Fermentation,Purification Of Human Type Ⅲ Collagen α1 Chain And Construction Of Hydroxylated α1 Chain System

Collagen is the most abundant protein in the body,which plays a role of structural support in the bones,skin,tendons and blood vessels.As a fibrous structural protein,collagen and collagen derived derivatives have a high demand for biomedical applications.At present,collagen derived from human and animal bones has a risk of contamination in applications such as HIV and mad cow viruses.Therefore,it is necessary to look for safer hosts to study the expression of collagen as an exogenous protein.Pichia pastoris is widely used in the expression of exogenous protein due to its low cost,high efficiency and purification of subsequent products.The purpose of this study is to investigate the fermentation of human type Ⅲ collagen α1 chain on the basis of recombinant strain GS115-COL3A1 which has been constructed before by our laboratory,and to separate and purify the fermentation broth containing the target protein.In addition,the post-translational modification process in the synthesis of collagen was studied by co-expressing the collagen gene with the prolyl 4-hydroxylase.The main works of this study are as follows.Firstly,the strain GS115-COL3A1 was cultured in a 20 L fermentor to determine the optimum conditions for the amplification process,on the basis of culture conditions such as temperature,p H,dissolved oxygen and rotational speed etc.,the optimized biomass of OD600 of 18,the halved BSM medium,and the mixed methanol and glycerol with the ratio of 1/2 in the induction of protein expression were determined.Finally to obtain the target protein concentration is as high as 2.38 g/L of yield.Secondly,the supernatant of the fermentation broth was purified by the four-step process,which containing salting out,ion exchange,gel filtration chromatography,and desalination using G25 desalting column.The purity of type Ⅲ collagen α1 chain was 95% and the recovery was 52.4%.The freeze-dried product was dissolved in a cell-specific medium to prepare a extraction solution for culturing BHK cells.The results suggest that the protein is capable of promoting BHK cell growth.Lastly,in order to improve the hydroxylation level of type Ⅲ collagen α1 chain,the two kinds of strongly inducible promoters p FLD1 and p AOX1 were used to control the P4 H and COL3A1 genes respectively.0.5% methanol and 0.25% methylamine can be used to obtain a type Ⅲ collagen α1 chain with higher degree of hydroxylation,which is superior to the double AOX1 promoter controlled co-expression system.The LC-MS/MS results showed that the proportion of Hyp covered Pro(in the Y position of Gly-X-Y repeats)in COL3A1 protein was 71.16%.