Characteristics of antibodies recognizing denatured proteins and application to separation

为了利用免疫亲和层析法纯化弱变性蛋白质，研究了针对目标蛋白质组成部分多肽的抗体的吸附特性。抗肽抗体被认为对具有高迁移率的蛋白质具有更高的亲和力，并且适合于纯化变性蛋白质。本工作取得的主要结果如下：1.通过将肌红蛋白、胰岛素和大米α-淀粉酶的C-末端肽免疫家兔制备抗肽抗体，并测量这些抗体对天然和变性蛋白以及肽的吸附特性。虽然天然肌红蛋白被针对肌红蛋白的C-末端肽的抗体轻微吸附，但在3 M尿素中的变性肌红蛋白以高亲和力吸附。另一方面，天然胰岛素和α-淀粉酶，以及rheir变性状态，被针对这些蛋白质的C-末端肽的抗肽抗体吸附。这些结果表明，抗肽抗体可用于弱变性剂如3 M尿素中变性蛋白的免疫亲和纯化.以溶菌酶为复性剂，研究了连续复性装置的性能和复性条件。完全变性的溶菌酶在3 M尿素下的松散折叠得到了高的复性产率。通过该装置进行连续重折叠。

To purify weakly denatured proteins by immunoaffinity chromatography, the adsorption characteristics of antibodies against peptides consisting a part of target protein were studied. Anti-peptide antibodies are considered to have higher affinity to proteins with high mobility and to be suitable for purification of denatured proteins. The results obtained in this work are as follows ;1. Anti-peptide antibodies against the C-terminal peptides of myoglobin, insulin and rice alpha-amylase were prepared by immunizing the C-terminal peptides to rabbits, and adsorption characteristics of these antibodies against native and denatured proteins, as well as the peptides, were measured. While native myoglobin was slightly adsorbed by the antibody against the C-terminal peptide of myoglobin, denatured myoglobin in 3M urea was adsorbed with high affinity. On the other hand, native insulin and alpha-amylase, as well as rheir denatured states, were adsorbed by the anti-peptide antibodies against the C-terminal peptides of these proteins. These results show that anti-peptide antibodies can be used for immunoaffinity purification of denatured proteins in weak denaturants such as 3M urea.2. Refolding conditions and the performance of a continuous refolding apparatus were studied by use of lysozyme. Loose folding of completely denatured lysozyme under 3M urea gave high refolding yields. Continuous refolding was performed by the apparatus.