Solution structure of Gaussia Luciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR

Wu, Nan; Kobayashi, Naohiro; Tsuda, Kazuhiko; Unzai, Satoru; Saotome, Tomonori; Kuroda, Yutaka; Yamazaki, Toshio

doi:10.1038/s41598-020-76486-4

Cited by 36 publications

(36 citation statements)

References 42 publications

(70 reference statements)

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“…Analytical RP-HPLC is a relatively fast and reliable method for analyzing the formation of SS bonds [40,41], and the difference in the retention time can resolve different disulfide bonding patterns in a multi-disulfide bonded protein [16,40,41]. The RP-HPLC elution profiles of the oxidized RBD-C9R expressed in the supernatant of T7 shuffle showed a single peak, suggesting that all of the protein had folded into a single species of S-S bond pairing.…”

Section: Discussionmentioning

confidence: 99%

A Multi-Disulfide Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike Protein Expressed in E. coli Using a SEP-Tag Produces Antisera Interacting with the Mammalian Cell Expressed Spike (S1) Protein

Brindha

Kuroda

2022

IJMS

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An Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 (isolate Wuhan-Hu-1) spike protein would significantly accelerate the search for anti-COVID-19 therapeutics because of its versatility and low cost. However, RBD contains four disulfide bonds and its expression in E. coli is limited by the formation of aberrant disulfide bonds resulting in inclusion bodies. Here, we show that a solubility-enhancing peptide (SEP) tag containing nine arginine residues (RBD-C9R) attached at the C-terminus can overcome this problem. The SEP-tag increased the expression in the soluble fraction and the final yield by five times (2 mg/L). The folding properties of the E. coli expressed RBD-C9R were demonstrated with biophysical characterization using RP-HPLC, circular dichroism, thermal denaturation, fluorescence, and light scattering. A quartz crystal microbalance (QCM) analysis confirmed the binding activity of RBD-C9R with ACE2, the host cell’s receptor. In addition, RBD-C9R elicited a Th-2 immune response with a high IgG titer in Jcl: ICR mice. The RBD-C9R antisera interacted with both itself and the mammalian-cell expressed spike protein (S1), as demonstrated by ELISA, indicating that the E. coli expressed RBD-C9R harbors native-like epitopes. Overall, these results emphasize the potential of our SEP-tag for the E.coli production of active multi-disulfide-bonded RBD.

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Section: Discussionmentioning

confidence: 99%

A Multi-Disulfide Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike Protein Expressed in E. coli Using a SEP-Tag Produces Antisera Interacting with the Mammalian Cell Expressed Spike (S1) Protein

Brindha

Kuroda

2022

IJMS

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“…ANSURR shows this region is highly flexible and so does not support the existence of the helix. However, 15 N relaxation dispersion and 1 H- 15 N heteronuclear NOE data suggest this region could potentially transiently adopt secondary structure 14 . Given that chemical shifts represent a population-weighted average, it seems an -helix in this position would not comprise the dominant conformation in solution, as suggested previously 4 .…”

Section: Target T1027mentioning

confidence: 96%

The accuracy of protein structures in solution determined by AlphaFold and NMR

Fowler

Williamson

2022

Preprint

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In the recent CASP (Critical Assessment of Structure Prediction) competition, AlphaFold2 performed outstandingly. Its worst predictions were for NMR structures, which has two alternative explanations: either the NMR structures were poor, implying that AlphaFold may be more accurate than NMR; or there is a genuine difference between crystal and solution structures. Here, we use the program ANSURR, which measures the accuracy of solution structures, and show that one of the NMR structures was indeed poor. We then compare AlphaFold predictions to NMR structures, and show that AlphaFold tends to be more accurate than NMR ensembles, in particular correctly more rigid in loops. There are however some cases where the NMR ensembles are more accurate. These tend to be dynamic structures where AlphaFold had low confidence. We suggest that AlphaFold could be used as the model for NMR structure refinements, and that AlphaFold structures validated by ANSURR require no further refinement.

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“…SEP tag has emerged as a reliable and versatile technique for controlling protein solubility [13,22]. In particular, we have shown their solubilization properties for bovine pancreatic trypsin inhibitor (BPTI) [23], dengue envelope protein [24], anti-epidermal growth factor receptor (EGFR)-ScFv [25], tobacco etch virus (TEV) protease [26], Gaussia luciferase (GLuc) [27,28] and the third domain of EGFR [29]. Here we assessed the effect of arginine and lysine tags to control protein solubility under thermal stress.…”

Section: Introductionmentioning

confidence: 99%

Anti-EGFR VHH Antibody under Thermal Stress Is Better Solubilized with a Lysine than with an Arginine SEP Tag

et al. 2021

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In this study, we assessed the potential of arginine and lysine solubility-enhancing peptide (SEP) tags to control the solubility of a model protein, anti-EGFR VHH-7D12, in a thermally denatured state at a high temperature. We produced VHH-7D12 antibodies attached with a C-terminal SEP tag made of either five or nine arginines or lysines (7D12-C5R, 7D12-C9R, 7D12-C5K and 7D12-C9K, respectively). The 5-arginine and 5-lysine SEP tags increased the E. coli expression of VHH-7D12 by over 80%. Biophysical and biochemical analysis confirmed the native-like secondary and tertiary structural properties and the monomeric nature of all VHH-7D12 variants. Moreover, all VHH-7D12 variants retained a full binding activity to the EGFR extracellular domain. Finally, thermal stress with 45-minute incubation at 60 and 75 °C, where VHH-7D12 variants are unfolded, showed that the untagged VHH-7D12 formed aggregates in all of the four buffers, and the supernatant protein concentration was reduced by up to 35%. 7D12-C5R and 7D12-C9R did not aggregate in Na-acetate (pH 4.7) and Tris-HCl (pH 8.5) but formed aggregates in phosphate buffer (PB, pH 7.4) and phosphate buffer saline (PBS, pH 7.4). The lysine tags (either C5K or C9K) had the strongest solubilization effect, and both 7D12-C5K and 7D12-C9K remained in the supernatant. Altogether, our results indicate that, under a thermal stress condition, the lysine SEP tags solubilization effect is more potent than that of an arginine SEP tags, and the SEP tags did not affect the structural and functional properties of the protein.

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Solution structure of Gaussia Luciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR

Cited by 36 publications

References 42 publications

A Multi-Disulfide Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike Protein Expressed in E. coli Using a SEP-Tag Produces Antisera Interacting with the Mammalian Cell Expressed Spike (S1) Protein

A Multi-Disulfide Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike Protein Expressed in E. coli Using a SEP-Tag Produces Antisera Interacting with the Mammalian Cell Expressed Spike (S1) Protein

The accuracy of protein structures in solution determined by AlphaFold and NMR

Anti-EGFR VHH Antibody under Thermal Stress Is Better Solubilized with a Lysine than with an Arginine SEP Tag

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