Akerström B, Björck L
Department of Medical and Physiological Chemistry, University of Lund, Sweden.
J Biol Chem. 1989 Nov 25;264(33):19740-6.
Protein L, a cell wall molecule of the bacterial species Peptostreptococcus magnus with affinity for immunoglobulin (Ig) light chains, was isolated after solubilization of the bacterial cell walls with mutanolysin or from the culture medium by a single affinity chromatography step on human IgG-Sepharose. A major protein band with an apparent molecular weight of 95,000 was obtained from both sources. The protein from the growth medium was size heterogeneous. From 1 ml of packed bacteria was prepared 0.92 mg of the mutanolysin-solubilized protein L (73% yield), whereas 4.1 mg of spontaneously released protein L (49% yield) was purified from the corresponding culture medium. The Mr of protein L was estimated to 76,000 by gel chromatography in 6 M guanidine HCl. Using this Mr value, the Stokes radius and the frictional ratio of protein L were determined to 4.74 nm and 1.70, respectively, suggesting an elongated fibrous molecule. No disulfide bond or subunit structure could be shown. The amino-terminal amino acid sequences of the whole protein and two internal non-IgG-binding tryptic fragments were determined and found to be unique. One of the tryptic fragments showed homology (40% identical residues) to a sequence within the cell wall-binding region of protein G, the Fc-binding protein of group C and G streptococci. The binding specificity of protein L was directed to the light chains of immunoglobulins; the affinity constant for polyacrylamide-coupled kappa-chains was 1.5 x 10(9) M-1 and for IgG, IgA, and IgM around 1 x 10(10) M-1. Maximal binding was achieved between pH 7 and 10. The binding to lambda-chains was too weak for determination of the affinity constant. 125I-Protein L was also shown to bind to mouse immunoglobulins. It could be used for detection of antigen-bound polyclonal and monoclonal antibodies in Western blots. This shows that the protein L/light chain reaction was not obstructed by occupation of the antigen-binding site. Finally, protein L and kappa-chains of human Ig formed precipitates upon double immunodiffusion analysis, an indication of at least two binding sites on protein L.
蛋白L是大消化链球菌细胞壁的一种分子，对免疫球蛋白（Ig）轻链具有亲和力。在用变溶菌素溶解细菌细胞壁后，或通过在人IgG-琼脂糖上进行单步亲和层析从培养基中分离得到该蛋白。从这两种来源都获得了一条表观分子量为95,000的主要蛋白带。来自生长培养基的蛋白在大小上是不均一的。从1 ml压实的细菌中制备出0.92 mg变溶菌素溶解的蛋白L（产率73%），而从相应的培养基中纯化出4.1 mg自发释放的蛋白L（产率49%）。在6 M盐酸胍中通过凝胶层析估计蛋白L的Mr为76,000。利用这个Mr值，确定蛋白L的斯托克斯半径和摩擦比分别为4.74 nm和1.70，表明它是一个细长的纤维状分子。未显示出二硫键或亚基结构。测定了整个蛋白以及两个内部非IgG结合胰蛋白酶片段的氨基末端氨基酸序列，发现它们是独特的。其中一个胰蛋白酶片段与C组和G组链球菌的Fc结合蛋白蛋白G的细胞壁结合区域内的一个序列具有同源性（40%相同残基）。蛋白L的结合特异性针对免疫球蛋白的轻链；与聚丙烯酰胺偶联的κ链的亲和常数为1.5×10⁹ M⁻¹，与IgG、IgA和IgM的亲和常数约为1×10¹⁰ M⁻¹。在pH 7至10之间实现最大结合。与λ链的结合太弱，无法测定亲和常数。¹²⁵I-蛋白L也显示与小鼠免疫球蛋白结合。它可用于在蛋白质印迹中检测抗原结合的多克隆和单克隆抗体。这表明蛋白L/轻链反应不会因抗原结合位点被占据而受阻。最后，在双向免疫扩散分析中，蛋白L与人Ig的κ链形成沉淀，这表明蛋白L上至少有两个结合位点。
