Improving the purification of NAD(+)-dependent formate dehydrogenase from Candida methylica

Ordu E., Karagueler N. G.

PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY, cilt.37, ss.333-341, 2007 (SCI-Expanded) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 37
  • Basım Tarihi: 2007
  • Doi Numarası: 10.1080/10826060701593233
  • Dergi Adı: PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Sayfa Sayıları: ss.333-341
  • Anahtar Kelimeler: NAD(+), formate dehydrogenase, Candida methylica, purification, ENZYME, GENE, TAG
  • Yıldız Teknik Üniversitesi Adresli: Evet

Özet

The Candida methylica ( cm) recombinant wild type formate dehydrogenase ( FDH) gene has been cloned into the pQE- 2 TAGZyme expression vector and the 6xHis- tagged FDH gene has been overexpressed in JM105 cells to purify the FDH protein more efficiently, by the use of exopeptidases, TAGZyme Purification System, which has allowed the complete removal of the small N- terminal His- tag. After the purification procedure, 1.2 mg/ mL cmFDH protein of >95% purity was obtained. The kinetic parameters of cmFDH have been determined by observing the oxidation of the nicotinamide coenzyme at 340 nm. The results have also been compared to the yield of standard vs. affinity purification of FDH.