Fast quantitation of recombinant plasminogen activator inhibitor type 1 in bacterial lysates by micropellicular reversed-phase liquid chromatography.
A rapid reversed-phase HPLC assay is described for quantitating recombinant plasminogen activator inhibitor type 1 (PAI-1) in cultures of Escherichia coli. The assay utilized a short nonporous micropellicular C18 column with an acetonitrile gradient in 0.1% trifluoroacetic acid. The selective resolution of recombinant PAI-1 from major host proteins occurred within 1.3 min. Regeneration of initial chromatography conditions was fast and allowed successive injections every 3 min. The assay's limit of detection for recombinant PAI-1 was 0.5 microg in 5-microl injections of bacterial lysates and the assay was linear to 20 microg. The assay's apparent recovery was between 94 and 108% throughout the quantitative range. In a direct comparison to gel electrophoresis the reversed-phase assay proved superior in monitoring recombinant PAI-1 titers in cultures of E. coli.
O'Keefe
D O
DO
Department of Macromolecular Structure and Biopharmaceuticals, Bristol-Myers Squibb, Princeton, NJ 08543, USA. okeefed@bms.com
Duke
G J
GJ
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry; D017395:Plasminogen Activator Inhibitor 1 / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis; D012680:Sensitivity and Specificity
D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins