Research On Purification Of Recombinant Human-like Collagen I

The optimum process parameters to purify recombinant human-like collagen â…  (HLC â…  ) were investigated in this paper. Harvested recombinant E.coli cell was lyses by High Pressure Homogenize, supernatant was treated with ammunition sulfate, after centrifugation, the crude product of HLC â…  was obtained. Consequently, two purification methods, which Ion Exchang Batch Chromatography & Gel Filtration Chromatography, and Ion Exchange Pole Chromatography were used in the down-steam processing. A better separation and purification process was obtained.1 Prepurification. The harvest cell was lyses by the High Pressure Homogenize for 20 minutes. It was shown that the lyses cell reached 78.7%, and the concentration of human-like collagen was 2.56g/L. The supernatant was salted out by 20⁵0% ammonium sulfate at the conditions of pH 2.9, 18℃ and 3g/L protein concentration according to the L₉(3³) orthogonal design. The purity of human-like collagen could reach 36.8%, and the recovery was 92.7% in this process.2 Purification. The crude product of HLC â…  was absorbed by DE52 batch chromatography, then the collected supernatant flowed through the Sephadex G-100 column for Gel Filtration Chromatography. The purity of human-like collagen could be 97.6%, and the total recovery was 63.0%. This is so-called the combination of Ion Exchange batch Chromatography & Gel Filtration Chromatography. The second method was Ion Exchange Pole Chromatography. The crude product of HLC â…  flowed through the DE-52 ion exchange pole,then the Tris-HCl buffer solution eluate the HLC â…  .The effluent volume was 12 multiple bed volume. The purity of human-likecollagen could reach 95.1%, and the total recovery was 77.0%. The purified product was run the SDS-PAGE electrophoretogram, it is observed that only one single band was appeared in gel and the molecular weight was around 97kD, which was consistent with gene sequencing. N terminus amino acids sequence was NH2-H-D-P-V-V-L-O-R -R-D-W-E-N-P-G, which consistent with that deduced from DNA sequence.The kinetics of Ion Exchange batch Chromatography and Ion Exchange Pole Chromatography was researched also in this paper. The kinetics of Ion Exchange6.916e081' +13.664 9.1eO81t-2.24batch Chromatography was c = oglt—^-^—, and the kinetics of breakthroughcurve was— = — 1 + er/l—1011. It could be seen that the kinetics were matchedwell with actual observation data by comparing the graphs of Ion Exchange batch Chromatography and breakthrough curve with those of the actual. The kinetics was testified by the analysis of zero average deviation, and the A values of the two models were lower than the Fm> n_i when the confidence probability was 99%, which proved that the kinetics models were effective.