Fermentation Conditions Study Of Producting Recombination Human-like Collagen In Pichia Pastoris

Collagen is a series of protein which is widely in the lower vertebrates and mammals. It is of high content in mammals' bodies, about 25%-30% in gross protein. Collagen is active, and is used as medical materials and hairdressing products. But nowadays the collagen from animals' coat and connective tissue by acid hydrolyzation, alkali hydrolyzation or enzyme hydrolyzation is always not active, of virus hidden trouble, and of rejection. These restrict its clinic and daily application. The collagen producted by gene engineering technology can overcome those disadvantages upwards. And on the basis of human source gene sequence of collagen, people can design and synthesize similar gene sequence transformed into host cell to express new protein which is called as human-like collagen.The aim of the present study was to screening factors that determining the production of recombinant collagen in Pichia Pastoris on the basis of previous successful construction recombinant microbe(GS115/pPIC9KG6) in our laboratory. The shake-flask fermentation conditions of recombination gene engineering microbe (Pichia pastoris GS115 Mut⁺) were optimized, which contained inducement temperature, pH value, methanol addition value, inducement period choice, shake-flask rotary speed, fermentation time, inoculation value and so on. The shake-flask fermentation technology that GS115 expresses human-like collagen was ulteriorly explored. By single factor test and orthogonal test, the results were showed, when the fermentation period induced with methanol is 84h, the optimum inducement temperature is 30℃, the methanol addition value is 2%/24h, the fermentation initial inducement pH value is 6.5, the yeast original concentration is 2 OD₆₀₀/mL, the expression value increases. And the yield of human-like collagen is about 98.06mg/L in the optimization inducement condition.On the basis of SDS-PAGE imaging analysis ration method, a rapid, handy analysis method—hydroxyproline ration method were explored primarily. And the transform formula of collagen content from two different ration analysis methods was educed.