Purification of His6-organophosphate hydrolase using monolithic supermacroporous polyacrylamide cryogels developed for immobilized metal affinity chromatography.
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.
Efremenko
E
E
Chemical Enzymology Department, Chemical Faculty, M.V. Lomonosov Moscow State University, Lenin's Hills 1/11, 119992 Moscow, Russia. efremenko@enzyme.chem.msu.ru
Votchitseva
Y
Y
Plieva
F
F
Galaev
I
I
Mattiasson
B
B
D000180:Acrylic Resins / Q000737:chemistry*; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D059985:Cryogels; D004926:Escherichia coli; D005353:Fibronectins / Q000737:chemistry*; D020100:Hydrogels; D007098:Imino Acids / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D000180:Acrylic Resins; D001798:Blood Proteins; D059985:Cryogels; D005353:Fibronectins; D020100:Hydrogels; D007098:Imino Acids; D008670:Metals; D011994:Recombinant Proteins; C016680:polyacrylamide gels; C045290:phosphorylphosphatase; D010744:Phosphoric Monoester Hydrolases; C008109:iminodiacetic acid