Recombinant Expression Of Human Type Ⅲ Collagen In Pichia Pastoris And Properties Study

Collagen is the most abundant series of proteins in mammals,which is distributed in cardiovascular,bone,skin and other tissues and organs,and has important physiological functions.the current mainstream means of obtaining collagen is the digestion of collagen peptide mixtures from animals such as bovine and porcine fur and connective tissues,by acid,alkaline and enzymatic hydrolysis.The collagen obtained by this process has problems of immunogenicity and animal virus potential,thus limiting its application in many fields.These problems can be well avoided by expressing fragments of type III collagen α1 chains from human by using Pichia pastoris as the expression host.In this study,peptide of the human type III collagen α1 chain(amino acids from position 908 to 1137)was selected as the expressed protein,and Pichia pastoris recombinant strains with collagen gene expression were constructed.Shake flask fermentation was optimized and High-density fermentation in 5L bioreactor was performed to improve the expression level of recombinant collagen.The antioxidant properties of recombinant collagen were tested and the structure of recombinant collagen was analyzed,which provided useful data for its potential application.The main research conclusions are as follows.(1)Construction of a high-copy recombinant collagen expression strain using Pichia pastoris as the host.Collagen gene was expressed under AOX1 promoter with α-factor signal peptide.The expression vector with two-copy collagen and four-copy collagen were successfully integrated into the HIS locus of Pichia pastoris by electrotransformation.The strains were cultivated in BMMY.After incubation by 0.5% methanol,the recombinant collagen was determined by SDS-PAGE electrophoresis,Western Blot and protein mass spectrometry,suggesting that the recombinant collagen was successfully expressed in Pichia pastoris.(2)Fermentation optimization of collagen recombinant strain.Using the high-density shake flask process,the strain was cultivated in BMGY medium and induced in BMMY medium under high-density of cell.For the four-copy collagen recombinant strain,by high-density shake flask process,the concentration of collagen concentration increased from a level that was difficult to detect by SDS-PAGE electrophoresis to a level where the protein bands were clearly visible.By optimizing the methanol induction concentration and fermentation days in the high-density shake flask process,the highest yield of collagen was finally achieved at 0.45mg/m L at 0.5% methanol induction concentration and 72-h induction time.High-density fermentation in 5-L bioreactor was performed with BSM medium.The maximum collagen concentration was reached at 120 h of fermentation time,and the yield of recombinant collagen was improved to be 1.98 mg/m L.(3)Purification and characterization of recombinant collagen.Results showed that the recombinant collagen could be concentrated at 40% ammonium sulfate concentration,but there were still too much other proteins.Nickel column affinity chromatography was much more effective in purifying the target protein,and 3.66 mg of collagen was purified with a recovery of 29.61%.The in vitro antioxidant properties of recombinant collagen was investigated,with 51.49% DPPH radical scavenging and 41.24% ABTS radical scavenging at the concentration of 6 mg/m L,showing effective antioxidant function.Circular dichroism data showed that the secondary structure of the recombinant collagen contained 49.5%β-folded,25.7% disordered structure and 22.7% β-turn,and only 1.7% α-helix.The recombinant collagen lyophilized powder was observed via scanning electron microscopy at the magnification of 250,500,1500 and 5000.Results showed that The recombinant collagen contained a spiral curl and filamentous structure,with spongy characteristic,owing the potential application in food and medicine as filling materials.In conclusion,in this study,a recombinant Pichia pastoris strain with high expression level of recombinant collagen was constructed via multi-copy expression strategy.The expression of recombinant collagen was further enhanced by fermentation optimization in flask and 5-L bioreactor.Antioxidant properties and protein structure of the lyophilized collagen powder were also analyzed,which could provide a foundation for the application of recombinant collagen.