Assay, purification and characterization of a recombinant malaria circumsporozoite fusion protein by high-performance liquid chromatography.
The immunodominant repeat region of the malaria circumsporozoite protein from Plasmodium falciparum was purified from a recombinant Escherichia coli to study as a potential subunit vaccine. The recombinant protein, R32Leu-Arg, is composed of 32 tetrapeptide repeat sequences from the circumsporozoite protein (R32) linked to the dipeptide, Leu-Arg. R32Leu-Arg was purified by a series of precipitation steps including temperature, ammonium sulfate, and acid pH treatments; followed by reversed-phase high-performance liquid chromatography (RP-HPLC). An automated RP-HPLC assay was developed to measure the R32Leu-Arg concentration during both fermentation and purification. This assay was used in a variety of applications including measurement of production levels of the antigen during fermentation, evaluation of the protein purification process, quantitation of protein recovery, and as one criterion of protein purity. With minimal changes, the assay conditions were easily adapted to the semi-preparative level to produce 200 mg of purified product. The purified product was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; amino acid composition; and analytical size-exclusion and RP-HPLC.
Folena-Wasserman
G
G
Department of Protein Biochemistry, Smith Kline and French Laboratories, King of Prussia, PA 19406.
Inacker
R
R
Rosenbloom
J
J
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005285:Fermentation; D008288:Malaria / Q000517:prevention & control; D010963:Plasmodium falciparum / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet; D014612:Vaccines
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014612:Vaccines