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  • Title: Protein L: an immunoglobulin light chain-binding bacterial protein. Characterization of binding and physicochemical properties.
    Author: Akerström B, Björck L.
    Journal: J Biol Chem; 1989 Nov 25; 264(33):19740-6. PubMed ID: 2479638.
    Abstract:
    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.
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