title: Purification of a recombinant protein expressed in yeast: optimization of analytical and preparative chromatography.
abstract: The industrial production of recombinant proteins requires control of both fermentation and purification steps. For the serodiagnosis of toxoplasmosis, the main antigen is a membrane protein of 30 kDa (P30). The P30 gene was cloned and expressed in Schizosaccharomyces pombe at 0.7 microg/ml in culture medium. Batch fermentation was optimized by the specific choice of peptones, which enabled optimum growth and protein expression without reducing the efficacy of the purification step. Analytical purification was then carried out using cation-exchange chromatography. For larger volumes, scaling up was performed on expanded mode by using a Streamline system (Pharmacia). This purification step allowed us to obtain a 67.5% recovery with a purification factor greater than 27-fold. Expanded bed adsorption technology is a convenient and effective technique for protein capture directly from feedstock, and the eluted fraction is ready for a second affinity chromatography step. This second step is performed with a yield of 40% and provides a final purification factor of 2000-fold.
authors.affiliation: BioMérieux S.A., Marcy l'Etoile, France.
authors.forename: F
authors.initials: F
authors.lastname: Raymond
authors.forename: D
authors.initials: D
authors.lastname: Rolland
authors.forename: M
authors.initials: M
authors.lastname: Gauthier
authors.forename: M
authors.initials: M
authors.lastname: Jolivet
mesh_terms: D000327:Adsorption; D000818:Animals; D000953:Antigens, Protozoan; D002412:Cations; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005285:Fermentation; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification; D012568:Schizosaccharomyces / Q000235:genetics*; D014122:Toxoplasma
chemical_list: D000953:Antigens, Protozoan; D002412:Cations; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C047168:SAG1 antigen, Toxoplasma