Haase-Pettingell C A, King J
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
J Biol Chem. 1988 Apr 5;263(10):4977-83.
The in vivo accumulation of polypeptide chains in the form of aggregated non-native states is a problem in many applications of biotechnology. In the maturation pathway of the thermostable P22 tailspike endorhamnosidase, the folding and chain association intermediates can be distinguished from the native tailspikes in crude extracts of phage-infected Salmonella cells. Temperature-sensitive folding mutations, at many sites in the chain, destabilize these conformational intermediates preventing the formation of native tailspikes at restrictive temperatures (Goldenberg, D. P., Smith, D. H., and King, J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7060-7064). We report here that both wild type and mutant tailspike polypeptide chains which fail to reach the native state accumulate in an aggregated state. These off-pathway aggregates form from a thermolabile intermediate in the productive folding pathway. These aggregation reactions are suppressed by lowering the temperature of maturation. Similar off-pathway steps from folding intermediates may account for the non-native aggregates often found in the expression of cloned genes in heterologous hosts.
以聚集的非天然状态形式存在的多肽链在体内的积累是生物技术许多应用中的一个问题。在热稳定的P22尾刺内鼠李糖苷酶的成熟途径中，折叠和链缔合中间体可以在噬菌体感染的沙门氏菌细胞的粗提物中与天然尾刺区分开来。在链中的许多位点上的温度敏感折叠突变使这些构象中间体不稳定，从而在限制温度下阻止天然尾刺的形成（戈尔登伯格，D.P.，史密斯，D.H.，和金，J.（1983年）《美国国家科学院院刊》80，7060 - 7064）。我们在此报告，未能达到天然状态的野生型和突变型尾刺多肽链都以聚集状态积累。这些偏离途径的聚集体由生产性折叠途径中的热不稳定中间体形成。通过降低成熟温度可以抑制这些聚集反应。来自折叠中间体的类似偏离途径步骤可能解释了在异源宿主中克隆基因表达时经常发现的非天然聚集体。
