Baculovirus expression, purification and evaluation of recombinant pneumococcal surface adhesin A of Streptococcus pneumoniae

Pneumococcal surface adhesin A (PsaA), with a molecular mass of similar to 37 kD by SDS-PAGE, is a common surface protein expressed by all 90 serotype s of Streptococcus pneumoniae. S. pneumoniae serotype 6B genomic DNA was am plified to generate a DNA fragment carrying the full-length psaA sequence a nd was cloned into a baculovirus expression system. We expressed either cel l-associated or cell-free nonfusion PsaA polypeptides using two insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni 5B1-4 (High-Five). Recombinant PsaA (rPsaA) polypeptides were partially purified by partitioni ng in PBS/Triton X-114 buffers and by weakly basic ion exchange filter chro matography. Membrane-bound 'hydrophobic rPsaA' (hrPsaA) expressed by either Sf9 or High-Five cells had a molecular mass of similar to 38 kD by SDS-PAG E and partitioned in a Triton X-114 phase, it reacted with both rabbit poly clonal and five monoclonal anti-PsaA antibodies by dot blot or Western blot analysis.