HYDROPHOBICITY ENGINEERING TO FACILITATE SURFACE DISPLAY OF HETEROLOGOUS GENE-PRODUCTS ON STAPHYLOCOCCUS-XYLOSUS

Protein engineering has been employed to investigate the effect of spe cific amino acid changes on the targeting of heterologous proteins to the outer cell surface of the Gram-positive bacterium Staphylococcus x ylosus. Three different variants, corresponding to a 101 amino acid re gion of the major glycoprotein (G protein) of human respiratory syncyt ial virus (RSV), were generated in which multiple hydrophobic phenylal anine residues were either substituted or deleted. The different G pro tein fragments were expressed as one part of recombinant receptors des igned for surface display on S. xylosus cells. The engineered variants of the RSV G protein hybrid receptors were, in contrast to a non-engi neered fragment, efficiently targeted to the outer cell surface of rec ombinant S. xylosus cells as determined by different methods, includin g fluorescence-activated cell sorting. In addition, immunization of mi ce with live recombinant S. xylosus demonstrated that surface exposure was required to generate receptor-specific antibodies. The presented strategy of hydrophobic engineering should be of general interest in s urface-display applications and for secretion of proteins otherwise di fficult to translocate through host cell membranes.