Research On Purification Of Recombinant Human-like Collagen HCB1

The optimum process parameters to purify recombinant human-like collagen HCB1 were investigated in this paper. HCB1 was separated and purified from recombinant genetically engineered E.coli produced by high-density fermentation.(1) Prepurification. The harvest cells were breaded by the ultrasonication for 20minutes. It was shown that the breaking ratio reached 87%, and the concentration of human-like collagen was 2.78g/L. The supernatant was salted out by 15~65% ammonium sulfate at the conditions of pH 2.9, 10 and 2g/L protein concentration, which was decided by the analysis of variance of Lg(34) orthogonal design. The purity of human-like collagen could reach 36.8%, and the recovery percent was 90.2% in this process.(2) Purification. Tow methods to be used were contrasted. The first was the combination of Ion Exchange Chromatography and Gel Filtration Chromatography. The heteroprotein was absorbed by DE52 batch chromatography. The supernatant was collected, and flowed through the Sephadex G-100 column for Gel Filtration Chromatography. And 20mmol/LTris-HCl (pH 7.5 20mmol/L sodium chloride) buffer was used as eluent. The purity of human-like collagen could be 98%, and the total recovery percent was 60.7%. The second method was the Immobilized Metal Ion Affinity Chromatography with Zinc chloride as the metal ion chelating agent, pH 7.5phosphate as the equilibrium buffer, and 100mmol/L ammonium chloride as the eluent. The purity of human-like collagen could reach 98%, and the total recovery percent was 77.0% by this process. It was shown only one single band in SDS-PAGE electrophoretogram, and the molecular weight was 90kD, which was consistent with gene sequencing. N terminus amino acids sequence was NH2-H-D-P-V-V-L-Q-R-R-D-W-E-N-P-G, which consistent with that deduced from DNA sequence. The kinetics of Immobilized Metal Ion Affinity Chromatography was researchedalso in this paper. The model of breakthrough curve was Inthe model of elution curve the mean retaining time was 26.36 minutes (tR = 26.36min), and spread degree was 203806( 2 = 203806). It could be seen that the models were matched well with actual observation data by comparing the graphs of breakthrough curve and elution curve models with those of the actual. The models were testified by the analysis of zero mean deviation, and the values of the two models were lower than the Fm, n-1 when the probability was 99%, which proved that the models were effective.