Baculovirus expression, purification and evaluation of recombinant pneumococcal surface adhesin A of Streptococcus pneumoniae.
Pneumococcal surface adhesin A (PsaA), with a molecular mass of approximately 37 kD by SDS-PAGE, is a common surface protein expressed by all 90 serotypes of Streptococcus pneumoniae. S. pneumoniae serotype 6B genomic DNA was amplified to generate a DNA fragment carrying the full-length psaA sequence and was cloned into a baculovirus expression system. We expressed either cell-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 partitioning in PBS/Triton X-114 buffers and by weakly basic ion exchange filter chromatography. Membrane-bound 'hydrophobic rPsaA' (hrPsaA) expressed by either Sf9 or High-Five cells had a molecular mass of approximately 38 kD by SDS-PAGE and partitioned in a Triton X-114 phase, it reacted with both rabbit polyclonal and five monoclonal anti-PsaA antibodies by dot blot or Western blot analysis. High-Five-cell-expressed 'soluble rPsaA' (srPsaA) with a molecular mass of approximately 37 kD by SDS-PAGE, was isolated from the serum-free culture medium and did not partition in the Triton X-114 phase; it reacted with anti-PsaA rabbit polyclonal and mouse monoclonal antibodies by ELISA and Western blot analysis. Both rPsaA polypeptide forms were immunogenic in Swiss-Webster adult female mice. In an infant mouse model of bacteremia, survival rates for mice given mouse anti-rPsaA immune serum (from mice immunized with High-Five-expressed srPsaA; 20 microl, 1:50,000 titer) 24 h before bacteremic challenge were greater than for the control group (48 h postchallenge, 20 vs. 90% survival rates) when challenged with S. pneumoniae serotype 6B. These results indicate that rPsaA is immunogenic and elicits protective antibody in mice similar to native protein.
De
B K
BK
Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA. bkd1@cdc.gov
Sampson
J S
JS
Ades
E W
EW
Johnson
S E
SE
Stinson
A R
AR
Crook
J
J
Tharpe
J A
JA
Huebner
R C
RC
Carlone
G M
GM
D018829:Adhesins, Bacterial; D000818:Animals; D001426:Bacterial Proteins; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D002460:Cell Line; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D007114:Immunization; D008074:Lipoproteins / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D026901:Membrane Transport Proteins; D051379:Mice; D011008:Pneumococcal Infections / Q000517:prevention & control; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D013296:Streptococcus pneumoniae / Q000235:genetics* / Q000276:immunology
D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D002352:Carrier Proteins; D008074:Lipoproteins; D026901:Membrane Transport Proteins; C085000:PsaA protein, Streptococcus; D011993:Recombinant Fusion Proteins