Study On High Cell Density Cultivation In Recombinant Escherichia Coli For Production Of Collagen Ⅵ

Collagen is a sort of biological macromolecules synthesis by animal desmocyte, with supporting organs and protect the organism function. CollagenVI which belongs to one member of collagen family and contains three subunits of α1, α2and α3, is a kind of extracellular function protein generally with the dominant characters of high molecular weight and a high content of cysteine. It can maintain integrity of organization because of perfect bioactiveproperties and well mechanical character promoting the interstitial cell and cartilage cell regenerate. Therefore, it can be used to make medical materials, artificial blood vessel and suture for surgery.In present paper, the HCDC fermentation culture medium and condition for recombinant E.coliBL21(pET22a-COL6A2) have been optimized from its characteris and metabolism system which is used in rocker experiment and slightly test. Through the Plackett-Burman experimental design and response surface method, the component of medium was optimized and established the relationship between the three primary components(glucose, mixed nitrogen sources and dipotassium hydrogen phosphate)and the expression rate of collagen. The percentage of collagen is defined through analysis of SDS-PAGE. The optimum medium was composed as follows(in g/L):glucose10, mixed nitrogen15.04, K2HPO411.91,(NH4)2SO44.5, NH4Cl4.5, NaH2PO44.5, MgSO41, NaCl4. The optimized operational conditions were initial pH7.0, temperature37℃, inoculating age5h and inoculums quantity with2.0%.When the absorptance A600of broth given by the recombinant stain up to3.8, IPTG was added so as to the final concentration to0.6mmol/L, and induction was conducted for5h at30℃, the dry cell weight and percentage of collagen reached the maximum to1.904g/L and30.5%, the cell density(at A600)did12.8, which is almost2.08times and2.01times of those under the initial fermentation conditions respectively. Influence of IPTG is very small through comparative trial of induction and no induction.Fed-batch process for E.coliBL21(pET22b-Co16A2) was carried out in a5L automatic fermentation cylinder. DO-star, pH-stat feeding, exponential feeding and the exponent-pH combination were comparatively investigated. The optimized operational conditions were initial DO30%, induction was conducted for6h, pH6.5and DO40%. The results indicated that the combined strategy was adoptable. The higher cell density and percentage of collagen were obtained with56(at A6oo)and35.9%.