Whiteley E M, Hsu T A, Betenbaugh M J
Department of Chemical Engineering, The Johns Hopkins University, Baltimore, MD 21218-2694, USA.
J Biol Chem. 1997 Sep 5;272(36):22556-63. doi: 10.1074/jbc.272.36.22556.
Coexpression of the enzyme, protein disulfide isomerase (PDI), has been shown to increase soluble and secreted IgG levels from baculovirus-infected insect cells (Hsu, T.-A., Watson, S., Eiden, J. J., and Betenbaugh, M. J. (1996) Protein Expression Purif. 7, 281-288). PDI is known to include catalytic active sites in two separate thioredoxin-like domains, one near the amino terminus and another near the carboxyl terminus. To examine the role of these catalytic active sites in enhancing immunoglobulin solubility, baculovirus constructs were utilized with cysteine to serine mutations at the first cysteine of one or both of the CGHC active site sequences. Trichoplusia ni insect cells were coinfected with a baculovirus vector coding for IgG in concert with either the wild-type human PDI virus, amino-terminal mutant (PDI-N), carboxyl-terminal mutant (PDI-C), or mutant with both active sites altered (PDI-NC). Western blot analysis revealed that both immunoglobulins and PDI protein were expressed in the coinfected cells. To evaluate the effect of the PDI variants on immunoglobulin solubility and secretion, the infected cells were labeled with 35S-amino-acids for different periods, and the soluble immunoglobulins were immunoprecipitated from clarified cell lysates and culture medium using anti-IgG antibodies. Only coinfections with the wild-type PDI and PDI-N mutant led to increased immunoglobulin solubility and higher IgG secretion. In contrast, infection with the PDI-C and PDI-NC variants actually lowered immunoglobulin solubility levels below those achieved with a negative control virus. Immunoprecipitation with anti-PDI antibody revealed that heterologous PDI-C and PDI-NC were insoluble, even though PDI-N and wild-type PDI protein were detected in soluble form. The capacity for PDI-N to increase immunoglobulin solubility whereas the PDI-C mutant lowered solubility indicates that the amino- and carboxyl-terminal thioredoxin domains of PDI are functionally distinct in vivo following mutations to the active site. Furthermore, mutations at the active site of the carboxyl-terminal thioredoxin domain result in PDI variants that can act as anti-chaperones of immunoglobulin solubility in vivo as has been observed in vitro for lysozyme aggregation by wild-type PDI and PDI mutants (Puig, A., and Gilbert, H. F. (1994) J. Biol. Chem. 269, 7764-7771).
已证明与酶蛋白二硫键异构酶（PDI）共表达可提高杆状病毒感染昆虫细胞中可溶性和分泌性IgG的水平（Hsu，T.-A.，Watson，S.，Eiden，J.J.和Betenbaugh，M.J.（1996年）《蛋白质表达与纯化》7，281 - 288）。已知PDI在两个独立的硫氧还蛋白样结构域中包含催化活性位点，一个靠近氨基末端，另一个靠近羧基末端。为了研究这些催化活性位点在增强免疫球蛋白溶解性中的作用，构建了杆状病毒，其CGHC活性位点序列中的一个或两个的第一个半胱氨酸发生了半胱氨酸到丝氨酸的突变。将粉纹夜蛾昆虫细胞与编码IgG的杆状病毒载体共感染，同时感染野生型人PDI病毒、氨基末端突变体（PDI - N）、羧基末端突变体（PDI - C）或两个活性位点都改变的突变体（PDI - NC）。蛋白质印迹分析表明，免疫球蛋白和PDI蛋白在共感染的细胞中均有表达。为了评估PDI变体对免疫球蛋白溶解性和分泌的影响，用35S - 氨基酸对感染的细胞进行不同时间段的标记，然后使用抗IgG抗体从澄清的细胞裂解物和培养基中免疫沉淀可溶性免疫球蛋白。只有与野生型PDI和PDI - N突变体共感染才导致免疫球蛋白溶解性增加和更高的IgG分泌。相反，用PDI - C和PDI - NC变体感染实际上使免疫球蛋白溶解性水平低于阴性对照病毒所达到的水平。用抗PDI抗体进行免疫沉淀显示，异源的PDI - C和PDI - NC是不溶性的，尽管以可溶形式检测到了PDI - N和野生型PDI蛋白。PDI - N增加免疫球蛋白溶解性而PDI - C突变体降低溶解性的能力表明，PDI的氨基和羧基末端硫氧还蛋白结构域在活性位点发生突变后在体内功能不同。此外，羧基末端硫氧还蛋白结构域活性位点的突变导致PDI变体在体内可作为免疫球蛋白溶解性的抗伴侣蛋白，正如在体外观察到野生型PDI和PDI突变体对溶菌酶聚集的作用一样（Puig，A.和Gilbert，H.F.（1994年）《生物化学杂志》269，7764 - 7771）。
