Design, preparation and directional insertion of peptides into lipid bilayer membrane and their application for the preparation of liposome of which surface could be coated by externally added antibody

Abstract

Pf3 coat protein, a protein coded by bacteriophage Pf3, is a short membrane spanning protein consisting from 44 amino acid residues. Its mutant protein PO-3L, having three additional Leu residues in its transmembrane region, was demonstrated to be spontaneously inserted into large unilamellar vesicle (LUV), even in the absence of protein machineries or membrane potential. However, detailed interaction manner of peptide with LUV are not yet fully understood. In the present study, we designed two PO derivatives of 3L-DR and 3L-RD in which His, T7 or Myc tags are incorporated. These mutants were expressed in bacteria and homogeneously purified by Ni Sepharose column chromatography. When they were incubated with preformed LUV, their spontaneous insertion into LUV was observed. Because both N- and C-terminal of newly designed peptides contain either T7 or Myc tag, even when one of N- or C-terminus was digested with trypsin, resulting truncated peptide could be detected using antibody against the tags present in the opposite C- or N-terminal. Using this property of the peptides, topologies of newly designed peptides embedded into LUV were clearly determined. Furthermore, we applied the membrane anchoring property of 3L-DR for development of a protein A derivative which could be spontaneously inserted into liposomal membrane. When parental small IgG binding unit of protein A was mixed with liposome, its binding to the liposome was not observed, however, its fusion peptide with 3L-DR was spontaneously incorporated into liposomal membrane. Furthermore, when antiserum was added to this liposome, significant binding of IgG to the liposome was observed. Thus, by using protein A derivative fused to the 3L-DR, we succeeded in the preparation of novel liposome of which surface could be modifiable with externally added antibodies.