批量离子交换层析和凝胶过滤层析在类人胶原蛋白I纯化中的应用

研究了利用批量离子交换层析与凝胶过滤层析相结合的方法纯化类人胶原蛋白I的最佳条件。分别考察了不同离子交换树脂、缓冲溶液pH、离子强度、进料蛋白浓度对批量离子交换的影响,以及不同凝胶过滤介质对凝胶过滤层析的影响。结果表明,采用阴离子交换树脂DEAE52吸附杂蛋白,缓冲液pH为7.0,NaCl浓度为0.2mol/L,进料蛋白浓度为40mg/mL,并采用凝胶过滤介质SephadexG-100进一步纯化后,类人胶原蛋白I的纯度可达到98.2%,总纯化倍数为3.1,总回收率为80.6%。SDS-PAGE分子量分析和N末端氨基酸序列分析均表明纯化后的类人胶原蛋白I与基因设计一致。     

Microcalorimetric Evaluation of the Effect of Kanamycin： An Analysis Based on the Median-Effect Principle

A study of the effect of drug, kanamycin, on the growth metabolism of recombinant Escherichia coli B 1 was carded out by microcalorimeter monitoring of the metabolic activity of treated cells. Power-time curves of growing recombinant Escherichia coli cell suspensions, treated with different kanamycin doses, were recorded. The extent of the effect was evaluated by changes in the slopes of the microcalorimetric curves and the kinetics of the drug action was interpreted from the time at which these changes reached their maximum values and maintained their maximum values. Experimental dose-effect relationships conform to the median-effect principle of the mass-action law： fa/（1-fa）=（D/D50）^m. A plot of y=lg[（fa）^1-1]^-1 versus x=lg D gives the slope m, D50 and R∞. The experimental results revealed that high concentration of kanamycin had an inhibitory effect on the growth of recombinant Escherichia coli B 1 in the lg phase, and had a promoting effect in the stationary period. Moreover, it was demonstrated that microcalorimetry was a reliable method for the detection of modulatory effects in biology.     

离子交换法纯化重组类人胶原蛋白Ⅱ的吸附动力学研究

为了确定重组类人胶原蛋白Ⅱ(Recombinant human-like collagen,RHLC Ⅱ)在CM52树脂上的动力学行为,考察温度、初始浓度等因素的影响,分别采用简单线性推动力模型(液膜扩散控制时)和均相扩散模型(颗粒扩散控制时)研究RHLC Ⅱ在不同工艺条件下的反应速率,确定离子交换过程的速率控制步骤,并计算模型参数.结果表明,(1)速率控制机理受转速和料液初始浓度的影响,在转速和料液初始浓度较低时,为液膜扩散控制(Film Diffusion Control,FDC);反之,则为颗粒扩散控制(Particle Diffusion Control,PDC);(2)以简单线性推动力模型拟合液膜扩散控制时的动力学数据,其线性关系良好,模型准确度较高;对于颗粒扩散控制的吸附,则以均相扩散模型拟合,求出有效扩散系数,模型较好地描述了CM52对RHLC Ⅱ的PDC吸附.(3)对于FDC,温度越高,溶液浓度越大,搅拌转速越高,外扩散速度常数越大,吸附越快;对于PDC,温度升高,有效扩散系数增大,吸附加快,料液浓度对有效扩散系数的影响不显著.