digested and recovered by enterokinase.5.Biological activity assayMTT assay was used to detect the proliferation or inhibitory effect of recombinant protein on mouse fibroblast 3T3.Research result:1.Construction and screening of engineering bacteria(1)The secretory expression vectors of TGF-β3,LAP-EK-hTGF-β3 and LAP-linker-EK-hTGF-β3 were successfully constructed.The three vectors were linearized by single enzyme digestion and successfully transferred into Pichia by electroporation method.(2)High-expressing strains of the three recombinants were successfully screened by two screening methods,in situ dot immunoblotting and conventional dot immunoblotting,but the expression level of hTGF-β3 was significantly lower than that of the other two constructs with LAP.Form.It is proved that LAP can effectively promote the secretion and expression of TGF-β3 in Pichia pastoris as in higher eukaryotic cells.(3)Through the comparative analysis of the intracellular and extracellular expression products of the three engineered bacteria,it was found that some of the intracellular expression products of the three recombinant progeny were not secreted to the outside of the cell.Among them,LAP-EK-hTGF-β3 and LAP-linker-EK-hTGF-β3 Although these two recombinants added a LAP precursor,there are still many expression products that are not secreted effectively outside the cell,which may be related to the weak ability of Pichia pastoris MF-α to guide the secretion of complex proteins such as hTGF-β3.2.Isolation and purification of expression productsTGF-β3,LAP-EK-TGF-β3 and LAP-Linker-EK-TGF-β3 The three recombinant proteins secreted by Pichia pastoris were all purified by DEAE-sephrose FF column and CM-sepharose FF.The two recombinant proteins,LAP-EK-TGF-β3 and LAP-Linker-EK-TGF-β3,were recovered by enterokinase and their purities were 0.32 mg/m L and 0.41 mg/m L,respectively,and both have a purity of more than 98%.3.Characterization of expression products(1)TGF-β3: The purified expression product is a stable monomer rather than a dimeric structure.(2)LAP-EK-TGF-β3: The size of the secreted and expressed recombinant protein far exceeded the theoretical size,suggesting that the expressed protein was highly glycosylated during secretion.Direct digestion with enterokinase alone fails to cut effectively,but when a reducing agent such as mercaptoethanol is added to the reaction system,it can be cut effectively,and the cut product still exists in the form of a stable monomer.Prove that the expression product structure is speculated,the intestinal kinase recognition site is covered by a sugar chain.(3)LAP-Linker-EK-TGF-β3: Its secreted expression product is also a highly glycosylated protein,which can be effectively cleaved by direct digestion with enterokinase alone.Obviously,by adding an elastic Linker composed of 15 amino acid residues,the problem of sugar chain masking can be effectively solved,and the enterokinase site can be effectively exposed.As with the first two constructs,the recovered product is also in the stable monomeric form.4.Biological activity assayThe monomers of the two recombinant proteins LAP-EK-TGF-β3 and LAP-Linker-EK-TGF-β3 recovered by enterokinase can inhibit the growth of mouse fibroblasts 3T3 to a certain extent,indicating that they have natural products activity.Conclusion and Outlook:Using the Pichia pastoris constitutive secretion expression system,high-expression engineering bacteria of TGF-β3 and two fusion proteins with precursor peptide LAP were successfully constructed,laying a solid foundation for the establishment of a green,efficient and inexpensive industrial production process for hTGF-β 3.The solid foundation also provides new ideas for the establishment of production processes for other members of the TGF-β family.Regardless of the form of construction in this study,the obtained hTGF-β 3 products all exist in the form of stable monomers,indicating that the cysteine that exists freely in such monomers is not affected by natural oxidation or redox agents.It exists and forms a disulfide bond with the monomer of another molecule.The related reasons and the relationship between structure and function need to be further studied.