Influence of His 6 Sequence on the Properties of Formate Dehydrogenase from Bacterium Pseudomonas sp. 101
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uence of His6 Sequence on the Properties of Formate Dehydrogenase from Bacterium Pseudomonas sp. 101 A. A. Pometuna, b, c, P. D. Parshinb, c, N. P. Galanichevab, I. V. Uporovb, D. L. Atroshenkoa, b, c, S. S. Savinb, c, and V. I. Tishkova, b, c, * aBach
Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences, Moscow, Russia b Department of Chemistry, Lomonosov Moscow State University, Moscow, Russia cInnovations and High Technologies MSU Ltd., Moscow, Russia *e-mail: [email protected] Received March 10, 2020; revised March 12, 2020; accepted March 20, 2020
Abstract—NAD(P)+-dependent formate dehydrogenase (FDH, EC 1.2.1.2.) is actively used in processes of chiral synthesis by oxidoreductases with systems of reduced cofactor regeneration. The efficient use of FDH in such systems requires simple and fast enzyme purification. Metal-chelate affinity chromatography is widely used for such purposes. The method requires the presence of at least six His residues at N- or C-terminus of protein. The addition of extra His residues can affect enzyme properties. The computer modeling of the structure of FDH from bacterium Pseudomonas sp. 101 with different positions of His6 sequence showed that the optimal case is His-tag at N-terminus. Three types of PseFDH with His6 were prepared: wild-type NAD+-dependent enzyme and two mutant NADP+-specific forms. New PseFDHs were obtained as homogeneous preparations through a one-step purification procedure. The comparison of PseFDHs with and without His-tag showed that they have similar kinetic properties. Keywords: formate dehydrogenase, Pseudomonas sp. 101, affinity chromatography, catalytic properties, thermal stability, His-tag DOI: 10.3103/S0027131420040057
Addition of polypeptide fragments (affinity tags) to a protein sequence makes it easy to purify proteins from both eukaryotic and prokaryotic organisms [1]. This method is extensively used for research and practical applications of different enzymes. Moreover, the addition of polypeptide tags can improve protein solubility, solve the protein folding problem and enhance the level of expression [2]. One of the most widely used approaches is the addition of a sequence of six histidine residues (His6tag) at the N- or C-terminus of the protein, although the sequences can be longer, e.g., His7–12 [3]. His6 is used for the purification of recombinant proteins by metal chelate affinity chromatography [1]. Note that His6 exhibits no immunogenicity, in contrast to many other polypeptide tags. The affinity of His6 to Cu2+, Ni2+, Co2+ or Zn2+ allows rapid separation of the His6-tagged protein– metal complex from all other bacterial proteins with up to 99% purity. The purification procedure includes the binding of the enzyme with the His-tag on a metal chelating carrier and the removal of unbound proteins from the carrier by washing. Buffer solutions with low imidazole concentrations were used for the elution of
low-affinity proteins from the column. Higher imidazole concentrations
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