[Mild solubilization, purification, and immunoreactivity characterization of VP1 inclusion body protein of serotype O foot-and-mouth disease virus based on sodium lauroyl sarcosinate].
Aug 2026· Sheng wu gong cheng xue bao = Chinese journal of biotechnology· Vol 42 8, pp.
3541-3549
· 0 citations
Medicine
TL;DR
The LSN-based mild solubilization and chromatographic purification process developed in this study successfully circumvents the destructive effects and complex refolding steps associated with conventional strong denaturants, and the prepared recombinant His-VP1 protein possesses the potential to serve as a high-quality serological diagnostic antigen.
Human noroviruses (HuNoVs) are one of the main causative agents of non-bacterial gastroenteritis. The viral capsid protein VP1 is the main target for diagnostics. Although eukaryotic expression systems can produce correctly assembled VP1, they are associated with high costs and complexity. The Escherichia coli prokaryotic expression system is attractive for large-scale expression; however, VP1 often aggregates into insoluble inclusion bodies in conventional strains (BL21 (DE3)). The ArcticExpress (DE3) strain co-expresses psychrophilic chaperonins to improve protein folding at low temperatures enhancing the soluble yield of difficult-to-express targets. In this study, we demonstrated that E. coli ArcticExpress (DE3) under low-temperature induction enabled the high-yield production of soluble full-length VP1 for HuNoVs GII.4 and GII.17. In BL21 (DE3), the mentioned antigens were aggregated into insoluble inclusion bodies. Using ArcticExpress(DE3), VP1 yield was identified as approximately 18-93 mg/L across the tested genotypes. In BL21 (DE3), the protein outcome was genotype-dependent: GII.4 could be recovered only via insoluble-fraction processing, GII.17 remained undetectable or present only at trace levels under the conditions tested. The ArcticExpress-derived viral antigen retained antigenic properties and was recognized by cross-reactive anti-GII antibodies. To our knowledge, this study provides evidence that ArcticExpress (DE3) enable the soluble production of full-length HuNoVs VP1 suitable for downstream immunochemical analysis.
I. Kazlouski, N. Paklonskaya, I. Belskaya et al.· FEMS Microbiology Letters· 0 citations
The receptor-binding domain (RBD) of SARS-CoV-2 spike glycoprotein is central to serological diagnostics and vaccine development, but production in eukaryotic expression systems is costly and technically demanding. Here, we describe an Escherichia coli platform to express the wild-type RBD, used as an initial model for the standardization of the methodology. Subsequently, the platform was applied to the alpha and beta variants, demonstrating its adaptability to different RBD variants. Inclusion bodies were purified under denaturing conditions and refolded by size-exclusion chromatography using a linear urea gradient and a redox/anti-aggregation buffer that promotes correct disulfide-bond formation and minimizes aggregation. The procedure yielded ∼36 mg of soluble protein per liter of culture. The refolded recombinant RBDs of wild-type, alpha and beta variants, as well as RDB wild-type produced in Expi293 cells as a control were evaluated in ELISA assays against sera from healthy, vaccinated and convalescent SARS-CoV-2 patients. All recombinant RBDs produced in E. coli were recognized by patients' sera, demonstrating that they retain antigenicity and supporting their use in serological assays as a rapid and scalable source of antigens.
Daniela Roa-Velázquez, J. Filisola-Villaseñor, D. I. Zavala-Vargas et al.· Protein Expression and Purif...· 0 citations
The heterologous expression of Bacillus thuringiensis (Bt) Cry toxins in Escherichia coli is frequently hindered by the formation of insoluble inclusion bodies, which severely limits subsequent functional and structural characterizations. In this study, we report an optimized in vitro solubilization and purification protocol utilizing a highly alkaline carbonate buffer (pH 11.3) to efficiently recover Cry toxins. Compared to the conventional method, this optimized strategy significantly enhanced the solubilization efficiency, increasing the yields of Cry1Ab and Cry1Fa by approximately 3.6-fold and 2.75-fold, respectively. Furthermore, the method proved highly effective for the structurally divergent, Coleopteran-specific Cry8Ea1 toxin. Following trypsin activation and size-exclusion chromatography, the purified toxins exhibited high purity and successfully retained their native biological functions. Specifically, purified Cry1Ab and Cry1Fa demonstrated potent receptor-dependent cytotoxicity against Sf9 cells heterologously expressing the BmABCC2 receptor, alongside distinct pore-forming activity in artificial liposomes. Utilizing the high-quality purified Cry8Ea1, we further investigated its oligomerization potential in membrane-mimetic environments, capturing specific early-stage assemblies-including detergent-induced "cloverleaf-shaped" trimers and nanodisc-reconstituted "wedge-shaped" dimers-via negative-stain electron microscopy. Overall, this highly efficient and versatile purification paradigm successfully overcomes the technical bottleneck of inefficient inclusion body solubilization during the in vitro preparation of Cry toxins, thereby laying a reliable material foundation for downstream functional applications and high-resolution structural characterization of their pore-forming mechanisms.
