Abstract
Low-cost bacterial production of the receptor binding domain (RBD) of the SARS-CoV-2 Omicron spike protein holds significant potential in expediting the development of therapeutics against COVID-19. However, RBD contains eight cysteines forming four disulfide bonds, and expression in E. coli using standard protocols produces insoluble RBD forming non-native disulfide bonds. Here, we expressed RBD in E. coli T7 SHuffle with high aeration, which enhanced disulfide formation in the cytoplasm and reshuffling of non-native disulfide bonds, and at a low temperature of 16°C, which stabilized the native conformation and thus the formation of the native disulfide bonds. The yield of RBD was as high as 3 mg per 200 mL culture. We analyzed the conformational and biophysical properties of our E. coli-expressed RBD. First, the RP-HPLC elution profile indicated a single peak, suggesting that RBD was folded with a single disulfide bond pairing pattern. Next, circular dichroism analysis indicated a secondary structure content very close to that computed from the crystal structure. RBD’s thermal denaturation monitored by CD was cooperative, strongly indicating a well-folded protein structure. Moreover, limited proteolysis showed that RBD was nearly as stable as RNase A, and the formation of native disulfide bonds was confirmed by LC-MS analysis. Furthermore, BLI analysis indicated a strong binding of RBD with the hACE2 with a dissociation constant of 0.83 nM, confirming the folded nature of RBD. Altogether, these results demonstrate that our E. coli-expression system can provide a large amount of highly purified RBD with correct disulfide bonds and native-like biochemical and biophysical properties.
Significance
Producing RBD in E. coli is difficult due to its four cysteines, which can lead to non-native bonds resulting in insoluble proteins. Here, we address these issues by expressing RBD in E. coli T7 SHuffle at a low temperature with high aeration. We achieved large quantities of highly purified RBD without fusion tags. This study represents the first comprehensive analysis of E. coli-expressed RBD, including carefully verifying the disulfide bond pairing by LC-MS analysis, validating its native-like biochemical and biophysical properties.
Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped, positive-sense RNA virus responsible for causing COVID-19 (coronavirus disease 2019) [1]. Initially reported in Wuhan, China, the disease quickly escalated into a global pandemic in early 2020. Currently, SARS-CoV-2 has infected over 750 million people and caused more than 6.8 million deaths globally [2].
Over the pandemic, SARS-CoV-2 has generated numerous variants. Among major variants, several strains are classified by the World Health Organization (WHO) as ‘variant of concerns (VOC)’ with Alpha, Beta, Gamma, Delta, and Omicron. Omicron has become the predominant variant and is classified as a VOC, while the others have been reclassified to ‘previous VOC’ [3]. First found in South Africa in November 2021 (BA.1), this omicron variant differs from other strains that mainly infect the lung. The Omicron variant primarily infects cells in the upper respiratory tract, which expresses fewer ACE2 receptors, presumably because the Omicron RBD binds to the ACE2 more strongly than the Wuhan strain [4]. Omicron BA.4/BA.5 is notable in escaping FDA-approved monoclonal antibodies developed against the original strain [5,6].
The spike protein (S protein) of SARS-CoV-2 plays a significant role in the infection of the host cell [7,8]. Especially the receptor binding domain (RBD), spanning residues Arg319–Phe541 of the S protein, is critical for cell entry [9], and it binds to the human angiotensin-converting enzyme 2 (hACE2). After binding, the transmembrane serine protease type II (TMPRSS2), expressed on the host cell, activates the S protein leading to a conformational change and allowing the virus into the cells [10,11]. Epitope mapping indicates that 90% of neutralizing antibodies target RBD [12], and hence, RBD is a promising target for developing therapeutics against SARS-CoV-2.
