Single-step purification of human C4b-binding protein (C4BP) by affinity chromatography on a peptide derived from a streptococcal surface protein.
Many Gram-positive bacteria express surface proteins that bind human plasma proteins. These bacterial proteins, and derivatives of them, are of interest for analysis of bacterial pathogenesis and as immunochemical tools. Well-characterized examples include the IgG-binding reagents staphylococcal protein A and streptococcal protein G, and the recently described streptococcal IgA-binding peptide Sap. Here, we show that a peptide derived from the streptococcal M22 protein can be used for single-step affinity purification of the human complement regulator C4b-binding protein (C4BP). Binding of C4BP was strongly enhanced by dimerization of the peptide via a C-terminal cysteine residue not present in the intact M22 protein. The purified C4BP had the expected binding characteristics, and acted as a cofactor for factor I in the degradation of C4b. Passage of serum through a peptide column under non-saturating conditions resulted in binding of >99.5% of serum C4BP, implying that such a column can be used to deplete serum of C4BP. These data indicate that the C4BP-binding peptide is a versatile tool that can be used for simple and rapid purification of biologically active human C4BP or for removal of C4BP from serum.
Persson
Jenny
J
Department of Medical Microbiology, Dermatology and Infection, Sölvegatan 23, 223 62 Lund, Sweden.
Lindahl
Gunnar
G
D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry*; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D050716:Complement C4b-Binding Protein; D019281:Dimerization; D006649:Histocompatibility Antigens / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011817:Rabbits
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; C055609:C4BPA protein, human; D002352:Carrier Proteins; D050716:Complement C4b-Binding Protein; D006649:Histocompatibility Antigens; D010455:Peptides; C014689:streptococcal M protein