Characteristics of antibodies recognizing denatured proteins and application to separation

为了用免疫亲和层析法纯化弱变性蛋白，研究了抗体对部分靶蛋白多肽的吸附特性。抗肽抗体被认为对高流动性的蛋白质有较高的亲和力，适合于变性蛋白质的纯化。本工作得到的结果如下：1。通过免疫兔，制备了针对肌红蛋白、胰岛素和大米α -淀粉酶c端肽的抗肽抗体，测定了这些抗体对天然蛋白和变性蛋白以及肽的吸附特性。天然肌红蛋白被针对肌红蛋白c端肽的抗体轻微吸附，而变性肌红蛋白在3M尿素中被高亲和力吸附。另一方面，天然胰岛素和α -淀粉酶及其变性状态被针对这些蛋白的c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3M脲）中变性蛋白的免疫亲和纯化。以溶菌酶为原料，研究了连续折纸装置的折纸条件和折纸性能。完全变性溶菌酶在3M尿素下松散折叠，可获得较高的再折叠率。用该仪器进行连续折纸。

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.