Organophosphate hydrolase containing hexahistidine tag at the N-terminus of recombinant protein (His6-OPH) and expressed in Escherichia coli cells was purified using supermacroporous polyacrylamide-based monolith columns with immobilized metal affinity matrices [Me2+-iminodiacetic acid (IDA)-polyacrylamide cryogel (PAA) and Me2+-N,N,N'-tris (carboxymethyl) ethylendiamine (TED)-PAA]. Enzyme preparation with 50% purity was obtained by direct chromatography of nonclarified cell homogenate, whereas the combination of addition of 10 mM imidazole to buffers for cell sonication and sample loading, the use of precolumn with IDA-PAA matrix noncharged with metal ions, and the application of high flow rate provided the 99% purity of enzyme isolated directly from crude cell homogenate. Co2+-IDA-PAA provided the highest level of selectivity for His6-OPH. Comparative analysis of purification using Co2+-IDA-PAA and Ni-nitrilotriacetic acid-agarose showed obvious advantages of the former in process time, specific activity of purified enzyme, and simplicity of adsorbent regeneration.

The catalytic properties of organophosphate hydrolase (OPH) containing a hexahistidine tag His6 (His6-OPH) and purified to 98% homogeneity were investigated. The pH optimum of enzymatic activity and isoelectric point of His6-OPH, which were shown to be 10.5 and 8.5, respectively, are shifted to the alkaline range as compared to the same parameters of the native OPH. The recombinant enzyme possessed improved catalytic activity towards S-containing substrates: the catalytic efficiency of methylparathion hydrolysis by His6-OPH is 4.2 x 10(6) M(-1) x sec(-1), whereas by native OPH it is 3.5 x 10(5) M(-1) x sec(-1).

Five Tyr residues present in the native organophosphate hydrolase (OPH) containing a hexahistidine tag at the N terminus of the protein molecule (His 6 OPH) were replaced by fluorine containing analogs using a biosynthetic approach. The modified enzyme had an extended pH optimum of action shifted to acidic pH and an enhanced thermal stability in the alkaline pH region.

We obtained recombinant variants of human antibody FI6 broadly specific to hemagglutinins of the influenza A virus. On the basis of a bi-promoter (CMV, hEF1-HTLV) vector, we developed genetic constructs for the expression of the heavy and light chains of the immunoglobulins of IgA1-, IgA2m1-, and IgG-isotypes. Following transfection and selection, stable Chinese hamster ovary (CHO) cell lines were produced. The antibodies of IgA1-, IgA2m1-, and IgG-isotypes were purified from culture media. We performed an immunochemical characterization and studied their interactions with influenza A strains of the H1N1- and H3N2-subtypes. It was shown that recombinant FI6 variants of the IgA-isotype retain the properties of the parental IgG antibody to demonstrate specificity to all the strains tested. The strongest binding was observed for the H1N1 subtype, which belongs to hemagglutinins of phylogenetic group I.