Protein Expression and Purification, volume 143, pages 77-82
Recombinant alpha-fetoprotein receptor-binding domain co-expression with polyglutamate tags facilitates in vivo folding in E. coli.
Mollaev Murad D
1
,
Gorokhovets Neonila
2
,
Nikolskaya Elena
3
,
Faustova Maria
1
,
Zabolotsky Arthur
1
,
Sokol Maria
4
,
Tereshenko Oksana
4
,
Zhunina Olga
4
,
Shvets Vitaliy
1
,
Severin Evgeniy
4
,
1
4
ANO Institute for Molecular Diagnostics, 117149, Moscow, Russia.
|
Publication type: Journal Article
Publication date: 2018-03-01
Journal:
Protein Expression and Purification
Quartile SCImago
Q3
Quartile WOS
Q4
Impact factor: 1.6
ISSN: 10465928, 10960279
Biotechnology
Abstract
A wide range of methods are known to increase the prokaryotic intracellular recombinant proteins solubility, for instance, growth at low temperature, supplementation of culture media with "chemical chaperones" (proline, glycine-betaine, and trehalose), co-expression with chaperones or highly soluble fusion partners. As an alternative, we have introduced the polyglutamate tag, which, as it has been shown, increased the protein solubility and facilitated folding. In this study we evaluated the minimal quantity of high density negatively charged EEEEVE amino acid repeats (pGlu) necessary to switch the recombinant receptor-binding domain of human alpha-fetoprotein (rbdAFP) expression almost entirely from the inclusion bodies to the soluble cytoplasmic fraction in E. coli. For this purpose, genetic constructs based on pET vectors coding rbdAFP and containing from 1 to 4 additional EEEEVE repeats at the C-terminus have been prepared. It was found that 3 pGlu repeats is the minimal number, that leads to a complete shift of the expression to the soluble cytoplasmic fraction in E. coli SHuffle Express T7 while 4 repeats were required for that in E. coli BL21(DE3). The rbdAFP contained 4 pGlu repeats was purified making use of ion-exchange chromatography and characterized by circular dichroism and ability to bind and accumulate in AFP receptor positive cancer cells in order to check for the structural and specific activity alterations related to the additional polyanionic sequence introduction.
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Mollaev M. D. et al. Recombinant alpha-fetoprotein receptor-binding domain co-expression with polyglutamate tags facilitates in vivo folding in E. coli. // Protein Expression and Purification. 2018. Vol. 143. pp. 77-82.
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Mollaev M. D., Gorokhovets N., Nikolskaya E., Faustova M., Zabolotsky A., Sokol M., Tereshenko O., Zhunina O., Shvets V., Severin E., Yabbarov N. G. Recombinant alpha-fetoprotein receptor-binding domain co-expression with polyglutamate tags facilitates in vivo folding in E. coli. // Protein Expression and Purification. 2018. Vol. 143. pp. 77-82.
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TY - JOUR
DO - 10.1016/j.pep.2017.11.001
UR - https://doi.org/10.1016%2Fj.pep.2017.11.001
TI - Recombinant alpha-fetoprotein receptor-binding domain co-expression with polyglutamate tags facilitates in vivo folding in E. coli.
T2 - Protein Expression and Purification
AU - Mollaev, Murad D
AU - Gorokhovets, Neonila
AU - Nikolskaya, Elena
AU - Faustova, Maria
AU - Zabolotsky, Arthur
AU - Sokol, Maria
AU - Tereshenko, Oksana
AU - Zhunina, Olga
AU - Shvets, Vitaliy
AU - Severin, Evgeniy
AU - Yabbarov, Nikita G
PY - 2018
DA - 2018/03/01 00:00:00
PB - Elsevier
SP - 77-82
VL - 143
SN - 1046-5928
SN - 1096-0279
ER -
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@article{2018_Mollaev,
author = {Murad D Mollaev and Neonila Gorokhovets and Elena Nikolskaya and Maria Faustova and Arthur Zabolotsky and Maria Sokol and Oksana Tereshenko and Olga Zhunina and Vitaliy Shvets and Evgeniy Severin and Nikita G Yabbarov},
title = {Recombinant alpha-fetoprotein receptor-binding domain co-expression with polyglutamate tags facilitates in vivo folding in E. coli.},
journal = {Protein Expression and Purification},
year = {2018},
volume = {143},
publisher = {Elsevier},
month = {mar},
url = {https://doi.org/10.1016%2Fj.pep.2017.11.001},
pages = {77--82},
doi = {10.1016/j.pep.2017.11.001}
}