The RBD is a 25 kDa protein domain made of a twisted five-stranded antiparallel β sheet, a two-stranded antiparallel β sheet, and five helices. RBD is thus small and is readily expressed in E. coli, the system of choice for expressing small proteins [13]. However, RBD contains nine cysteines, eight forming four disulfide bonds [14], making it challenging to refold into its native state after expression in E. coli. Namely, the reducing environment of the E. coli cytoplasm often results in misfolded disulfide bond proteins [15,16]. To solve this issue, we expressed the RBD protein at a low temperature of 16°C with high aeration using the E. coli T7 SHuffle strain, which is engineered to enhance and reshuffle disulfide bonds in its cytoplasm [17]. Here, we report the first E. coli-expression of the SARS-CoV-2 Omicron BA.5 variant’s RBD without any fusion tag and having native-like properties.
Materials and Methods
Plasmid Construction, Protein Expression, and Purification
A DNA sequence encoding SARS-CoV-2 RBD Omicron BA.5 was designed with His-tag at the N-terminal for protein purification. The synthesized gene was inserted into pET15b at the NdeI/BamH1 with ampicillin resistance (Supplementary Figure S1a). The RBD was expressed in E. coli T7 SHuffle cells and purified from inclusion bodies as explained elsewhere [18]. The RBD proteins tagged by 6-Histidines were purified using denaturing open nickel-nitrilotriacetic acid (Ni-NTA) (Wako, Japan) chromatography. The proteins were further purified using reversed-phase (RP)-HPLC. The purified RBD were lyophilized and stored at –30°C.
Spectroscopic Measurements
The measurements were carried out under the condition of 10 mM Hepes buffer (pH 6.5) at a protein concentration of 0.3 mg/mL. The spectroscopic measurements: Far-UV circular dichroism (CD), Tryptophan fluorescence, Dynamic light scattering (DLS), and Static light scattering (SLS) were carried out, in essence, as reported previously [18].
Analytical Ultracentrifugation
The sedimentation coefficients were determined by sedimentation velocity experiments using an Optima XL-I analytical ultracentrifuge (Beckman-Coulter) with AN-50 Ti analytical 8-place titanium rotor. RBD samples were dialyzed overnight against 10 mM MES pH 6.5 and diluted to 0.3 mg/mL. The experiments were carried out at 4°C with a rotor speed of 50,000 rpm. Data were collected using SEDFIT software [19]. The sedimentation coefficient was fitted and calculated using the continuous sedimentation coefficient distribution model [c(s)]. The c(s) distribution was converted into c(M), a molar mass distribution. Solvent density, viscosity, and protein partial specific volumes were calculated using SEDNTERP software [20].
Limited Proteolysis
Limited proteolysis of E. coli-expressed SARS-CoV-2 Omicron BA.5 RBD was assessed by digesting 0.2 mg/mL (8.2 μM) folded state (oxidized form) and unfolded state (reduced form) of Omicron BA.5 RBD by 20 ng/mL of pepsin in 10 mM acetate buffer at pH 3.5. The folded RBD protein solution was prepared by centrifuging dissolved protein in MQ at 20000×g 4°C for 20 min and filtered through a 0.2 μm filter. The unfolded (reduced form) of RBD protein was prepared by incubating the RBD protein solution with 0.3 M dithiothreitol (DTT) for 2 h. To eliminate DTT and keep RBD in reduced form by 15 h dialysis against 1 L of 2 mM DTT in reverse osmosis (RO) water with two times changes of outer solution. The digestion mixing solutions were incubated for 30, 60, and 120 minutes at 37°C. We aliquoted the sample after each incubation period and immediately stopped the reaction by mixing it with loading buffer containing β-mercaptoethanol, then heating it at 95°C for 3 minutes. The proteolysis monitored by SDS-PAGE gel and band intensities were analyzed using ATTO Image Analysis Software (CS Analyzer 4). The comparison was assessed by limited proteolysis of RNase A (RNase A from Bovine pancreas) at the same molarity of 8.2 μM (0.12 mg/mL) digested by 20 ng/mL of pepsin. The sample was prepared the same as the RBD sample.
