High-Efficiency and Robust Expression System for Stable Isotope-Labeled Peptides

Kohno, Toshiyuki; Liu, Xiang; Inaoka, Yoshihiko; Hayashi, Kazuhiko; Suzuki, Chisa; Kusunoki, Hideki; Tanaka, Takeshi; Sugai, Mariko; Sato, Kazuki; Wakamatsu, Kaori

doi:10.1007/s10989-008-9125-4

Cited by 4 publications

(4 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The HBx(101–136) and HBx(101–120) constructs were expressed in BL21(DE3) pLysS cells using a ubiquitin fusion system. ,− For the expression of the unlabeled sample, cells were grown in LB medium. The purification protocol described previously was followed for both samples .…”

Section: Methodsmentioning

confidence: 99%

“…The purification protocol described previously was followed for both samples . His 10 -Ub-HBx(101–136) and His 10 -Ub-HBx(101–120) were solubilized in denaturant, either 6 M urea or 6 M guanidine hydrochloride, and purified using a His-accept column (Nacalai Tesque Inc., Kyoto, Japan) or a Ni-NTA agarose column (FUJIFILM-Wako Pure Chemical Corporation, Osaka, Japan) with the on-column refolding method. ,, Subsequently, HBx(101–136) and HBx(101–120) were cleaved from the fusion protein using yeast ubiquitin hydrolase at 37 °C and further purified using a Resource Q column (Cytiva, MA, USA) followed by a Hiprep Sephacryl S-200 column (Cytiva, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Structural Insights into the Interaction between the C-Terminal-Deleted BH3-like Motif Peptide of Hepatitis B Virus X Protein and Bcl-x_L

Kusunoki,

Sakamoto,

Kobayashi

et al. 2024

Biochemistry

Self Cite

View full text Add to dashboard Cite

Hepatitis B virus X protein (HBx) plays a crucial role in the development of hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) infection. The full-length HBx protein interacts with Bcl-x L and is involved in the HBV replication and cell death processes. The three hydrophobic residues Trp120, Leu123, and Ile127 of the HBx BH3-like motif are essential for the Bcl-x L -binding. On the other hand, various lengths of C-terminal-truncated HBx mutants are frequently detected in HCC tissues, and these mutants, rather than the full-length HBx, appear to be responsible for HCC development. Notably, the region spanning residues 1−120 of HBx [HBx(1 and 120)] has been strongly associated with an increased risk of HCC development. However, the mode of interaction between HBx(1−120) and Bcl-x L remains unclear. HBx(1−120) possesses only Trp120 among the three hydrophobic residues essential for the Bcl-x L -binding. To elucidate this interaction mode, we employed a C-terminal-deleted HBx BH3-like motif peptide composed of residues 101−120. Here, we present the NMR complex structure of Bcl-x L and HBx(101−120). Our results demonstrate that HBx(101−120) binds to Bcl-x L in a weaker manner. Considering the high expression of Bcl-x L in HCC cells, this weak interaction, in conjunction with the overexpression of Bcl-x L in HCC cells, may potentially contribute to HCC development through the interaction between C-terminal-truncated HBx and Bcl-x L .

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Structural Insights into the Interaction between the C-Terminal-Deleted BH3-like Motif Peptide of Hepatitis B Virus X Protein and Bcl-x_L

Kusunoki,

Sakamoto,

Kobayashi

et al. 2024

Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Uniformly 15 N‐labeled yeast ubiquitin was expressed and purified, as described , and concentrated in buffer A [25 mM sodium phosphate, pH 6.5, 50 mM NaCl, 10% D 2 O, and 0.1 mM 4,4‐dimethyl‐4‐silapentane‐1‐sulfonate sodium salt (DSS)] by ultrafiltration using Amicon Ultra‐5 (Millipore, Billerica, MA, USA) at 4 °C. NDSB‐195 was purchased from Merck (Tokyo, Japan).…”

Section: Methodsmentioning

confidence: 99%

Protein stabilizer, NDSB-195, enhances the dynamics of the β₄-α₂loop of ubiquitin

et al. 2016

Self Cite

View full text Add to dashboard Cite

Non-detergent sulfobetaines (NDSBs) are a new group of small, synthetic protein stabilizers, which have advantages over classical compatible osmolytes, such as polyol, amines, and amino acids: they do not increase solution viscosity, unlike polyols, and they are zwitterionic at all pH ranges, unlike amines and amino acids. NDSBs also facilitate the crystallization and refolding of proteins. The mechanism whereby NDSBs exhibit such activities, however, remains elusive. To gain insight into this mechanism, we studied, using nuclear magnetic resonance (NMR), the effects of dimethylethylammonium propane sulfonate (NDSB-195) on the dynamics of ubiquitin, on which a wealth of information has been accumulated. By analyzing the line width of amide proton resonances and the transverse relaxation rates of nitrogen atoms, we found that NDSB-195 enhances the microsecond-millisecond dynamics of a β4 -α2 loop of ubiquitin. Although those compounds that enhance protein dynamics are generally considered to destabilize protein molecules, NDSB-195 enhanced the stability of ubiquitin against guanidium chloride denaturation. Thus, the simultaneous enhancement of stability and flexibility by a single compound can be attained.

