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Nucleotide sequence and high-level expression of the major Escherichia coli phosphofructokinase.

Publication ,  Journal Article
Hellinga, HW; Evans, PR
Published in: Eur J Biochem
June 3, 1985

The gene for the major phosphofructokinase enzyme in Escherichia coli, pfkA, has been sequenced. Comparison of the amino acid sequence with other phosphofructokinases showed that this enzyme is related to the Bacillus stearothermophilus and rabbit muscle enzymes, but is different from the second, minor phosphofructokinase found in E. coli. The region which has been sequenced comprises the complete pfkA--tpi interval on the E. coli genetic map. Two other genes have been identified from the nucleotide sequence: a gene for a periplasmic sulphate-binding protein, sbp, and for a membrane-bound enzyme, CDP-diglyceride hydrolase, cdh. This establishes the complete gene arrangement in this region as pfkA-sbp-cdh-tpi. The pfkA gene has been subcloned into a high-copy-number plasmid under the control of a strong, chimaeric promoter which arose as an artefact in the construction of the plasmid gene bank from which the original pfkA recombinant was isolated. A specialised recombinant has been constructed which carries a 1.4 X 10(3)-nucleotide insert containing just the pfkA gene flanked by two HindIII recognition sites providing a simple system for the recloning of this gene into different vectors. This recombinant expresses the enzyme at high levels (40-50% of total cell protein is active, soluble phosphofructokinase). This expression system is now being used to study the enzyme using 'reverse genetics'.

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Published In

Eur J Biochem

DOI

ISSN

0014-2956

Publication Date

June 3, 1985

Volume

149

Issue

2

Start / End Page

363 / 373

Location

England

Related Subject Headings

  • Species Specificity
  • Recombination, Genetic
  • Rabbits
  • Phosphofructokinase-1
  • Mutation
  • Muscles
  • Geobacillus stearothermophilus
  • Genes
  • Gene Expression Regulation
  • Escherichia coli
 

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Hellinga, H. W., & Evans, P. R. (1985). Nucleotide sequence and high-level expression of the major Escherichia coli phosphofructokinase. Eur J Biochem, 149(2), 363–373. https://doi.org/10.1111/j.1432-1033.1985.tb08934.x
Hellinga, H. W., and P. R. Evans. “Nucleotide sequence and high-level expression of the major Escherichia coli phosphofructokinase.Eur J Biochem 149, no. 2 (June 3, 1985): 363–73. https://doi.org/10.1111/j.1432-1033.1985.tb08934.x.
Hellinga HW, Evans PR. Nucleotide sequence and high-level expression of the major Escherichia coli phosphofructokinase. Eur J Biochem. 1985 Jun 3;149(2):363–73.
Hellinga, H. W., and P. R. Evans. “Nucleotide sequence and high-level expression of the major Escherichia coli phosphofructokinase.Eur J Biochem, vol. 149, no. 2, June 1985, pp. 363–73. Pubmed, doi:10.1111/j.1432-1033.1985.tb08934.x.
Hellinga HW, Evans PR. Nucleotide sequence and high-level expression of the major Escherichia coli phosphofructokinase. Eur J Biochem. 1985 Jun 3;149(2):363–373.

Published In

Eur J Biochem

DOI

ISSN

0014-2956

Publication Date

June 3, 1985

Volume

149

Issue

2

Start / End Page

363 / 373

Location

England

Related Subject Headings

  • Species Specificity
  • Recombination, Genetic
  • Rabbits
  • Phosphofructokinase-1
  • Mutation
  • Muscles
  • Geobacillus stearothermophilus
  • Genes
  • Gene Expression Regulation
  • Escherichia coli