Recombinant Glutamine Synthetase (GS) from <Emphasis Type="Italic">C. glutamicum</Emphasis> Existed as Both Hexamers &; Dedocamers and C-terminal His-tag Enhanced Inclusion Bodies Formation in <Emphasis Type="Italic">E. coli</Emphasis> |
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Authors: | Ye Li Guang Yang Xing Huang Bo Ye Ming Liu Zhanglin Lin Chun Li Zhu-an Cao |
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Institution: | (1) Department of Chemical Engineering, Tsinghua University, Beijing, 100084, People’s Republic of China;(2) School of Life Science and Technology, Beijing Institute of Technology, Beijing, 100081, People’s Republic of China; |
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Abstract: | In order to investigate the effect of his-tag on glutamine synthetase (GS, EC 6.3.1.2) from Corynebacterium glutamicum, recombinant Escherichia coli strains overexpressing GSIM, HGSIM (GS fused with N-terminal his-tag), GSIMH (GS fused with C-terminal his-tag), and HGSIMH
(GS fused with N-terminal & C-terminal his-tags) were constructed, respectively. Under similar expression conditions, GSIM
and HGSIM were partially solubly expressed; no soluble GSIMH and HGSIMH were observed, based on the result of SDS-PAGE. Gel
filtration of purified soluble HGSIM showed that hexamers and dedocamers coexisted in the quaternary structure of GS from
C. glutamicum. Combined this result with the analysis of two GS crystal structure models, we hypothesized that C-terminal residues participated
in GS folding after translation on ribosome. After the folding process, C-terminal residues were released again and exposed
to solvent. Fused C-terminal his-tag interrupted the GS to fold into its correct conformation so that inclusion bodies formed. |
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