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© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.

Abstract

The production of heterologous proteins is an important procedure for biologists in basic and applied sciences. A variety of cell-based and cell-free protein expression systems are available to achieve this. The expression system must be selected carefully, especially for target proteins that require post-translational modifications. In this study, human Src family kinases were prepared using six different protein expression systems: 293 human embryonic kidney cells, Escherichia coli, and cell-free expression systems derived from rabbit reticulocytes, wheat germ, insect cells, or Escherichia coli. The phosphorylation status of each kinase was analyzed by Phos-tag SDS-PAGE. The kinase activities were also investigated. In the eukaryotic systems, multiple phosphorylated forms of the expressed kinases were observed. In the rabbit reticulocyte lysate system and 293 cells, differences in phosphorylation status between the wild-type and kinase-dead mutants were observed. Whether the expressed kinase was active depended on the properties of both the kinase and each expression system. In the prokaryotic systems, Src and Hck were expressed in autophosphorylated active forms. Clear differences in post-translational phosphorylation among the protein expression systems were revealed. These results provide useful information for preparing functional proteins regulated by phosphorylation.

Details

Title
Characterization of Phosphorylation Status and Kinase Activity of Src Family Kinases Expressed in Cell-Based and Cell-Free Protein Expression Systems
Author
Kinoshita-Kikuta, Emiko 1   VIAFID ORCID Logo  ; Kinoshita, Eiji 2   VIAFID ORCID Logo  ; Suga, Misaki 3 ; Higashida, Mana 3 ; Yamane, Yuka 3 ; Nakamura, Tomoka 3 ; Koike, Tohru 1 

 Department of Functional Molecular Science, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan; [email protected] 
 Department of Human Nutrition, Faculty of Human Sciences, Hiroshima Bunkyo University, Hiroshima 732-0295, Japan; [email protected] 
 School of Pharmaceutical Science, Hiroshima University, Hiroshima 734-8553, Japan; [email protected] (M.S.); [email protected] (M.H.); [email protected] (Y.Y.); [email protected] (T.N.) 
First page
1448
Publication year
2021
Publication date
2021
Publisher
MDPI AG
e-ISSN
2218273X
Source type
Scholarly Journal
Language of publication
English
ProQuest document ID
2584327163
Copyright
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.