Porcine pyridoxal kinase c-DNA cloning, expression and confirmation of its primary sequence

Z. G. Gao, C. K. Lau, Chun Lap Samuel Lo, S. Y. Choi, J. E. Churchich, F. Kwok

Research output: Journal article publicationJournal articleAcademic researchpeer-review

13 Citations (Scopus)

Abstract

Porcine brain pyridoxal kinase has been cloned. A 1.2 kilo-based cDNA with a 966-base pair open reading frame was determined from a porcine brain cortex cDNA library using PCR technique. The DNA sequence was shown to encode a protein of 322 amino acid residues with a molecular mass of 35.4kDa. The amino acid sequence deduced from the nucleotide sequence of the cDNA was shown to match the partial primary sequence of pyridoxal kinase. Expression of the cloned cDNA in E. coli has produced a protein which displays both pyridoxal kinase activity and immunoreactivity with monoclonal antibodies raised against natural enzyme from porcine brain. With respect to the physical properties, it is shown that the recombinant protein exhibits identical kinetic parameters with the pure enzyme from porcine brain. Although the primary sequence of porcine pyridoxal kinase has been shown to share 87% homology with the human enzyme, we have shown that the porcine enzyme carries an extra peptide of ten amino acid residues at the N-terminal domain.
Original languageEnglish
Pages (from-to)1379-1388
Number of pages10
JournalInternational Journal of Biochemistry and Cell Biology
Volume30
Issue number12
Publication statusPublished - 1 Dec 1998

Keywords

  • CDNA cloning
  • Expression
  • Monoclonal antibody
  • Pig
  • Primary sequence
  • Pyridoxal kinase

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

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