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.
Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA. bkd1@cdc.gov
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BK
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J S
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Sampson
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Ades
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Johnson
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Stinson
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Crook
J A
JA
Tharpe
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Huebner
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Carlone
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