Bacterial expression and purification of recombinant Plasmodium yoelii circumsporozoite protein.
We report the expression and purification of recombinant rodent malarial Plasmodium yoelii circumsporozoite surface protein (PyCSP) in Escherichia coli. To facilitate purification of the recombinant protein, the PyCSP was expressed as an amino-terminal fusion protein to glutathione S-transferase and as a carboxy-terminal fusion protein to a hexahistidyl tag. The expression of the fusion protein was controlled by the inducible tac promoter. Under optimal conditions the immunoreactive PyCSP represented approximately 0.04% of the total cell lysate. Western blot analysis probing with an anti-PyCSP antibody revealed a wide array of immunoreactive bands. Material isolated by affinity purification on glutathione-Sepharose 4B resin also contained multiple bands indicative of premature termination or carboxyl-terminal degradation. Analysis of protein retained on a nickel nitrilotriacetic acid resin revealed evidence of amino-terminal deleted material. Combining the two mild affinity purifications resulted in isolation of a single immunoreactive protein of approximate molecular weight of 96 kDa. We anticipate that the approach described in this study will facilitate the production of highly purified recombinant proteins.
Stratmann
T
T
Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037, USA.
Schmid
S R
SR
Harper
J F
JF
Kang
A S
AS
D000818:Animals; D000953:Antigens, Protozoan / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D015153:Blotting, Western; D004926:Escherichia coli; D008970:Molecular Weight; D010967:Plasmodium yoelii / Q000235:genetics*; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping
D000953:Antigens, Protozoan; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C041595:circumsporozoite protein, Protozoan