Exploration Of Recombinant Human Gelatin Expression Condition In Pichia Pastoris

Gelatin is a collagen loss of activity. For its excellent physical andproperties, gelatin has been widely used in food, cosmetic, medical,photosensitive materials and so on. At present, gelatin is mainly derived fromanimal. But these gelatins possess the risk of potential contamination withviruses and prisons. Recombinant gelatins can have stable and predictablecharacteristics, and have the exact molecular composition. To date, allrecombinant gelatins have no hydroxylation or the level of hydroxylation waslowIn our research, we used the Pichia pastoris recombinant gelatin andprolyl hydroxylase gene, and achieve co-expression. We detectedbothβsubunit of prolyl hydroxylase protein disulfide isomerase and gelatinby SDS gel electrophoresis. We take N terminal sequencing of gelatin and theresult march perfectly the theory N sequence of recombinant gelatin.Through the optimization of induction conditions and observation ofelectrophoresis of supernatant, we find out condition of the maximize induction production. The conclusion is, in250mL shake flask, the volume ofliquid less than50mL adding2%methanol each12h is the best choice underthe temperature of25, the shaking speed of250rpm. When the wet yeastdestiny less than0.5g/L the higher destiny will bring the more production.And we also know the expression quantity ofβsubunit of prolyl hydroxylasereach a peak in the forth day, while the expression quantity of gelatin reachesa peak in the second day.In addition, we also detect the quantity of hydroxylation gelatin byhydroxyproline color method. The data show that the hydroxylation gelatineven less than10%of total gelatin in supernatant. Adding ketoglutaric acidand vitamin C cannot increase the quantity of hydroxylation gelatin; evenworse inhibit the expression of all induced production.