A novel expression system for surface display of heterologous proteins on Staphylococcus carnosus cells has been developed. Taking advantage of the promoter and secretion signals, including a propeptide region, from the lipase gene of Staphylococcus hyicus and the cell wall-spanning and membrane-binding region of protein A from Staphylococcus aureus, efficient surface display of an 80-amino-acid peptide from a malaria blood stage antigen could be achieved. A serum albumin binding protein from streptococcal protein G was used both as a general reporter molecule and to increase the accessibility of the surface-displayed proteins. Immunoblotting, immunogold staining, and immunofluorescence on intact recombinant S. carnosus cells verified the presence of the propeptide, the malaria antigen, and the albumin-binding reporter protein on the bacterial surface. For the first time, fluorescence-activated cell sorting was used to analyze the presence of surface-displayed hybrid receptors on gram-positive bacteria.
Two different host‐vector expression systems, designed for cell surface display of heterologous receptors on Staphylococcus xylosus and Staphylococcus carnosus , respectively, were compared for the surface display of four variants of a 101 amino acid region derived from the G glycoprotein of human respiratory syncytial virus (RSV). Surface localization of the different chimeric receptors was evaluated by a colorimetric assay and by fluorescence‐activated cell sorting. It was concluded that the S. carnosus system was better both in the ability to translocate inefficiently secreted peptides and in the number of exposed hybrid receptors. The potential use of the described staphylococci as live bacterial vaccine vehicles or alternatives to filamentous phages for surface display of protein libraries is discussed.
Expression of receptors for IgA (Fc alpha Rs) was investigated on a panel of 35 human B cell lines by labelling with human secretory IgA (0.5 mg/ml) and flow cytometry analysis after staining with fluoresceinated goat anti-human secretory component and/or anti-alpha chain F(ab')2 fragments. Receptors for IgA could be demonstrated on one out of nine Burkitt's lymphoma cell lines, three out of five myeloma cell lines and five out of 21 lymphoblastoid cell lines. The percentage of Fc alpha R-positive cells within the same B cell line varied upon repeated examination. Human dimeric IgA1 lambda myeloma protein revealed the same number of IgA receptor positive cells as did secretory IgA, whereas monomeric IgA did not bind to Fc alpha R. Detection of Fc alpha R was not inhibited when the tests were carried out in the presence of human dimeric IgG, IgM, asialo-orosomucoid, and secretory component but it was abrogated by pre-treatment of the cells with trypsin. The binding characteristics of Fc alpha Rs were studied on the myeloma cell line Esteve, using 125I-labelled human dimeric IgA and secretory IgA. The binding was dose-dependent with rapid kinetics and specific inhibition by unlabelled secretory IgA. Scatchard plot analysis resulted in an equilibrium constant K ranging from 3.2 to 4.7 x 10(6) M/l. No correlation was observed between Fc alpha R expression and differentiation stage, monoclonality, polyclonality of the cell lines, or Ig class produced by the B cells.