Liquid Chromatography-Mass Spectrometry (LC-MS)
We performed the trypsin digestion of RBD by incubating 250 μL of the sample at 37°C for 24 hours in 50 mM Tris-HCl pH 7.0 with 0.3 mg/mL of RBD, 0.03 mg/mL of trypsin, 2 M of urea and 1 mM of CaCl2. After the incubation, 100 μL of the sample was aliquoted for the LC-MS measurement. The reduced RBD sample was prepared by the same preparation of limited proteolysis and trypsin digestion was performed using the same condition of oxidized RBD. LC-MS analysis was performed using a Shimadzu LCMS-8040 triple quadruple mass spectrometer (Kyoto, Japan) equipped with a Shimadzu model LC-20AD liquid chromatography system (Kyoto, Japan). Chromatography was done by binary gradient system with 0.1% formic acid (A) and methanol (B). The sample was loaded onto an Intrada (IMTAKT Corp, Kyoto) 5WP-RP column equilibrated with 0.1% formic acid with a flow rate of 0.2 mL/min. The injection volume was 5 μL.
Binding Activity by Bio-Layer Interferometry
The RBD binding affinity with hACE2 (purity >90% SDS-PAGE) (Bioworld Tech, St Louis Park, Minnesota, USA) was measured using an Octet-N1 Bio-Layer Interferometry (BLI) (Sartorius, Goettingen, Germany). A 5 μg/mL concentration of SARS-CoV-2 RBD was immobilized on the Ni-NTA biosensor surface for 180 s. The baseline interference phase was measured for 30 s in a kinetics buffer (KB: 10 mM Hepes pH 7.4 and 0.005% Tween-20). Then, the sensors were subjected to association phase immersion for 300 s in wells containing recombinant hACE2 diluted in KB. Then, the sensors were immersed in KB for up to 300 s in the dissociation step. The RBD binding affinities for hACE2 were calculated from global fitting with a 1:1 Langmuir binding model.
The unfolded RBD sample was prepared by incubating 0.3 mg/mL of RBD with 6 M urea in 10 mM Hepes buffer for 2 hours, followed by 0.3 M DTT for 2 h incubation. The protein sample was dialyzed for 15 h against 1 L of 2 mM DTT in RO water with two times changes of the outer solution.
Results and Discussion
Expression and Purification
E. coli is a powerful expression system for producing small to mid-size proteins. It is cost-effective, high-yield, and fast [13]. E. coli would be ideal for expressing Omicron RBD, but the reducing environment in the cytoplasm of the standard E. coli strain hinders disulfide bond formation, resulting in the misfolding of multi-disulfide bonded proteins. Here, we used E. coli T7 SHuffle, in which the reduction pathway genes (TrxB and gor) are suppressed, allowing for the formation of disulfide bonds within the cytoplasm [17]. Furthermore, the DsbC (disulfide bond isomerase) gene assists in the formation of correct disulfide bonds [21]. Another essential element of our strategy is the low expression temperature of 16°C, which stabilizes the native structure and consequently favors the formation of native disulfide bonds [22,23]. An alternative effect of a low expression temperature is that it decreases the protein production speed, providing time for the protein to fold [24].
