title: Recombinant glutamine synthetase (GS) from C. glutamicum existed as both hexamers & dedocamers and C-terminal His-tag enhanced inclusion bodies formation in E. coli.
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.
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, People's Republic of China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Li
authors.forename: Guang
authors.initials: G
authors.lastname: Yang
authors.forename: Xing
authors.initials: X
authors.lastname: Huang
authors.forename: Bo
authors.initials: B
authors.lastname: Ye
authors.forename: Ming
authors.initials: M
authors.lastname: Liu
authors.forename: Zhanglin
authors.initials: Z
authors.lastname: Lin
authors.forename: Chun
authors.initials: C
authors.lastname: Li
authors.forename: Zhu-an
authors.initials: ZA
authors.lastname: Cao
mesh_terms: D002850:Chromatography, Gel; D048230:Corynebacterium glutamicum / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D005974:Glutamate-Ammonia Ligase