Highly efficient expression and purification system of small-size protein domains in Escherichia coli for biochemical characterization

Bao, Wen Jing; Gao, Yong-Guang; Chang, Yong Gang; Zhang, Tie Ying; Lin, Xiao; Yan, Xian Zhong; Hu, Hong Yu

doi:10.1016/j.pep.2005.11.021

Cited by 64 publications

(61 citation statements)

References 16 publications

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“…We used PCR to amplify different lengths of cDNA coding for human 4E-BP1 (residues 1-118, 17-118, 35-87, 44-118, 44-87, 44-84, 44-63, 51-64, and 64-87) and subcloned them into a pH-GB1 Escherichia coli expression vector. 4E-BP1 fragments were inserted such that they are expressed as fusion proteins with a hexahistidine, a protein G B1 domain solubility enhancement tag (GB1) (44), and a TEV cleavage site at their N terminus, generating pH-GB1-Tev-4E-BP1 constructs. We also subcloned the above-described constructs of 4E-BP1 into a pcDNA3-eGFP [enhanced green fluorescent protein (eGFP)] mammalian expression vector using HindIII-XhoI restriction sites (Addgene plasmid 13031).…”

Section: Methodsmentioning

confidence: 99%

“…To study the importance of the C-terminal loop in the inhibition of translation initiation, we expressed GFP-fusion proteins comprising full-length 4E-BP1 (4E-BP1 FL ), 4E-BP1 , 4E-BP1 [44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63] (including M1), or 4E-BP1 64-87 (including M2 and M3), in mammalian cells. m 7 GTP pull-down experiments showed that 4E-BP1 FL and 4E-BP1 44-87 both bound eIF4E and disrupted its interaction with eIF4G (Fig.…”

Section: The C-terminal Loop Of 4e-bp1 Is Required To Inhibit Cap-depmentioning

confidence: 99%

“…3C). Cells expressing 4E-BP1 [44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63] or 4E-BP1 64-87 fragments progressed through the cell cycle just like cells expressing WT GFP. Although 4E-BP1 FL reduced cell cycle progression to G2 by twofold, it did not completely arrest cells in the G1 phase as observed with 4E-BP1 44-87 fragment.…”

Section: The C-terminal Loop Of 4e-bp1 Is Required To Inhibit Cap-depmentioning

confidence: 99%

“…4EGI-1 therefore displaces a short 4E-BP1 peptide (4E-BP1 51-64 ) but not a long fragment of 4E-BP1 (4E-BP1 ) . We conducted the same 4EGI-1 titration experiments as described above, but where eIF4E is 15 N-labeled. We observed that the addition of 4EGI-1 to the 15 N-eIF4E/4E-BP1 15 N-eIF4E/4E-BP1 [44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63] complex (devoid of the C-terminal loop) caused severe peak broadenings in the eIF4E spectrum ( Fig. S8 A and B).…”

Section: The C-terminal Loop Of 4e-bp1 Is Required To Inhibit Cap-depmentioning

confidence: 99%

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Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1

Sekiyama

Arthanari

Papadopoulos

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

111

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The eIF4E-binding protein (4E-BP) is a phosphorylation-dependent regulator of protein synthesis. The nonphosphorylated or minimally phosphorylated form binds translation initiation factor 4E (eIF4E), preventing binding of eIF4G and the recruitment of the small ribosomal subunit. Signaling events stimulate serial phosphorylation of 4E-BP, primarily by mammalian target of rapamycin complex 1 (mTORC1) at residues T 37 /T 46 , followed by T 70 and S 65 . Hyperphosphorylated 4E-BP dissociates from eIF4E, allowing eIF4E to interact with eIF4G and translation initiation to resume. Because overexpression of eIF4E is linked to cellular transformation, 4E-BP is a tumor suppressor, and up-regulation of its activity is a goal of interest for cancer therapy. A recently discovered small molecule, eIF4E/eIF4G interaction inhibitor 1 (4EGI-1), disrupts the eIF4E/eIF4G interaction and promotes binding of 4E-BP1 to eIF4E. Structures of 14-to 16-residue 4E-BP fragments bound to eIF4E contain the eIF4E consensus binding motif, 54 YXXXXLΦ 60 (motif 1) but lack known phosphorylation sites. We report here a 2.1-Å crystal structure of mouse eIF4E in complex with m 7 GTP and with a fragment of human 4E-BP1, extended C-terminally from the consensus-binding motif (4E-BP1 50-84 ). The extension, which includes a proline-turn-helix segment (motif 2) followed by a loop of irregular structure, reveals the location of two phosphorylation sites (S 65 and T 70 ). Our major finding is that the C-terminal extension (motif 3) is critical to 4E-BP1-mediated cell cycle arrest and that it partially overlaps with the binding site of 4EGI-1. The binding of 4E-BP1 and 4EGI-1 to eIF4E is therefore not mutually exclusive, and both ligands contribute to shift the equilibrium toward the inhibition of translation initiation.translation initiation | phosphorylation | protein-protein interaction inhibitor | structure

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Section: Methodsmentioning

confidence: 99%

Section: The C-terminal Loop Of 4e-bp1 Is Required To Inhibit Cap-depmentioning

confidence: 99%

Section: The C-terminal Loop Of 4e-bp1 Is Required To Inhibit Cap-depmentioning

confidence: 99%

Section: The C-terminal Loop Of 4e-bp1 Is Required To Inhibit Cap-depmentioning

confidence: 99%

See 2 more Smart Citations

Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1

Sekiyama

Arthanari

Papadopoulos

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

111

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“…The mouse FBP11 gene was from Fulengen. The cDNAs for the tandem WW domains (residues 91-178, and renumbered as 3-90) and the two single domains (3-43, 52-84) were subcloned into pGBTNH (14) and pGEX-4T-3. Mutation of PYYY to TYYY in WW2 was produced according to the corresponding sequence of WW1 and denoted as P67T for the sake of structural analysis.…”

Section: Methodsmentioning

confidence: 99%

Interaction with Polyglutamine-expanded Huntingtin Alters Cellular Distribution and RNA Processing of Huntingtin Yeast Two-hybrid Protein A (HYPA)

Jiang

Xia

Yuan

et al. 2011

Journal of Biological Chemistry

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Strategies to Optimize Protein Expression in E. coli

2010

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Recombinant protein expression in Escherichia coli (E. coli) is simple, fast, inexpensive, and robust, with the expressed protein comprising up to 50 percent of the total cellular protein. However, it also has disadvantages. For example, the rapidity of bacterial protein expression often results in unfolded/misfolded proteins, especially for heterologous proteins that require longer times and/or molecular chaperones to fold correctly. In addition, the highly reductive environment of the bacterial cytosol and the inability of E. coli to perform several eukaryotic post-translational modifications results in the insoluble expression of proteins that require these modifications for folding and activity. Fortunately, multiple, novel reagents and techniques have been developed that allow for the efficient, soluble production of a diverse range of heterologous proteins in E. coli. This overview describes variables at each stage of a protein expression experiment that can influence solubility and offers a summary of strategies used to optimize soluble expression in E. coli.

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Highly efficient expression and purification system of small-size protein domains in Escherichia coli for biochemical characterization

Cited by 64 publications

References 16 publications

Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1

Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1

Interaction with Polyglutamine-expanded Huntingtin Alters Cellular Distribution and RNA Processing of Huntingtin Yeast Two-hybrid Protein A (HYPA)

Strategies to Optimize Protein Expression in E. coli

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