Speed M A, Wang D I, King J
Biotechnology Process Engineering Center, Massachusetts Institute of Technology, Cambridge 02139, USA.
Protein Sci. 1995 May;4(5):900-8. doi: 10.1002/pro.5560040509.
The failure of newly synthesized polypeptide chains to reach the native conformation due to their accumulation as inclusion bodies is a serious problem in biotechnology. The critical intermediate at the junction between the productive folding and the inclusion body pathway has been previously identified for the P22 tailspike endorhamnosidase. We have been able to trap subsequent intermediates in the in vitro pathway to the aggregated inclusion body state. Nondenaturing gel electrophoresis identified a sequential series of multimeric intermediates in the aggregation pathway. These represent discrete species formed from noncovalent association of partially folded intermediates rather than aggregation of native-like trimeric species. Monomer, dimer, trimer, tetramer, pentamer, and hexamer states of the partially folded species were populated in the initial stages of the aggregation reaction. This methodology of isolating early multimers along the aggregation pathway was applicable to other proteins, such as the P22 coat protein and carbonic anhydrase II.
在生物技术领域，新合成的多肽链因积聚形成包涵体而无法达到天然构象是一个严重问题。先前已确定P22尾刺内鼠李糖苷酶在有效折叠途径和包涵体途径交界处的关键中间体。我们已能够在体外途径中捕获后续中间体，直至其形成聚集的包涵体状态。非变性凝胶电泳确定了聚集途径中一系列连续的多聚体中间体。这些代表了由部分折叠中间体的非共价缔合形成的离散物种，而非类似天然三聚体物种的聚集。在聚集反应的初始阶段，部分折叠物种的单体、二聚体、三聚体、四聚体、五聚体和六聚体状态均有出现。这种沿着聚集途径分离早期多聚体的方法适用于其他蛋白质，如P22外壳蛋白和碳酸酐酶II。
