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Purification of His(6)-organophosphate hydrolase using monolithic supermacroporous polyacrylamide cryogels developed for immobilized metal affinity chromatography

Efremenko, E ; Votchitseva, Y ; Plieva, Fatima LU ; Galaev, Igor LU and Mattiasson, Bo LU (2006) In Applied Microbiology and Biotechnology 70(5). p.558-563
Abstract
Organophosphate hydrolase containing hexahistidine tag at the N-terminus of recombinant protein (His(6)-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... (More)
Organophosphate hydrolase containing hexahistidine tag at the N-terminus of recombinant protein (His(6)-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 His(6)-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. (Less)
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organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Applied Microbiology and Biotechnology
volume
70
issue
5
pages
558 - 563
publisher
Springer
external identifiers
  • pmid:16088350
  • wos:000236624500007
  • scopus:33645708122
ISSN
1432-0614
DOI
10.1007/s00253-005-0103-x
language
English
LU publication?
yes
id
786344bd-7d85-4551-bb78-d699d381bbd6 (old id 414672)
date added to LUP
2016-04-01 16:28:46
date last changed
2020-05-20 02:59:03
@article{786344bd-7d85-4551-bb78-d699d381bbd6,
  abstract     = {Organophosphate hydrolase containing hexahistidine tag at the N-terminus of recombinant protein (His(6)-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 His(6)-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.},
  author       = {Efremenko, E and Votchitseva, Y and Plieva, Fatima and Galaev, Igor and Mattiasson, Bo},
  issn         = {1432-0614},
  language     = {eng},
  number       = {5},
  pages        = {558--563},
  publisher    = {Springer},
  series       = {Applied Microbiology and Biotechnology},
  title        = {Purification of His(6)-organophosphate hydrolase using monolithic supermacroporous polyacrylamide cryogels developed for immobilized metal affinity chromatography},
  url          = {http://dx.doi.org/10.1007/s00253-005-0103-x},
  doi          = {10.1007/s00253-005-0103-x},
  volume       = {70},
  year         = {2006},
}