Extracellular expression, purification, and characterization of a winter flounder antifreeze polypeptide from Escherichia coli

HPLC6 is the major component of liver-type antifreeze polypeptides (AFPs) f rom the winter flounder, Pleuronectes americanus. To facilitate mutagenesis studies of this protein, a gene encoding the 37-amino acid mature polypept ide was chemically synthesized and cloned into the Tac cassette immediately after the bacterial ompA leader sequence for direct excretion of the AFP i nto the culture medium. Escherichia coli transformant with the construct pl acIQpar8AF was cultured in M9 medium. The recombinant AFP (rAFP) was detect ed by a competitive enzyme-linked immunosorbent assay (ELISA). After IPTG i nduction, a biologically active rAFP was expressed. The majority of the rAF P was excreted into the culture medium with only trace amounts trapped in t he periplasmic space and cytoplasm. After 18 h of induction, the accumulate d rAFP in the culture medium amounted to about 16 mg/L. The excreted AFP wa s purified from the culture medium by a single-step reverse-phase HPLC. Mas s spectrometric and amino acid composition analyses confirmed the identity of the purified product. The rAFP, which lacked amidation at the C-terminal , was about 70% active when compared to the amidated wild-type protein, thu s confirming the importance of C-terminal cap structure in protein stabilit y and function. (C) 2000 Academic Press. Press