Zi-Bo Cui, Lian-Lian Cui, Kai-Yue Wang et al.· Protein Expression and Purif...· 0 citations
Findings support the feasibility of the multi-epitope construct and provide a foundation for future investigations of its immunogenicity and protective efficacy studies in animal models.
Nadálin Yandra Botton, Guilherme Feijó de Sousa, Marina Sturbelle Garcia et al.· Journal of Biotechnology· 0 citations
Introduction: The box jellyfish Chironex fleckeri is a toxin-producing species whose cytotoxic and neurotoxic effects have been well demonstrated in living organisms. Due to the presence of various bioactive proteins within its toxin, acute exposure can lead to temporary paralysis or even death under severe conditions. This study aimed to solubilize and evaluate the immunogenicity of the engineered SUMO-tagged recombinant c-CfTXB protein as an antigen, by comparing its immune response to the untagged form in animal model.
Methods: The StXB gene fragment was excised from the pET-28aSUMO-c-CfTXB-StXB vector using enzymatic digestion. Subsequently, the recombinant proteins SUMO-c-CfTXB and c-CfTXB were expressed in competent prokaryotic hosts following IPTG induction, and purified through Ni-NTA affinity chromatography. Protein identity was confirmed by Western blot analysis. Following immunization of mouse groups, antibody titers were measured using indirect ELISA, and protective efficacy was evaluated through a toxin challenge. Data analysis was performed using One-way ANOVA combined with survival analysis.
Results: Successful protein expression and separation were confirmed by SDS-PAGE and Western blot analysis, demonstrating that the SUMO tag enhanced the solubility of the target protein. In the toxin challenge test, only the vaccinated groups survived (100%) due to a significant increase in antibody titers (Pvalue c-CfTXB›0.0001 and Pvalue SUMO-c-CfTXB›0.0003).
Conclusion: Fusion of the SUMO tag to the engineered c-CfTXB protein improved its folding, expression yield, purification efficiency, and solubility. These findings suggest that the successful immunogenicity of the antigen in the mouse model may pave the way for the design of recombinant vaccines to neutralize box jellyfish venom.
Mehdi Bakhshian, H. Honari, H. S. Abianeh et al.· Journal of Shahid Sadoughi U...· 0 citations
Mycobacterium tuberculosis
is extremely dependent upon lipid-hydrolyzing enzymes to utilize the resources of host lipids and survive within the cell. Rv1063c is a conserved hypothetical protein predicted to share sequence homology with the patatin-like family.
In the present study, the
rv1063c
gene was cloned and heterologously expressed in Escherichia coli BL21 (DE3). Functional enzyme was obtained by solubilizing inclusion bodies in 8 mol·L
−1
urea, refolding via gradient dialysis, and purifying through Ni-affinity chromatography.
Bioinformatic homology analysis indicates Rv1063c is homologous to members of the patatin-like family containing a putative Ser52-Asp166 catalytic dyad, though this residue pair has not been experimentally validated in the present study. The recombinant Rv1063c was mainly expressed as inclusion bodies without significant soluble cytoplasmic expression. Biochemical assays demonstrated the recombinant protein exclusively hydrolyzes medium-chain pnitrophenyl esters (C8–12), with maximum hydrolytic activity toward p-NP-C8 (caprylate), followed by C10 and C12, and exhibited the highest activity at 40 °C and pH 7.0. The enzyme exhibited moderate catalytic efficiency but poor thermal stability. All catalytic data in this paper were obtained from artificial p-nitrophenyl ester substrates, which cannot fully reflect natural lipid substrate preference of mycobacteria in vivo.
This work biochemically characterizes Rv1063c as a medium-chain-specific esterase encoded by a protein homologous to the patatin superfamily. Its physiological and pathogenic roles remain undetermined, and further multi-level experiments are required to clarify its biological functions.
Yuming Song, Songsong Dong, Rumeng Zhai et al.· Frontiers in Microbiology· 0 citations
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