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Deuteration of human carbonic anhydrase for neutron crystallography : Cell culture media, protein thermostability, and crystallization behavior

Koruza, K. LU ; Lafumat, B. LU ; Végvári, LU ; Knecht, W. LU and Fisher, S. Z. LU (2018) In Archives of Biochemistry and Biophysics 645. p.26-33
Abstract

Deuterated proteins and other bio-derived molecules are important for NMR spectroscopy, neutron reflectometry, small angle neutron scattering, and neutron protein crystallography. In the current study we optimized expression media and cell culture conditions to produce high levels of 3 different deuterated human carbonic anhydrases (hCAs). The labeled hCAs were then characterized and tested for deuterium incorporation by mass spectrometry, temperature stability, and propensity to crystallize. The results show that is possible to get very good yields (>10 mg of pure protein per liter of cell culture under deuterated conditions) and that protein solubility is unaffected at the crystallization concentrations tested. Using unlabeled... (More)

Deuterated proteins and other bio-derived molecules are important for NMR spectroscopy, neutron reflectometry, small angle neutron scattering, and neutron protein crystallography. In the current study we optimized expression media and cell culture conditions to produce high levels of 3 different deuterated human carbonic anhydrases (hCAs). The labeled hCAs were then characterized and tested for deuterium incorporation by mass spectrometry, temperature stability, and propensity to crystallize. The results show that is possible to get very good yields (>10 mg of pure protein per liter of cell culture under deuterated conditions) and that protein solubility is unaffected at the crystallization concentrations tested. Using unlabeled carbon source and recycled heavy water, we were able to get 65–77% deuterium incorporation, sufficient for most neutron-based techniques, and in a very cost-effective way. For most deuterated proteins characterized in the literature, the solubility and thermal stability is reduced. The data reported here is consistent with these observations and it was clear that there are measurable differences between hydrogenous and deuterated versions of the same protein in Tm and how they crystallize.

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organization
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type
Contribution to journal
publication status
published
subject
keywords
Carbonic anhydrase, Deuteration, Minimal media, Neutron scattering, Thermal stability
in
Archives of Biochemistry and Biophysics
volume
645
pages
8 pages
publisher
Academic Press
external identifiers
  • pmid:29524429
  • scopus:85043590348
ISSN
0003-9861
DOI
10.1016/j.abb.2018.03.008
language
English
LU publication?
yes
id
b0c0e0f1-4642-49c6-b2f6-5e90eef64e4f
date added to LUP
2018-03-23 13:50:28
date last changed
2020-07-08 04:14:10
@article{b0c0e0f1-4642-49c6-b2f6-5e90eef64e4f,
  abstract     = {<p>Deuterated proteins and other bio-derived molecules are important for NMR spectroscopy, neutron reflectometry, small angle neutron scattering, and neutron protein crystallography. In the current study we optimized expression media and cell culture conditions to produce high levels of 3 different deuterated human carbonic anhydrases (hCAs). The labeled hCAs were then characterized and tested for deuterium incorporation by mass spectrometry, temperature stability, and propensity to crystallize. The results show that is possible to get very good yields (&gt;10 mg of pure protein per liter of cell culture under deuterated conditions) and that protein solubility is unaffected at the crystallization concentrations tested. Using unlabeled carbon source and recycled heavy water, we were able to get 65–77% deuterium incorporation, sufficient for most neutron-based techniques, and in a very cost-effective way. For most deuterated proteins characterized in the literature, the solubility and thermal stability is reduced. The data reported here is consistent with these observations and it was clear that there are measurable differences between hydrogenous and deuterated versions of the same protein in T<sub>m</sub> and how they crystallize.</p>},
  author       = {Koruza, K. and Lafumat, B. and Végvári,  and Knecht, W. and Fisher, S. Z.},
  issn         = {0003-9861},
  language     = {eng},
  month        = {05},
  pages        = {26--33},
  publisher    = {Academic Press},
  series       = {Archives of Biochemistry and Biophysics},
  title        = {Deuteration of human carbonic anhydrase for neutron crystallography : Cell culture media, protein thermostability, and crystallization behavior},
  url          = {http://dx.doi.org/10.1016/j.abb.2018.03.008},
  doi          = {10.1016/j.abb.2018.03.008},
  volume       = {645},
  year         = {2018},
}