TY  - JOUR
AU  - Macinkovic, Igor S.
AU  - Abughren, Mohamed
AU  - Mrkić, Ivan
AU  - Grozdanović, Milica M.
AU  - Prodanović, Radivoje
AU  - Gavrović-Jankulović, Marija
PY  - 2013
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/1449
AB  - High levels of recombinant protein expression can lead to the formation of insoluble inclusion bodies. These complex aggregates are commonly solubilized in strong denaturants, such as 6-8 M urea, although, if possible, solubilization under milder conditions could facilitate subsequent refolding and purification of bioactive proteins. Commercially available GST-tag assays are designed for quantitative measurement of GST activity under native conditions. GST fusion proteins accumulated in inclusion bodies are considered to be undetectable by such assays. In this work, solubilization of recombinantly produced proteins was performed in 4 M urea. The activity of rGST was assayed in 2 M urea and it was shown that rGST preserves 85% of its activity under such denaturing conditions. A colorimetric GST activity assay with 1-chloro-2, 4-dinitrobenzene (CDNB) was examined for use in rapid detection of expression targeted to inclusion bodies and for the identification of inclusion body proteins which can be solubilized in low concentrations of chaotropic agents. Applicability of the assay was evaluated by tracking protein expression of two GST-fused allergens of biopharmaceutical value in E. coli, GST-Der p 2 and GST-Mus a 5, both targeted to inclusion bodies.
PB  - Elsevier Science Bv, Amsterdam
T2  - Journal of Biotechnology
T1  - Employment of colorimetric enzyme assay for monitoring expression and solubility of GST fusion proteins targeted to inclusion bodies
VL  - 168
IS  - 4
SP  - 506
EP  - 510
DO  - 10.1016/j.jbiotec.2013.09.019
ER  - 

@article{
author = "Macinkovic, Igor S. and Abughren, Mohamed and Mrkić, Ivan and Grozdanović, Milica M. and Prodanović, Radivoje and Gavrović-Jankulović, Marija",
year = "2013",
abstract = "High levels of recombinant protein expression can lead to the formation of insoluble inclusion bodies. These complex aggregates are commonly solubilized in strong denaturants, such as 6-8 M urea, although, if possible, solubilization under milder conditions could facilitate subsequent refolding and purification of bioactive proteins. Commercially available GST-tag assays are designed for quantitative measurement of GST activity under native conditions. GST fusion proteins accumulated in inclusion bodies are considered to be undetectable by such assays. In this work, solubilization of recombinantly produced proteins was performed in 4 M urea. The activity of rGST was assayed in 2 M urea and it was shown that rGST preserves 85% of its activity under such denaturing conditions. A colorimetric GST activity assay with 1-chloro-2, 4-dinitrobenzene (CDNB) was examined for use in rapid detection of expression targeted to inclusion bodies and for the identification of inclusion body proteins which can be solubilized in low concentrations of chaotropic agents. Applicability of the assay was evaluated by tracking protein expression of two GST-fused allergens of biopharmaceutical value in E. coli, GST-Der p 2 and GST-Mus a 5, both targeted to inclusion bodies.",
publisher = "Elsevier Science Bv, Amsterdam",
journal = "Journal of Biotechnology",
title = "Employment of colorimetric enzyme assay for monitoring expression and solubility of GST fusion proteins targeted to inclusion bodies",
volume = "168",
number = "4",
pages = "506-510",
doi = "10.1016/j.jbiotec.2013.09.019"
}

Macinkovic, I. S., Abughren, M., Mrkić, I., Grozdanović, M. M., Prodanović, R.,& Gavrović-Jankulović, M.. (2013). Employment of colorimetric enzyme assay for monitoring expression and solubility of GST fusion proteins targeted to inclusion bodies. in Journal of Biotechnology
Elsevier Science Bv, Amsterdam., 168(4), 506-510.
https://doi.org/10.1016/j.jbiotec.2013.09.019

Macinkovic IS, Abughren M, Mrkić I, Grozdanović MM, Prodanović R, Gavrović-Jankulović M. Employment of colorimetric enzyme assay for monitoring expression and solubility of GST fusion proteins targeted to inclusion bodies. in Journal of Biotechnology. 2013;168(4):506-510.
doi:10.1016/j.jbiotec.2013.09.019 .

Macinkovic, Igor S., Abughren, Mohamed, Mrkić, Ivan, Grozdanović, Milica M., Prodanović, Radivoje, Gavrović-Jankulović, Marija, "Employment of colorimetric enzyme assay for monitoring expression and solubility of GST fusion proteins targeted to inclusion bodies" in Journal of Biotechnology, 168, no. 4 (2013):506-510,
https://doi.org/10.1016/j.jbiotec.2013.09.019 . .