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) from the winter flounder, Pleuronectes americanus. To facilitate mutagenesis studies of this protein, a gene encoding the 37-amino acid mature polypeptide was chemically synthesized and cloned into the Tac cassette immediately after the bacterial ompA leader sequence for direct excretion of the AFP into the culture medium. Escherichia coli transformant with the construct placIQpar8AF was cultured in M9 medium. The recombinant AFP (rAFP) was detected by a competitive enzyme-linked immunosorbent assay (ELISA). After IPTG induction, a biologically active rAFP was expressed. The majority of the rAFP was excreted into the culture medium with only trace amounts trapped in the periplasmic space and cytoplasm. After 18 h of induction, the accumulated rAFP in the culture medium amounted to about 16 mg/L. The excreted AFP was purified from the culture medium by a single-step reverse-phase HPLC. Mass 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, thus confirming the importance of C-terminal cap structure in protein stability and function.
Tong
L
L
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Lin
Q
Q
Wong
W K
WK
Ali
A
A
Lim
D
D
Sung
W L
WL
Hew
C L
CL
Yang
D S
DS
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D021301:Antifreeze Proteins; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005432:Flounder / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D000596:Amino Acids; D021301:Antifreeze Proteins; D006023:Glycoproteins; D011994:Recombinant Proteins