show abstract

“…Hence, it is necessary to test several fusion strategies in search for a highly efficient protocol that works for a given peptide. Several different proteins have been described in the literature as fusion tags for peptide production [18] , [19] . We tested glutathion S-transferase (GST), the immunoglobulin-binding domain of streptococcal protein G (GB1), and yeast ubiquitin as fusion partners of NS4A(1-48) in our quest for an effective production strategy of this peptide in E. coli .…”

Section: Introductionmentioning

confidence: 99%

Recombinant Production of the Amino Terminal Cytoplasmic Region of Dengue Virus Non-Structural Protein 4A for Structural Studies

et al. 2014

View full text Add to dashboard Cite

BackgroundDengue virus (DENV) is a mosquito-transmitted positive single strand RNA virus belonging to the Flaviviridae family. DENV causes dengue fever, currently the world's fastest-spreading tropical disease. Severe forms of the disease like dengue hemorrhagic fever and dengue shock syndrome are life-threatening. There is no specific treatment and no anti-DENV vaccines. Our recent data suggests that the amino terminal cytoplasmic region of the dengue virus non-structural protein 4A (NS4A) comprising amino acid residues 1 to 48 forms an amphipathic helix in the presence of membranes. Its amphipathic character was shown to be essential for viral replication. NMR-based structure-function analysis of the NS4A amino terminal region depends on its milligram-scale production and labeling with NMR active isotopes.Methodology/Principal FindingsThis report describes the optimization of a uniform procedure for the expression and purification of the wild type NS4A(1-48) peptide and a peptide derived from a replication-deficient mutant NS4A(1-48; L6E, M10E) with disrupted amphipathic nature. A codon-optimized, synthetic gene for NS4A(1-48) was expressed as a fusion with a GST-GB1 dual tag in E. coli. Tobacco etch virus (TEV) protease mediated cleavage generated NS4A(1-48) peptides without any artificial overhang. Using the described protocol up to 4 milligrams of the wild type or up to 5 milligrams of the mutant peptide were obtained from a one-liter culture. Isotopic labeling of the peptides was achieved and initial NMR spectra were recorded.Conclusions/SignificanceSmall molecules targeting amphipathic helices in the related Hepatitis C virus were shown to inhibit viral replication, representing a new class of antiviral drugs. These findings highlight the need for an efficient procedure that provides large quantities of the amphipathic helix containing NS4A peptides. The double tag strategy presented in this manuscript answers these needs yielding amounts that are sufficient for comprehensive biophysical and structural studies, which might reveal new drug targets.

show abstract

High-Efficiency and Robust Expression System for Stable Isotope-Labeled Peptides

Cited by 4 publications

References 33 publications

Structural Insights into the Interaction between the C-Terminal-Deleted BH3-like Motif Peptide of Hepatitis B Virus X Protein and Bcl-x_L

Structural Insights into the Interaction between the C-Terminal-Deleted BH3-like Motif Peptide of Hepatitis B Virus X Protein and Bcl-x_L

Protein stabilizer, NDSB-195, enhances the dynamics of the β₄-α₂loop of ubiquitin

Recombinant Production of the Amino Terminal Cytoplasmic Region of Dengue Virus Non-Structural Protein 4A for Structural Studies

Contact Info

Product

Resources

About

High-Efficiency and Robust Expression System for Stable Isotope-Labeled Peptides

Cited by 4 publications

References 33 publications

Structural Insights into the Interaction between the C-Terminal-Deleted BH3-like Motif Peptide of Hepatitis B Virus X Protein and Bcl-xL

Structural Insights into the Interaction between the C-Terminal-Deleted BH3-like Motif Peptide of Hepatitis B Virus X Protein and Bcl-xL

Protein stabilizer, NDSB-195, enhances the dynamics of the β4-α2loop of ubiquitin

Recombinant Production of the Amino Terminal Cytoplasmic Region of Dengue Virus Non-Structural Protein 4A for Structural Studies

Contact Info

Product

Resources

About

Structural Insights into the Interaction between the C-Terminal-Deleted BH3-like Motif Peptide of Hepatitis B Virus X Protein and Bcl-x_L

Structural Insights into the Interaction between the C-Terminal-Deleted BH3-like Motif Peptide of Hepatitis B Virus X Protein and Bcl-x_L

Protein stabilizer, NDSB-195, enhances the dynamics of the β₄-α₂loop of ubiquitin