Purification Of The Recombinant Human-like Collagen (Ⅱ) In E.coli

Protein purification is one of the biochemical purification processes. The optimal process parameters to purify recombinant human-like collagen II were investigated in this paper. High Pressure Homogenizer lysed the harvested recombinant E. coli cell;the supernatant was purified by sedimentation, after ultrafiltration then through CM52 chromatography. The purity of human-like collagen II reached 96.4%, recovery was 71.6%. By SDS-PAGE analysis, the molecular weight was 90kD.In cells lysate phase, harvested recombinant E. coli cell was lysed by a high-pressure homogenizes. A systematic study was carried out on the influence of physical and chemical factors including homogenizes cycle, operation pressure and characteristics of the suspension solution during homogenization processes. The experimental data from the following conditions: pressure: 70MPa, cell mass: water volume=l:10 suspended in deionized water with 2 cycles.In sedimentation phase, through the single factor experiment, the operation parameter was confirmed. After that according to the L₁₆(4⁵) orthogonal design ,through the ANOVA analysis ,the sedimentation process was run at pH 2.8, 5% NaCl concentration, 18°C and 5g/L protein concentration. The purity of human-like collagen II could reach 37.68%, and the recovery was 88.73% after treatment.Then, the supernatant was operated by ultrafiltration with cut-off molecular weight 30kD. The ultrafiltration operation was carried out at 0.2Mpa with pH value 7.0 ,3% protein concentration, 25 °C and 2L/min protein volume flux. The mathematics model ofultrafiltration is:Thepurity of human-like collagen II could reach 47.62%, and the recovery was 91.04% in thisprocess.In chromatography phase, the purification ability of the negative ion exchange chromatography (DE52) was compared with the positive ion exchange chromatography (CM52). The crude product of human-like collagen II was absorbed by CM52 chromatography. The purity of human-like collagen II could reach 96.4%, recovery was 71.6%. By SDS-PAGE analysis, the molecular weight was 97kD.