Hsu T A, Watson S, Eiden J J, Betenbaugh M J
Department of Chemical Engineering, Johns Hopkins University, Baltimore, Maryland 21218-2694, USA.
Protein Expr Purif. 1996 May;7(3):281-8. doi: 10.1006/prep.1996.0040.
A substantial fraction of immunoglobulin heavy and light chain polypeptides were insoluble when expressed in the baculovirus-insect cell expression system. In the presence of coexpressed heterologous protein disulfide isomerase (PDI), however, the solubility of the immunoglobulins was enhanced and IgG was secreted at higher levels from baculovirus-infected Trichoplusia ni insect cells. Pulse-chase experiments indicated that some immunoglobulin polypeptides were initially insoluble in the presence of PDI but subsequently were rescued in a soluble form competent for IgG assembly and secretion. Recovery of the insoluble immunoglobulins was not observed in the absence of coexpressed PDI. Even after treatment of insect cells with tunicamycin to inhibit N-glycosylation of immunoglobulin heavy chains, coexpressed PDI was able to salvage insoluble immunoglobulins and secrete these modified glycoforms. The capacity for PDI to rescue immunoglobulins was also demonstrated in vitro where immunoglobulin heavy chains and light chain dimers were salvaged from aggregates of denatured IgG. PDI-mediated rescue of proteins, perhaps assisted by chaperones and other foldases, may be important in vivo where insolubility is a common occurrence for newly synthesized polypeptides.
当在杆状病毒-昆虫细胞表达系统中表达时，相当一部分免疫球蛋白重链和轻链多肽是不溶的。然而，在共表达异源蛋白二硫键异构酶（PDI）的情况下，免疫球蛋白的溶解度得到提高，并且杆状病毒感染的粉纹夜蛾昆虫细胞分泌的IgG水平更高。脉冲追踪实验表明，一些免疫球蛋白多肽最初在PDI存在的情况下是不溶的，但随后以能够进行IgG组装和分泌的可溶形式被挽救。在没有共表达PDI的情况下，未观察到不溶性免疫球蛋白的回收。即使在用衣霉素处理昆虫细胞以抑制免疫球蛋白重链的N-糖基化后，共表达的PDI仍能够挽救不溶性免疫球蛋白并分泌这些修饰的糖型。在体外也证明了PDI挽救免疫球蛋白的能力，其中免疫球蛋白重链和轻链二聚体从变性IgG的聚集体中被挽救。PDI介导的蛋白质挽救，可能在伴侣蛋白和其他折叠酶的协助下，在体内可能很重要，因为新合成的多肽不溶性是常见现象。
