Site-directed lipid modification of IgG-binding protein by intracellular bacterial lipoprotein process.
IgG-binding protein was genetically expressed and lipid-modified in a site-directed manner in Escherichia coli. The DNA sequence encoding the signal peptide and the nine N-terminal amino acid residues of the major lipoprotein of E. coli (lpp) was fused to the sequence of B-domain which was one of the IgG binding domains of Staphylococcal Protein A (SpA). The N-terminal cysteine residue of the resulting protein was enzymatically linked with lipids in the bacterial membrane. The lipid-modified protein was translocated at the bacterial membrane in a manner similar to native bacterial lipoprotein, and it was purified with IgG-Sepharose by affinity chromatography. The lipid modified proteins (lppB1 and lppB5) showed a similar IgG binding activity to unmodified proteins, which was estimated by competitive ELISA. Proteoliposomes of lipid modified proteins were prepared in an elegant fashion so that the IgG binding site should be properly oriented on the surface of an individual liposome by anchoring the lipid-tail into the hydrophobic layer of the liposome membrane. As compared with the unmodified one, the lipid modified protein incorporated into the proteoliposome exhibited higher IgG binding activity.
Shigematsu
H
H
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan.
Ebihara
T
T
Yanagida
Y
Y
Haruyama
T
T
Kobatake
E
E
Aizawa
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001709:Biotechnology; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism*; D066298:In Vitro Techniques; D008055:Lipids / Q000737:chemistry; D011510:Proteolipids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013205:Staphylococcal Protein A / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
D002352:Carrier Proteins; D007074:Immunoglobulin G; D008055:Lipids; D011510:Proteolipids; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; C020485:proteoliposomes