N-terminus deletion affecting the preparation of soluble cyanobacterial glutaredoxin in <Emphasis Type="Italic">Escherichia coli</Emphasis> |
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Authors: | Min Li Qing Yang Yang Gao Qingyu Wu |
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Institution: | (1) Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, 100084, P. R. China;(2) Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian, 116023, P. R. China |
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Abstract: | Glutaredoxin has been implicated in maintenance of a normal cellular thiol/disufide ratio and the regeneration of oxidatively
damaged proteins. In order to obtain more information about these important regulatory proteins in cyanobacteria, we have
previously cloned and expressed the first cyanobacterial glutaredoxin gene ssr2061 in Escherichia coli. In this work, the second glutaredoxin gene slr1562 was studied. About 90% of Grx2061 coded by ssr2061 was produced in a soluble form while 90% of Grx1562 coded by slr1562 was found in inclusion bodies. To improve the production of soluble Grx1562, we constructed two mutants: Grx1562NC with cysteines
in conserved site substituted by serines, and Grx1562M with N-terminus hydrophobic region deletion. Only the latter mutant
was successfully expressed in soluble form with increased glutaredoxin activity and showed less sensitivity in oxidative stress.
Spectroscopic analysis shows that the structure of Grx1562M with less hydrophobic nature could give more opportunity for protein
solubility and could improve the substrate catalytic efficiency. These results suggest that hydrophobic N-terminus determines
the insolubility of Grx1562 and may provide another strategy for increasing expression level of soluble heterologous proteins
in E. coli.
Published in Russian in Biokhimiya, 2007, Vol. 72, No. 3, pp. 383–391. |
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Keywords: | inclusion body glutaredoxin N-terminus deletion Synechocystis sp PCC 6803 |
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