Expression and purification of selenoprotein M
Date
2014
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
The unusual amino acid selenocysteine (Sec) contains the component Se-lenium (Se), which is an essential element that can be utilized by organisms throughout the three-domain system. Sec is also involved in the redox reaction as a constituent of selenoproteins. The specific functions of numerous human selenoproteins remain unknown; among them is the focus of this thesis, Seleno-protein M.(SelM). ☐ In order to determine the structure and function of SelM, our objective was to express SelM in E.coli. An engineered Sec-tRNAUTu was used to successfully insert Sec residue into SelM. To increase the protein stability, solubility and yield, a maltose binding protein (MBP) was used for SelM expression. The second chapter details the tests of two protein factors involved in Sec incorporation for co-expression with SelM. SelM was expressed in E. coli BL21 (DE3) and MH5 competent cells. The expression level was analyzed by 16% Tris-glycine SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE), and Sec incorporation was evaluated by mass spectrometry. ☐ To further optimize the SelM expression, variables that affect cell growth and induction were tested. As the third chapter reports, these variables included the different ingredients of medium and induction conditions. The highest SelM expression level was achieved by co-expression with 2 SelA/PSTK in MH5 competent cells. In the condition of growing in LB medium with induction for 16 hours, the yield and Sec incorporation for SelM was around 0.6 mg/L and 50%, respectively.
Description
Keywords
Expression, Selenoprotein, Seleno-protein M