We optimized RBD’s expression and purification protocols to maximize the production of RBD with native disulfide bonds. A small-scale 5-mL expression indicated that a substantial amount of RBD was expressed in the inclusion body 16–18 h after induction with IPTG, as observed by SDS-PAGE analysis (Supplementary Figure S1b). We hypothesize that the cysteines are not fully oxidized, leading to expression in the precipitate. The 200 mL large-scale expression also showed that the RBD was predominantly expressed in the pellet fraction (Supplementary Figure S1c). The insoluble fractions were solubilized with 6 M GnHCl. RBD was purified by denaturing Ni-NTA chromatography and RP-HPLC. We assessed the disulfide bond formation during the air oxidation process at pH 8.8, 25°C, utilizing RP-HPLC. The RP-HPLC elution profile of non-oxidized RBD exhibited a broad peak (Supplementary Figure S2a), implying the formation of mixed disulfide bond patterns. In contrast, by subjecting the RBD to oxidation over varying time intervals, the broad peak collapsed to a sharp single peak. We observed a full oxidation state at 72 h as monitored by analytical RP-HPLC (Supplementary Figure S2b). After RP-HPLC purification, the elution profile showed a single sharp peak (Figure 1a), indicating that the purified RBD formed a single and most likely native species of disulfide bond pairing [18,25–27]. The elution time of the fully oxidized and reduced RBD differed by about 2 minutes (Supplementary Figure S3). MALDI-TOF MS measurements confirmed the identity of the RBD (Supplementary Figure S4). The final yield of SARS-CoV-2-RBD after RP-HPLC was 3 mg for a 200 mL culture.
The E. coli-expressed RBD conformational structure was characterized using several spectroscopic techniques. The secondary structures of the RBD were assessed by far-UV CD spectroscopy. The secondary structure content of the RBD computed using BeStSel [28] was 29.8% (26.9%) of β-sheet, and α-helix content was 9.9% (9.1%) (Supplementary Figure S5) in line with the crystal structure [PDB ID: 7ZXU] (values in the parentheses indicate the X-ray structure values) [29]. CD spectra from 25°C to 70°C indicated a reversible thermal denaturation (Figure 1b). An S-shaped thermal denaturation curve was observed with a melting temperature (Tm) of 53.6°C monitored by CD at 222 nm, which showed the cooperative reversible [30], indicating a well-folded structure (Figure 1c).
The tertiary structures of the RBD were assessed by fluorescence spectroscopy. The tryptophan fluorescence spectrum intensity of the RBD exhibited a peak with a maximum at 342 nm. When the sample was heated to 70°C, we observed a 5 nm redshift (347 nm) [31] and a 2.5-fold decrease in fluorescence intensity, likely to originate from quenching with water molecules accessing the chromophores [32]. The changes were reversible (Figure 1d), in line with observations reported for small proteins undergoing thermal unfolding and refolding.
Particle Size and Solubility
The particle size and the solubility of the RBD were assessed by SLS and DLS, respectively. SLS measures the intensity of scattered light, which is affected when protein aggregates [33]. The SLS intensities remained low between 25°C and 37°C (Figure 1e), indicating the absence of any aggregation, and DLS analysis revealed a hydrodynamic radius [Rh] ranging from 2 to 3 nm (Figure 1f), consistent with a monomeric RBD of 24 kDa [34]. Further, analytical ultracentrifugation (AUC) indicated that all of the RBD had a molecular weight of 24.41 kDa, close to the theoretical value of 24.3 kDa (Figure 1g). These results confirmed the monomeric form of the RBD.
Biochemical Stability
Well-folded proteins are stable against proteolysis degradation [35–37]. The biochemical stability of our RBD was evaluated using limited proteolysis with pepsin digestion [38]. As a control, we used RNase A, a stably folded protein [39,40], and the unfolded (reduced form) of Omicron RBD.
All fragments of digested RBD produced through pepsin digestion, calculated using PeptideCutter [41] (Supplementary Figure S6), are under 2 kDa in size, making them undetectable through SDS PAGE gel (15% Acrylamide) analysis. In Figure 2a, the oxidized RBD display bands corresponding to digested fragments at approximately 15 kDa and 6–7 kDa that are larger than the calculated fragment sizes. The large size of the digested fragments suggests incomplete digestion by pepsin, potentially attributed to the robust stability of the oxidized RBD’s structure.
The undigested fractions were determined by quantifying the SDS band intensities and setting the initial incubation (0 min) band intensity to 100%. The results showed that 55% of RBD and 57% of RNase remained undigested after 120 min. (Figures 2a and b).
