Recombinant bovine dihydrofolate reductase produced by mutagenesis and nested PCR of murine dihydrofolate reductase cDNA

Vivian Cody, Qilong Mao, Sherry Queener

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Recent reports of the slow-tight binding inhibition of bovine liver dihydrofolate reductase (bDHFR) in the presence of polyphenols isolated from green tea leaves has spurred renewed interest in the biochemical properties of bDHFR. Earlier studies were done with native bDHFR but in order to validate models of polyphenol binding to bDHFR, larger quantities of bDHFR are necessary to support structural studies. Bovine DHFR differs from its closest sequence homologue, murine DHFR, by 19 amino acids. To obtain the bDHFR cDNA, murineDHFR cDNA was transformed by a series of nested PCRs to reproduce the amino acid coding sequence for bovine DHFR. The bovine liver DHFR cDNA has an open reading frame of 561 base pairs encoding a protein of 187 amino acids that has a high level of conservation at the primary sequence level with other DHFR enzymes, and more so for the amino acid residues in the active site of the mammalian DHFR enzymes. Expression of the bovine DHFR cDNA in bacterial cells produced a stable recombinant protein with high enzymatic activity and kinetic properties similar to those previously reported for the native protein.

Original languageEnglish
Pages (from-to)104-110
Number of pages7
JournalProtein Expression and Purification
Volume62
Issue number1
DOIs
StatePublished - Nov 2008

Fingerprint

Tetrahydrofolate Dehydrogenase
Mutagenesis
Complementary DNA
Polymerase Chain Reaction
Liver
Polyphenols
Amino Acids
Tea
Enzymes
Sequence Homology
Recombinant Proteins
Base Pairing
Open Reading Frames
Amino Acid Sequence
Catalytic Domain
Proteins

Keywords

  • Bovine
  • Crystallization
  • Dihydrofolate reductase
  • Kinetics
  • Murine
  • PCR

ASJC Scopus subject areas

  • Biotechnology

Cite this

Recombinant bovine dihydrofolate reductase produced by mutagenesis and nested PCR of murine dihydrofolate reductase cDNA. / Cody, Vivian; Mao, Qilong; Queener, Sherry.

In: Protein Expression and Purification, Vol. 62, No. 1, 11.2008, p. 104-110.

Research output: Contribution to journalArticle

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