EXPRESSION OF ESCHERICHIA-COLI HOMOSERINE KINASE IN MOUSE 3T3 CELLS

W D REES, S M HAY, H J FLINT

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

The Escherichia coli gene for homoserine kinase (thrB) has been cloned into a simian-virus-40-based eukaryotic expression vector which also includes a neomycin-resistance gene. Mouse 3T3 cells transfected with this plasmid were selected for resistance and screened for homoserine kinase activity. It has thus been possible to isolate clones which are capable of accumulating homoserine O-phosphate when supplied with homoserine. In broken-cell preparations the kinetic constants for the production of homoserine O-phosphate were similar to those of the wild-type E. coli enzyme. These experiments demonstrate that E. coli homoserine kinase can be expressed in an animal cell and that it can successfully phosphorylate L-homoserine in the intact cell utilizing endogenous ATP.

Original languageEnglish
Pages (from-to)865-870
Number of pages6
JournalBiochemical Journal
Volume281
Publication statusPublished - 1 Feb 1992

Keywords

  • MAMMALIAN-CELLS
  • RECOMBINANT GENOMES
  • GENE
  • TRANSCRIPTION
  • BIOSYNTHESIS
  • INHIBITION
  • SEQUENCES
  • THREONINE
  • VIRUS
  • RNA

Cite this

EXPRESSION OF ESCHERICHIA-COLI HOMOSERINE KINASE IN MOUSE 3T3 CELLS. / REES, W D ; HAY, S M ; FLINT, H J .

In: Biochemical Journal, Vol. 281, 01.02.1992, p. 865-870.

Research output: Contribution to journalArticle

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abstract = "The Escherichia coli gene for homoserine kinase (thrB) has been cloned into a simian-virus-40-based eukaryotic expression vector which also includes a neomycin-resistance gene. Mouse 3T3 cells transfected with this plasmid were selected for resistance and screened for homoserine kinase activity. It has thus been possible to isolate clones which are capable of accumulating homoserine O-phosphate when supplied with homoserine. In broken-cell preparations the kinetic constants for the production of homoserine O-phosphate were similar to those of the wild-type E. coli enzyme. These experiments demonstrate that E. coli homoserine kinase can be expressed in an animal cell and that it can successfully phosphorylate L-homoserine in the intact cell utilizing endogenous ATP.",
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N2 - The Escherichia coli gene for homoserine kinase (thrB) has been cloned into a simian-virus-40-based eukaryotic expression vector which also includes a neomycin-resistance gene. Mouse 3T3 cells transfected with this plasmid were selected for resistance and screened for homoserine kinase activity. It has thus been possible to isolate clones which are capable of accumulating homoserine O-phosphate when supplied with homoserine. In broken-cell preparations the kinetic constants for the production of homoserine O-phosphate were similar to those of the wild-type E. coli enzyme. These experiments demonstrate that E. coli homoserine kinase can be expressed in an animal cell and that it can successfully phosphorylate L-homoserine in the intact cell utilizing endogenous ATP.

AB - The Escherichia coli gene for homoserine kinase (thrB) has been cloned into a simian-virus-40-based eukaryotic expression vector which also includes a neomycin-resistance gene. Mouse 3T3 cells transfected with this plasmid were selected for resistance and screened for homoserine kinase activity. It has thus been possible to isolate clones which are capable of accumulating homoserine O-phosphate when supplied with homoserine. In broken-cell preparations the kinetic constants for the production of homoserine O-phosphate were similar to those of the wild-type E. coli enzyme. These experiments demonstrate that E. coli homoserine kinase can be expressed in an animal cell and that it can successfully phosphorylate L-homoserine in the intact cell utilizing endogenous ATP.

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KW - RECOMBINANT GENOMES

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KW - SEQUENCES

KW - THREONINE

KW - VIRUS

KW - RNA

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JF - Biochemical Journal

SN - 0264-6021

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