In contrast, the unfolded RBD underwent complete digestion within 60 minutes, displaying faint bands that aligned with the position of oxidized RBD fragments. This observation suggests that the reduced RBD was entirely digested by pepsin. The oxidized RBD was significantly more resistant to proteolysis than the reduced one. This is most probably because the disulfide bonds in the reduced RBD are cleaved, unfolding the protein and increasing the flexibility of the peptide backbone resulting in the exposure of the proteolytic sites [42,43]. This result thus suggests that the proteolytic stability of the oxidized RBD arises from its native-like structure.
Disulfide Bond Determination
The native disulfide bond pairing of RBD was characterized by limited proteolysis using trypsin, followed by liquid chromatography-mass spectrometry (LC-MS) analysis. Our RBD contains eight cysteines that could form over a hundred disulfide bond pairing patterns. The molecular weight of the native pairing pattern digested fragments was calculated with all cysteines oxidized, forming intramolecular disulfide bonds. The LC-MS spectra exhibited all of the peaks corresponding to the calculated mass of the fragments containing the native disulfide bonds [44] (Figures 3a and b). As a control, we assessed that these peaks disappeared upon the addition of DTT for reducing the cysteines, from the LC-MS spectra at the same retention time (Figures 3a and c). These results indicate that the disulfide bond pairings of the E. coli-expressed RBD are identical to that observed in the crystal structure [PDB ID: 7ZXU, 29].
Binding of RBD Omicron BA.5 to hACE2
The hACE2 binding activity of our E. coli-expressed RBD was measured using a Bio-Layer Interferometry. The strength of binding affinity was measured through interaction with different concentrations of hACE2. The dissociation constant (KD) of RBD-hACE2 was 0.83 nM, calculated using the global fitting to all concentrations of ACE2 (Figure 4a), suggesting that RBD retains the necessary conformation required for its receptor recognition. A KD of 0.83 nM indicates a strong interaction comparable with mammalian-expressed S protein and ACE2 [45]. Furthermore, we investigated the specificity of the RBD/ACE2 binding by comparing it to the binding with ovalbumin, an unrelated protein. The binding between RBD and ovalbumin was extremely weak, even at a high equimolar concentration of >200 nM for both ovalbumin and RBD (see Supplementary Figure S7). Additionally, the unfolded RBD showed no or negligible binding activity with hACE2 (Figure 4b), emphasizing the importance of the conformation for recognition by its receptor, further confirming the folded conformation of E. coli-expressed RBD.
Conclusion
Following our strategy, we obtained up to 3 mg/200 mL culture of E. coli-produced SARS-CoV-2-RBD Omicron BA. 5 protein without any fusion tags. The RBD exhibited native-like biochemical and biophysical properties and formed native disulfide bonds. This strategy can be adapted to a wide range of multi-disulfide bond proteins, enabling rapid and low-cost production.
Conflict of Interest
The author declares no conflicts of interest.
Author Contributions
RW, SB, and YK designed the project. RW, SB, and YK wrote the manuscript. RW, SB, TY, and KM performed the experiments. RW, SB, and SO analyzed and compiled the data. All authors read and approved the manuscript.
Data Availability
The evidence data generated and analyzed during the current study are available from the corresponding author on reasonable request.
Acknowledgments
We thank Prof. Jeremy R. H. Tame for the analytical ultracentrifugation analysis, Dr. Seketsu Fukuzawa for the LC-MS analysis, Mr. Zhirui Cheng for graphics, and all members of Kuroda’s laboratory for advice and discussion.
References
- [1] Coronaviridae study group of the international committee on taxonomy of viruses. The species severe acute respiratory syndrome-related coronavirus: Classifying 2019-nCoV and naming it SARS-CoV-2. Nat. Microbiol. 5, 536–544 (2020). https://doi.org/10.1038/s41564-020-0695-z
- [2] World health organization COVID-19 dashboard. https://covid19.who.int/ (accessed on 19 February 2023).
