Study Of Expression Recombinant Hydroxylated Human-Derived Gelatin In Pichia Pastoris

Gelatin is a class of collagen thermal denaturation and irreversible chemical degradation products, and it can be categorized as photographic gelatin, medicine gelatin, edible gelatin, and industrial gelatin according to different uses, and used widely in various fields as an ingredient agent, suspending agent, adhesive agent and forming agent. As a slaughterhouse by-products, the commercialization gelatin in the traditional sense was mainly extracted by physical and chemical methods, therefore, different production batches of gelatin is generally not the same and have different characteristics, coupled with the extracted gelatin possesses the risk of potential contamination with viruses and prions, which make it difficult to fully guarantee their product safety, while recombinant DNA technology is another strategy to obtain gelatin.This strategy is produce gelatin directly by expressing collagen gene fragments with a specific length and composition, which can provide a gelatin with stable and predictable characteristics, and having the exact molecular composition. To date, all recombinant gelatins have no hydroxylation without the introduction of the exogenous prolyl 4-hydroxylase genes, and even some species have the endogenous hydroxylation capacity, but the level of hydroxylation was low, and thus can not form a solid three-helix structure, and it is easily degradable in cells that can not be rapidly secreted out of the cell.In this thesis, expression of recombinant gelatin was achieved in Pichia pastoris, and a certain hydroxylation of gelatin was gain by introducing exogenous prolyl 4-hydroxylase genes. During the process of constructing the vector, three independent expression cassettes were connected one after another, and each expression cassette has its own promoter and terminator, respectively. In this way, their mRNAs were transcripted, respectively and translated into different proteins. In each expression cassette, we used two strong inducible promoter (PAOX1 and PFLD1), respectively as promoting elements of these genes. The main works are the following aspects:1. Synthesized a length of about 300bp encoding 100 amino acids (No.531-630) of the human typeⅠcollagen alpha (1) gelatin chain gene (Genebank login:P02452) by overlap extension PCR method.2. The components of expression cassette were successfully cloned. PFLD1 promoter was amplified with Pichia pastoris genomic DNA as a template, and the expression of green fluorescent protein was used to detect the function of the promoter; aMF signal peptide and TT (terminator) is amplified, separately with the expression vector pPIC9K as a template; prolyl 4-hydroxylase genes (αP4H andβ) was amplified, separately with the cDNA clones containing their coding gene as a template.3. We constructed multi-gene expression vector with the gelatin gene and prolyl 4-hydroxylase gene (αP4H andβ) introducing into the same yeast expression vector pPIC9K. First, the components were cloned, then connect every two adjacent vectors containing the components by digestion and connection method in order to assemble the cloning vector onto the same pGEM-T vector, and finally the entire expression cassette was cut from the T-vector and inserted into the expression vector pPIC9K.4. Expression vector was linearized with Sal I, and homologous exchange by electroporation, the entire expression cassette was integrated into the chromosome of Pichia pastoris KM71. All transformants were Muts, and MD plate containing 4mg/L G418 was used to screen high copy strains. After methanol induction, there is a protein band showing a strongly induced expression patterns. The molecular weight of the protein is about three times size of the monomer (non-hydroxylated gelatin). The protein was detected by LC-MS/MS, which indicated it matches with human type I collagen, and 66% hydroxylation of proline in the Y position of Gly-X-Y was occurred.5. The fermentation conditions of microbial production of recombinant gelatin were preliminary optimized. The yield on the sixth day is still presented a growth trend-thus there is room for further increase in the yield of recombinant gelatin; The methanol concentration of 1% (once every 12 hours) is the most optimal concentration. The expressed recombinant gelatins were identified and characterizated. Circular dichroism detection indicated the secondary structure of gelatin contains 61.5%β-turn and 38.5% random coil without the traditional a-helix and P-sheet. The nuclear magnetic resonance spectroscopy of gelatin test results show that the 1H and 13C spectra have many corresponding characteristic displacement peaks. UV spectra of gelatin shows its maximum absorption peak is 225nm.