看一看：蛋白质纯化及复性

重组蛋白在大肠杆菌（E. coli）高效表达时，常常以不溶的、无活性的蛋白聚集体，即包涵体(inclusion body)的情势存在于细胞内。必须从细胞内分离出包涵体，采取高浓度变性剂（如7.0mol/L盐酸胍、8.0mol/L脲）溶解包涵体，然后除去变性剂或降落变性剂的浓度，使包涵体蛋白得以复性，最后再用色谱法使目标蛋白质得到纯化。其中包涵体蛋白的复性和纯化是全部进程中的核心。 目前重组蛋白生产中普遍存在的问题是：（1）复性效率低。传统的复性方法稀释法和透析法。稀释复性法对样品几10倍，乃至上百倍的稀释会使样品的体积急剧增大，给后续的分离纯化带来很大的困难，而且复性进程中需要较大的复性容器。透析法耗时较长，而且要多次更换透析溶液。这两种方法的共同缺点是蛋白质在复性进程中会产生聚集而产生大量沉淀，复性效率低养猪强拆合法吗，通常蛋白质的活性回收率只有5~20%，而且复性后的蛋白质溶液中含有大量的杂蛋白，需要进行进1步的分离纯化。（2）工艺线路烦琐，生产周期长。在传统的重组蛋白质分离纯化工艺中，大多采取经典的软凝胶分离介质，由于这类介质的颗粒较大，分离效率较差，因此常常需要采取多种不同模式的色谱操作联用对目标蛋白质进行纯化，才能得到纯度符合1定标准的目标蛋白质。另外，这类色谱介质的耐压性很差，只能在流速较低的情况下进行操作，分离纯化时间较长。分离纯化步骤多和分离时间长使得蛋白质的质量回收率和活性回收率很低。而且在传统的重组蛋白质生产工艺中，蛋白质的复性和纯化是生产进程中两个独立的单元操作，也在很大程度上制约着生产效率。（3）生产本钱高，设备投资大。由于复性和分离纯化分别单独进行，而且分离纯化步骤多，每步都需要有与之配套的设备，致使设备投资大，生产本钱高。随着生产范围的增加，这类弊端会愈来愈严重。 1991年耿信笃教授首先将高效疏水相互作用色谱(HPHIC)用于变性蛋白的复性，很好的解决了上述问题，现已成功用于重组人干扰素-g(rhIFN-g)、重组人干扰素-a(rhIFN-a)、人粒细胞集落刺激因子(rhG-CSF)、重组人胰岛素原(proinsulin)、重组牛朊病毒(prion)等重组蛋白和溶菌酶和核搪核酸酶等标准模型蛋白的复性与同时纯化中违建抢建为什么不被强拆。目前，排阻色谱法、离子交换色谱法和亲合色谱法也已用于蛋白质的复性和同时纯化中。与传统的稀释法及透析法比较，用色谱法进行蛋白复性的优点是：①在进样后可很快除去变性剂；②由于色谱固定相对变性蛋白质的吸附，可明显地减少、乃至完全消除复性进程中蛋白质聚集体和沉淀的产生，从而提高蛋白质复性的质量和活性回收率；③在蛋白质复性的同时可使目标蛋白质与杂蛋白分离以到达纯化的目的，使复性和纯化同时进行；④便于回收变性剂，以降落废水处理本钱。简言之，色谱法复性可以提高蛋白质的活性和质量回收率，将蛋白复性和纯化集成在1步操作完成，缩短了操作步骤和生产时间，减少了设备投资，使生产本钱大大降落，已引发了全球范围内许多生化研究者和重组蛋白药物生产厂家的关注。由于高效液相色谱（HPLC）分离效率高，常常在1步操作中便可得到纯度符合要求的蛋白质，而且分离速度快，在利用方面具有更大的优势。 精纯化2精纯化1 除盐 成品 复性（稀释或透析） 提取 发酵 粗纯化 图1用制备型USRPP缩短重组蛋白下游工艺示意图 以下介绍1些相干文献的摘要，全文可来函索要，陕西西大科林基因药业有限公司 Purification of recombinant bovine normal prion protein PrP(104–242) by HPHIC Chaozhan Wang, Xindu Geng, Dawei Wang, Bo Tian, Journal of Chromatography B, 806 (2004) 185–190 Purification of the prion protein (PrP) is a major concern for biological or biophysical analysis as are the structural specificities of this protein in relation to infectivity. A simple and efficient method for purification of recombinant bovine normal prion protein containing residues 104–242, PrP(104–242) expressed in Escherichia coli by high performance hydrophobic interaction chromatography (HPHIC) was presented in this work. The solution containing denatured and reduced protein in 8.0 mol/L urea extracted from the inclusion body was directly injected into the HPHIC column, aggregates were prevented by the interaction between the denatured PrP(104–242) molecules and the stationary phase during the chromatographic process, the soluble form of PrP(104–242) in aqueous solution was obtained after desorbed from the column. Several factors, including pH value, types of stationary phase and salt, and gradient mode, influencing the purification results were investigated. Optimal conditions were obtained for the purification of PrP(104–242) by HPHIC. This procedure yield PrP(104–242) of a purity of 96% with a recovery of 87%, respectively, for a single step purification of 40 min. Refolding of Denatured/Reduced Lysozyme Using Weak-Cation Exchange Chromatography Yan WANG, Bo Lin GONG, Xin Du GENG， Chinese Chemical Letters, 14 (2003) 828 – 831 Oxidative refolding of the denatured/reduced lysozyme was investigated by usingweak-cation exchange chromatography (WCX). The stationary phase of WCX binds to the reduced lysozyme and prevented it from forming intermolecular aggregates. At the same time urea and ammonium sulfate were added to the mobile phase to increase the elution strength for lysozyme. Ammonium sulfate can more stabilize the native protein than a common eluting agent, sodium chloride. Refolding of lysozyme by using this WCX is successfully. It was simply carried out to obtain a completely and correctly refolding of the denatured lysozyme at high concentration of 20.0 mg/mL.High-performance hydrophobic interaction chromatography as a tool for protein refoldingXindu Geng and Xiaoqing Chang, J. Chromatogr., 599 (1992) 185⑴94 A method for the refolding of previously unfolded proteins with a concentrated solution of denaturing agent is presented, involving the use of high-performance hydrophobic interaction chromatography (HPHIC) to separate the denaturing agent completely from the unfolded protein and to provide a suitable environment for its refolding. The retention, peak shape and peak height in HPHIC and size-exclusion chromatography, UV spectra, circular dichroic spectra and bioactivity were used to test the possibility and the completeness of the protein refolding. The proposed method permits the extracted solution from Escherichia coli cells to be injected directly into the HPHIC column and, at the same time, the refolding and purification of the proteins to be effected. The renaturation and purification of recombinant human interferon­‑g form E. coli cells is one example of the application of the method in biotechnology. 疏水色谱法的进展及其在生化研究中的利用 刘彤, 耿信笃, 色谱, 1998, 16 (1) : 30⑶4 侧重评述了疏水色谱法的理论研究及疏水固定相中无机填料、有机填料、非多孔填料和大孔膜的新发展，并对疏水色谱法在生物大分子的分离、纯化和生化研究中的利用,包括在蛋白质复性、折叠和分子构象变化等方面的利用作了介绍，全文包括62篇文献和1张表格。 多功能蛋白增活器的研制及性能研究资讯分类行业动态帮助文档展会专题报道5金人物商家文章