- [3] World health organization tracking SARS-CoV-2 variants. https://www.who.int/activities/tracking-SARS-CoV-2-variants/ (accessed on 19 February 2023).
- [4] Geng, Q., Shi, K., Ye, G., Zhang, W., Aihara, H., Li, F. Structural basis for human receptor recognition by SARS-CoV-2 Omicron variant BA.1. J. Virol. 96, e00249-22 (2022). https://doi.org/10.1128/jvi.00249-22
- [5] Tegally, H., Moir, M., Everatt, J., Giovanetti, M., Scheepers, C., Wilkinson, E., et al. Emergence of SARS-CoV-2 Omicron lineages BA.4 and BA.5 in South Africa. Nat. Med. 28, 1785–1790 (2022). https://doi.org/10.1038/s41591-022-01911-2
- [6] Takashita, E., Yamayoshi, S., Simon, V., van Bakel, H., Sordillo, E. M., Pekosz, A., et al. Efficacy of antibodies and antiviral drugs against Omicron BA.2.12.1, BA.4, and BA.5 subvariants. N. Engl. J. Med. 387, 468–470 (2022). https://doi.org/10.1056/NEJMc2207519
- [7] Dai, L., Gao, G. F. Viral targets for vaccines against COVID-19. Nat. Rev. Immunol. 21, 73–82 (2021). https://doi.org/10.1038/s41577-020-00480-0
- [8] Salvatori, G., Luberto, L., Maffei, M., Aurisicchio, L., Roscilli, G., Palombo, F., et al. SARS-CoV-2 spike protein: An optimal immunological target for vaccines. J. Transl. Med. 18, 222 (2020). https://doi.org/10.1186/s12967-020-02392-y
- [9] Tai, W., He, L., Zhang, X., Pu, J., Voronin, D., Jiang, S., et al. Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell. Mol. Immunol. 17, 613–620 (2020). https://doi.org/10.1038/s41423-020-0400-4
- [10] Astuti, I., Ysrafil. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): An overview of viral structure and host response. Diabetes. Metab. Syndr. 14, 407–412 (2020). https://doi.org/10.1016/j.dsx.2020.04.020
- [11] Shang, J., Wan, Y., Luo, C., Ye, G., Geng, Q., Auerbach., A., et al. Cell entry mechanisms of SARS-CoV-2. Proc. Natl. Acad. Sci. U.S.A. 117, 11727–11734 (2021). https://doi.org/10.1073/pnas.2003138117
- [12] Piccoli, L., Park, Y. J., Tortorici, M. A., Czudnochowski, N., Walls, A. C., Beltramello, M., et al. Mapping neutralizing and immunodominant sites on the SARS-CoV-2 spike receptor-binding domain by structure-guided high-resolution serology. Cell 183, 1024–1042 (2020). https://doi.org/10.1016/j.cell.2020.09.037
- [13] Yin, J., Li, G., Ren, X., Herrler, G. Select what you need: a comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes. J. Biotechnol. 127, 335–347 (2007). https://doi.org/10.1016/j.jbiotec.2006.07.012
- [14] Lan, J., Ge, J., Yu, J., Shan, S., Xhou, H., Fan, S., et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 581, 215–220 (2020). https://doi.org/10.1038/s41586-020-2180-5
- [15] Bessette, P. H., Aslund, F., Beckwith, J., Georgiou, G. Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc. Natl. Acad. Sci. U.S.A. 96, 13703–13708 (1999). https://doi.org/10.1073/pnas.96.24.13703
- [16] Rathnayaka, T., Tawa, M., Nakamura, T., Sohya, S., Kuwajima, K., Yohda, M., et al. Solubilization and folding of a fully active recombinant Gaussia luciferase with native disulfide bonds by using a SEP-Tag. Biochim. Biophys. Acta Proteins Proteom. 1814, 1775–1778 (2011). https://doi.org/10.1016/j.bbapap.2011.09.001
- [17] Lobstein, J., Emrich, C. A., Jeans, C., Faulkner, M., Riggs, P., Berkmen, M. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microb. Cell Fact. 11, 753 (2012). https://doi.org/10.1186/1475-2859-11-56
- [18] Brindha, S., Yoshizue, T., Wongnak, R., Takemae, H., Oba, M., Mizutani, T., et al. An Escherichia coli expressed multi-disulfide bonded SARS-CoV-2 RBD shows native-like biophysical properties and elicits neutralizing antisera in a mouse model. Int. J. Mol. Sci. 23, 15744 (2022). https://doi.org/10.3390/ijms232415744
- [19] Schuck, P. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys. J. 78, 1606–1619 (2000). https://doi.org/10.1016/S0006-3495(00)76713-0
- [20] Laue, T. M. Computer-aided interpretation of analytical sedimentation data for proteins. analytical ultracentrifugation in biochemistry and polymer science. pp. 90–125 (R. Soc. Chem, Cambridge, 1992).
