Björck L, Kastern W, Lindahl G, Widebäck K
Hagedorn Research Laboratory, Gentofte, Denmark.
Mol Immunol. 1987 Oct;24(10):1113-22. doi: 10.1016/0161-5890(87)90080-0.
Protein G is expressed at the cell surface of certain group C and group G streptococcal strains. The protein shows a unique and specific affinity for the Fc region of mammalian polyclonal and monoclonal immunoglobulin G (IgG). We have cloned the streptococcal gene coding for protein G into E. coli, using phage lambda as the vector. The protein G produced by E. coli infected with this phage was detected and analysed in Western blot experiments using radiolabelled IgG Fc fragments as a probe. Three major IgG Fc-binding bands were obtained corresponding to apparent mol. wts of 47,000, 57,000 and 65,000, respectively. Analysis of the expression in E. coli indicates that this heterogeneity is caused by a post-translational degradation of the molecule before lysis of the lambda infected E. coli cells occurred. The protein G produced in E. coli was purified by affinity chromatography on IgG-Sepharose followed by gel-filtration on Sephadex G-200. This highly purified E. coli-produced protein G was compared to protein G solubilized by papain from streptococci, in direct binding experiments and in a competitive binding assay. The two protein G variants were found to interact with polyclonal IgG from different species in a similar way. Streptococcal strains expressing protein G also show affinity for human albumin, and at the molecular level protein G was found to be responsible also for the binding of albumin. Thus, both E. coli-produced protein G and the proteolytic fragment of protein G obtained from streptococci, bound albumin. On the protein G molecule, two different and separate sites were found to bind IgG and albumin. Finally, when whole streptococci were incubated with human plasma, the interactions with protein G caused a coating of the bacteria with albumin and IgG, whereas other plasma proteins showed no affinity for protein G.
G蛋白在某些C组和G组链球菌菌株的细胞表面表达。该蛋白对哺乳动物多克隆和单克隆免疫球蛋白G（IgG）的Fc区域表现出独特而特异的亲和力。我们使用噬菌体λ作为载体，将编码G蛋白的链球菌基因克隆到大肠杆菌中。用放射性标记的IgG Fc片段作为探针，在蛋白质印迹实验中检测和分析了感染该噬菌体的大肠杆菌产生的G蛋白。获得了三条主要的IgG Fc结合带，其表观分子量分别为47,000、57,000和65,000。对大肠杆菌中表达的分析表明，这种异质性是由λ感染的大肠杆菌细胞裂解前分子的翻译后降解引起的。通过在IgG-琼脂糖上进行亲和层析，然后在Sephadex G-200上进行凝胶过滤，纯化了大肠杆菌中产生的G蛋白。在直接结合实验和竞争性结合试验中，将这种高度纯化的大肠杆菌产生的G蛋白与木瓜蛋白酶从链球菌中溶解的G蛋白进行了比较。发现这两种G蛋白变体以相似的方式与来自不同物种的多克隆IgG相互作用。表达G蛋白的链球菌菌株也对人白蛋白表现出亲和力，并且在分子水平上发现G蛋白也负责白蛋白的结合。因此，大肠杆菌产生的G蛋白和从链球菌获得的G蛋白的蛋白水解片段都结合白蛋白。在G蛋白分子上，发现两个不同且分开的位点结合IgG和白蛋白。最后，当将完整的链球菌与人血浆一起孵育时，与G蛋白的相互作用导致细菌被白蛋白和IgG覆盖，而其他血浆蛋白对G蛋白没有亲和力。
