High-density Fermentation Of Genetically Engineered Pichia Pastoris Expressing Recombinant Human-source Collagen

Collagen is widely used in various fields, such as medical and health, tissue engineering, food, hairdressing, etc., because of its good biological compatibility, decomposability and low immunogenic idiosyncrasy. Currently, most commercial collagens and gelatins are mainly prepared by acid or alkali hydrolysis and enzyme-treating method from animal tissue such as skins and bones, etc. However, the inherent defects of these products prepared by the above methods greatly limit their application as medicine, biomedical material or drug delivery system, etc. In this dissertation, based on modern biological technology, recombinant human-source collagen was expressed and produced by genetically engineered strain.1. Based on the successful construction of genetically engineered Pichia pastoris expressing recombinant human-source collagen, yeast cell pellets were disrupted intermittently by ultrasonic under low-temperature to obtain cell lysate. SDS-PAGE analysis was used to confirm the expression form of recombinant human-source collagen.2. Response surface methodology (RSM) was used to optimize the culture conditions of genetically engineered Pichia pastoris in shake flask cultivation.3. In view of the characteristic of methylotropic yeast Pichia pastoris, using the fermentation conditions optimized by RSM, the optimization of high-density fermentation techniques was studied in a3.7L fermenter and a12.5L fermenter. And then, kinetic analysis was actualized aiming at the high-density fermentation under optimal fermentation technique. Cell growth, product synthesis and substance consumption model at methanol inducement phase were established.4. Three approaches including ammonium sulfate precipitation, combination separation technique with Q-Sepharose Fast Flow anion exchange chromatography and Sephadex G-100gel filtration chromatography, and single-step purification with Sephadex G100gel filtration chromatography were adopted to separate and purify the expression product.5. The structure of recombinant human-source collagen was characterized by amino acid analyse, MALDI-TOF-MS analyse, ultraviolet-visible spectroscopy, FTIR spectroscopy, circular dichroism and scanning electron microscope. And the potential as biomedicine material was evaluated by the determination of water-solubility, film forming ability, viscosity and biological activity.6. The biological effects of genetically engineered Pichia pastoris expressing recombinant human-source collagen under simulated space environment were explored using high-magnetogravitational environment and modeled microgravity. 7. Recombinant human-source collagen and chitosan were blended to produce biomedical nanomaterial, and its structure characterization was actualized by analyses of FTIR, SEM and TEM, etc.As a result, the optimized fermentation conditions and techniques were obtained. Using eukaryotic expression system to express recombinant human-source collagen, the expression level is in the domestic and international leading position. The best purification methods of recombinant human-source collagen were obtained, thereinto, the single-step purification with Sephadex G100gel filtration chromatography is simple and easy in operation, large in capacity, and so, it could be treat as a perfect method in the purification of recombinant human-source collagen dimer. The biological effects of genetically engineered Pichia pastor is expressing recombinant human-source collagen under simulated space environment and the methods of producing biomedical nanomaterial using recombinant human-source collagen were explored.