Extraction of the outer membrane protein pertactin from Bordetella pertussis with urea for the production of acellular pertussis vaccine

Abstract

Pertactin (PRN), a non-fimbrial outer membrane protein of Bordetella pertussis, is the limiting component of the acellular pertussis vaccine because of its low concentration. This study aimed to develop a large-scale urea-based process for PRN extraction from B. pertussis. Cell pellet processing conditions, including freezing and thawing, were found to substantially affect PRN yield. A single cycle of rapid freezing of the cell pellet at - 30 degrees C with slow thawing at 5 +/- 3 degrees C resulted in up to fivefold higher PRN yield than condition without freezing and thawing. The search for urea treatment conditions was also conducted, and 5 M urea treatment for 2 h was the optimal condition. The developed urea-based process was applied to 50 L culture scale, and residual impurities were removed by sequential anion exchange, hydrophobic interaction and gel filtration chromatography and resulted in PRN with a purity of over 95% at a yield of 33.2%. From 50 L culture broth, the final yield of PRN per cell pellet was 0.23 mg/g (wet weight). Thus, a large-scale production process for high-quality PRN from B. pertussis was developed based on urea extraction process. The results may serve as a reference for production of other membrane proteins.