Expression,purification,and Bioactivity Of A Novel Recombinant Human Fibronectin Peptide In Pichia Pastoris X33

Objective: Alone the develpment of the biomaterials,the third generation of “tissue inducing” material became mainstream.The “tissue inducing” materials should have excellent bioactivity,which induces cell attachment,growth and proliferation to achieve complete regeneration of tissues.These advances have led to clinical successes for inducing some simple tissues such as skin and cartilage.Collagen and fibronectin are the main components of extracellular matrix(ECM),which are widely used in regenerative medicine as biodegradable biomaterials and meet characters of above.However,animal-derived proteins would be immunogenic,while at the same time,problems related to the processing techniques and degradation rate of animal-derived proteins,limit its application.It is a research hotspot to acquire more excellent regenerative medicine materials by the technique of genetic engineering and synthetic biology.In this study,we designed a novel fusion protein(FNC)composed of recombinant human fibronectin and recombinant human collagen using gene recombination technology,and successfully expressed it.FNC has better ability of recruiting and sticking stem cells,which can be developed into a implantable regenerative medicine materials.Methods:(1)After yeast codon optimization for gene FNC and FN10(fibronectin fragment of the fusion protein,as the reference in the subsequent experiments),the recombinant plasmids of p PICZ?A-FNC-his and p PICZ?A-FN10-his were constructed and transformed into Pichia pastoris expression system for recombinant expression and purification.Through the screening and optimization of expression strains and expression conditions,the engineering strains X33 with high expression of FNC and FN10 were obtained.Both of them were purified using Ni-NTA Sepharose affinity chromatography column.We also conducted a 2L scale-up process study.(2)The secondary structure of FNC protein was predicted by the folding recognition method using software of PSIPRED 4.0.Moreover,FNC was docked with cytointegrin ?3(ZDOCK docking,the docking result is optimized by IRa PPA)to predict the cell-binding ability of FNC.(3)Primary rat bone marrow mesenchymal stem cells(BMSCs)were extracted,and the multidifferentiation ability and expression of integrin ?3 of BMSCs were identified respectively.The effects of FNC,FN10 and collagen fragments(CP)on the proliferation and adhesion of BMSCs were respectively studied by the methods of crystal violet staining,confocal microscopy,immunofluorescence,and scratch test.Results:(1)p PICZ?A-FNC and p PICZ?A-FN10 recombinant expression vectors were constructed,and X33/FNC and X33/FN10 engineering strains were successfully obtained.At the same time,the expression conditions of recombinant human proteins,FNC and FN10,were selected and optimized.The results showed that the expression of the fusion protein was dose-independent with zeocin.The optimum fermentation conditions were 1)the best medium was YPM,2)methanol was 1.0%,3)induction time was 72 h.The HPLC purity of FNC and FN10 was 96% and 97%,respectively.The molecular weight of FNC is 33.87 k Da determined by LC-MS/MS,which is consistent with the theoretical value.The expression of FNC was above 63.65% of the total protein;the p H of the fermentation broth was stable at about 7.2.(2)The structure of FNC was successfully simulated by threading method.The dock of FNC and integrin ?3 was carried out by software ZDOCK.The docking results showed that FNC would interacte with integrin ?3 to form a relatively stable complex.(3)Flow cytometry was applied to characterize the immunophenotype of the BMSCs,and they have the ability of adipogenic and osteogenesis differentiation.The effects of FNC on the promoting adhesion and migration of BMSCs were studied.Compared with FN10 and CP,FNC has a better ability in promoting the adhesion and migration of BMSCs.Conclusion: In this study,a novel fusion protein of rh FNC and rh FN10 protein were successfully expressed by Pichia pastoris X33,and their structure and function were verified.The HPLC purity of both was more than 95%.Compared with independent fragments of fusion proteins,FNC significantly promoted the adhesion and migration of primary BMSCs.As a new ECM-like protein,FNC has the potential to further studied.