title: Intein mediated hyper-production of authentic human basic fibroblast growth factor in Escherichia coli.
abstract: Human basic fibroblast growth factor is a functionally versatile but very expensive polypeptide. In this communication, employing a novel amplification method for the target gene and genetic optimization of a previously engineered expression construct, pWK3R, together with a refined fed-batch fermentation protocol, we report an achievement of a phenomenal yield of 610 mg/L of the 146 aa authentic human basic fibroblast growth factor (bFGF) in Escherichia coli. Construct pWK3R was first modified to form plasmid pWK311ROmpAd, which was devoid of the ompA leader sequence and possessed two copies of a DNA segment encoding a fusion product comprising an intein, Saccharomyces cerevisiae vascular membrane ATPase (VMA), and bFGF. When E. coli transformant JM101 [pWK311ROmpAd] was cultivated using the refined fed-batch fermentation protocol, superb expression resulting in a total yield of 610 mg/L of bFGF was detected. Despite existing in high levels, the bFGF remained to be soluble and highly bioactive.
authors.affiliation: Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.
authors.forename: Keith W Y
authors.initials: KW
authors.lastname: Kwong
authors.affiliation: Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.
authors.forename: T
authors.initials: T
authors.lastname: Sivakumar
authors.affiliation: Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.
authors.forename: W K R
authors.initials: WK
authors.lastname: Wong