- [21] Bushweller, J. H. Protein disulfide exchange by the intramembrane enzymes DsbB, DsbD, and CcdA. J. Mol. Biol. 432, 5091–5103 (2020). https://doi.org/10.1016/j.jmb.2020.04.008
- [22] Vasina, J. A., Baneyx, F. Expression of aggregation-prone recombinant proteins at low temperatures: A comparative study of the Escherichia coli cspA and tac promoter systems. Protein Expr. Purif. 9, 211–218 (1997). https://doi.org/10.1006/prep.1996.0678
- [23] Rathnayaka, T., Tawa, M., Sohya, S., Yohda, M., Kuroda, Y. Biophysical characterization of highly active recombinant Gaussia luciferase expressed in Escherichia coli. Biochim. Biophys. Acta Proteins Proteom. 1804, 1902–1907 (2010). https://doi.org/10.1016/j.bbapap.2010.04.014
- [24] Chen, X., Li, C., Liu, H. Enhanced recombinant protein production under special environmental stress. Front. Microbiol. 12, 630814 (2021). https://doi.org/10.3389/fmicb.2021.630814
- [25] Weissman, S. J, Kim, S. P. Reexamination of the folding of BPTI: Predominance of native intermediates. Science 253, 1386–1393 (1991). https://doi.org/10.1126/science.1716783
- [26] Brindha, S., Kuroda, Y. 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. Int. J. Mol. Sci. 23, 1703 (2022). https://doi.org/10.3390/ijms23031703
- [27] Argentinian AntiCovid Consortium. Structural and functional comparison of SARS-CoV-2-spike receptor binding domain produced in Pichia pastoris and mammalian cells. Sci. Rep. 10, 21779 (2020). https://doi.org/10.1038/s41598-020-78711-6
- [28] Micsonai, A., Wien, F., Bulyáki É., Kun, J., Moussong, É., Lee, Y. H., et al. BeStSel: A web server for accurate protein secondary structure prediction and fold recognition from the circular dichroism spectra. Nucleic Acids. Res. 46, W315–W322 (2018). https://doi.org/10.1093/nar/gky497
- [29] Tuekprakhon, A., Nutalai, R., Dijokaite-Guraliuc, A., Zhou, D., Ginn, H. M., Selvaraj, M., et al. Antibody escape of SARS-CoV-2 Omicron BA.4 and BA.5 from vaccine and BA.1 serum. Cell 185, 2422–2433 (2022). https://doi.org/10.1016/j.cell.2022.06.005
- [30] Malhotra, P., Udgaonkar, J. B. How cooperative are protein folding and unfolding transitions? Protein Sci. 25, 1924–1941 (2016). https://doi.org/10.1002/pro.3015
- [31] Lehrer, S. S. Solute perturbation of protein fluorescence. Quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion. Biochemistry 10, 3254–3263 (1971). https://doi.org/10.1021/bi00793a015
- [32] Vivian, J. T., Callis, P. R. Mechanisms of tryptophan fluorescence shifts in proteins. Biophys. J. 80, 2093–2109 (2001). https://doi.org/10.1016/S0006-3495(01)76183-8
- [33] Waters Wyatt Technology. Light Scattering Methodologies Developed for the Characterisation of Protein Aggregate Composition. AzoM (2023). https://www.azom.com/article.aspx?ArticleID=10365
- [34] Nobbmann, Ulf. Characterization down to Nanometers: Light Scattering from Proteins and Micelles. Mesoscale Phenomena in Fluid Systems. Chapter 4, pp. 44–59 (2003). https://doi.org/10.1021/bk-2003-0861.ch004
- [35] Parsell, D. A., Sauer, R. T. The structural stability of a protein is an important determinant of its proteolytic susceptibility in Escherichia coli. J. Biol. Chem. 264, 7590–7595 (1989).
