Expression Of Recombinant Collagen ?-?1 In Human Embryonic Kidney 293 Cells

With the rapid development of the cosmetic industry and tissue engineering,recombinant human collagen based on the genetic engineering has long-term perspective opportunities and huge potential of industry products because of its excellent biological properties.This paper have established transient expression system and stable expression system about the recombinant human collagen ?-?1.The growth characteristics of its stable cell lines were fully understand by the analysis of amino acid metabolism.We also found the copper ion concentration effect of lactic acid metabolism in HEK293F cell.We have established recombinant human collagen ?-?1 protein purification process and studied the physical and chemical properties of recombinant ?-?1 protein.The results of the study are as follows:Firstly,the expression plasmid pCDNA3.1(+)-?-?1 was constructed and the transient expression of the recombinant ?-?1 in HEK293F cell line was studied.By expressing green fluorescent protein to optimize the transfection efficiency.The expression plasmid was transferred into HEK293F cells to achieve recombinant ?-?1.Then,we have successfully transferred the plasmid into HEK293F cell and completed recombinant ?-?1 stable cell line selection.Determine the minimum lethal concentration of G418 to cell line HEK 293F at 0.25 mg/mL.The highest production of the 7 minipools is 1.84 mg/L.Furthermore,7L scale up bioreactor culture of C09 pool cell lines result in excess lactic acid accumulation and cause death.Then,8 single clone cell line have been obtained by LDC method which yield up to 6.28 mg/L.Meanwhile,the copper ions concentration within the range of 0.05 to 1.6?mol/L can effectively promote the transformation from lactate production to lactate consumption,and significantly prolong the cell growth cycle.Eventually,The recombinant collagen ?-?1 can be completely eluted with 500mM imidazole buffer with good purity.The distribution range of isoelectric points was 4.0-5.6,the main peak was 4.8,and the relative content was 41.2%.The molecular weight of the monomer was 201.7 kDa and the molecular weight of the polymer was 696.0 kDa.