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A. A. Pometun, K. M. Boyko, S. A. Zubanova, A. Yu. Nikolaeva, D. L. Atroshenko, S. S. Savin, V. I. Tishkov
Preparation of recombinant formate
dehydrogenase from thermotolerant yeast Ogataea Parapolymorpha and
crystallization of apo- and holo- forms of the enzyme
Abstract
NAD+-dependent formate
dehydrogenase from thermotolerant yeast Ogataea parapolymorpha DL-1
(OpaFDH, EC 1.2.1.2) with additional Gly residue at N-terminus and it double
mutant OpaFDH_AD were over expressed in E. colicells with yield 6000 and
6200 U per liter of cultivation medium, respectively. Purified enzymes were
obtained as homogeneous preparations with activity yield 62%. Purification
procedure included ultrasonic cell disruption, heat treatment of cell free
extract at 55 oC for 15 min and hydrophobic chromatography on Phenyl
Sepharose Fast Flow. Crystallization experiments with wild-type OpaFDH resulted
in preparation of crystals of apo- but not holo- form. Crystals of holo-form
were obtained with mutant OpaFDH_AD only in the presence of 7 mM NAD+ and 10 mM
sodium azide. Size and quality of crystals were enough for collection of X-ray
diffraction data.
Key words: formate dehydrogenase, Ogataea
parapolymorpha DL-1,expression, purification, crystallization, apo-
and holo- forms.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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