- [36] Fontana, A., Polverino de Laureto, P., De Filippis, V., Scaramella, E., Zambonin, M. Probing the partly folded states of proteins by limited proteolysis. Fold. Des. 2, R17–R26 (1997). https://doi.org/10.1016/S1359-0278(97)00010-2
- [37] Miki, Y., Endo, S., Giga-Hama, Y., Tanji, M., Wada, A. Probing stability and dynamics of proteins by protease digestion. II: Identification of the initial chymotryptic cleavage sites of homologous cytochromes c. J. Biomol. Struct. Dyn. 6, 1–21 (1988). https://doi.org/10.1080/07391102.1988.10506479
- [38] Kabir, Md. G., Islam, M. M., Kuroda, Y. Reversible association of proteins into sub-visible amorphous aggregates using short solubility controlling peptide tags. Biochim. Biophys. Acta Proteins Proteom. 1866, 366–372 (2018). https://doi.org/10.1016/j.bbapap.2017.09.012
- [39] Neira, J. L., Rico, M. Folding studies on ribonuclease A, a model protein. Fold. Des. 2, R1–R11 (1997). https://doi.org/10.1016/S1359-0278(97)00001-1
- [40] Gagné, D., Doucet, N. Structural and functional importance of local and global conformational fluctuations in the RNase A superfamily. FEBS. J. 280, 5596–5607 (2013). https://doi.org/10.1111/febs.12371
- [41] Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M. R., Appel R. D., et al. Protein Identification and Analysis Tools on the Expasy Server. in The Proteomics Protocols Handbook (John, M. Walker ed.) (Copyright Humana Press, 2005).
- [42] Minde, D. P., Maurice, M. M., Rüdiger, G. D. Determining biophysical protein stability in lysates by a fast proteolysis assay, FAST pp. PLoS One 7, 0046147 (2012). https://doi.org/10.1371/journal.pone.0046147
- [43] Arnold, U., Köditz, J., Markert, Y., Ulbrich-Hofmann, R. Local fluctuations vs. global unfolding of proteins investigated by limited proteolysis. Biocatal. Biotransformation 23, 159–167 (2005). https://doi.org/10.1080/10242420500183287
- [44] Wu, N., Kobayashi, N., Tsuda, K., Unzai, S., Saotome, T., Kuroda, Y., et al. Solution structure of Gaussia Luciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR. Sci. Rep. 10, 20069 (2020). https://doi.org/10.1038/s41598-020-76486-4
- [45] Wang, Q., Iketani, S., Li, Z., Guo, Y., Yey, A. Y., Liu, M., et al. Antigenic characterization of the SARS-CoV-2 Omicron subvariant BA.2.75. Cell Host Microbe 30, 1512–1517 (2022). https://doi.org/10.1016/j.chom.2022.09.002
