Generation of antibodies to an extracellular region of the transporters Glut1/Glut4 by immunization with a designed antigen.

Monoclonal antibodies are one of the fastest growing class of drugs. Nevertheless, relatively few biologics target multispanning membrane proteins because of technical challenges. To target relatively small extracellular regions of multiple membrane-spanning proteins, synthetic peptides, which are composed of amino acids corresponding to an extracellular region of a membrane protein, are often utilized in antibody discovery. However, antibodies to these peptides often do not recognize parental membrane proteins. In this study, we designed fusion proteins in which an extracellular helix of the membrane protein glucose transporter 1 (Glut1) was grafted onto the scaffold protein Adhiron. In the initial design, the grafted fragment did not form a helical conformation. Molecular dynamics simulations of full-length Glut1 suggested the importance of intramolecular interactions formed by surrounding residues in the formation of the helical conformation. A fusion protein designed to maintain such intramolecular interactions did form the desired helical conformation in the grafted region. We then immunized an alpaca with the designed fusion protein and obtained VHH (variable region of heavy-chain antibodies) using the phage display method. The binding of these VHH antibodies to the recombinant Glut1 protein was evaluated by surface plasmon resonance, and their binding to Glut1 on the cell membrane was further validated by flow cytometry. Furthermore, we also succeeded in the generation of a VHH against another integral membrane protein, glucose transporter 4 (Glut4) with the same strategy. These illustrates that our combined biochemical and computational approach can be applied to designing other novel fusion proteins for generating site-specific antibodies.

Biophysical insight into protein-protein interactions in the Interleukin-11/Interleukin-11Rα/glycoprotein 130 signaling complex.

Interleukin-11 (IL-11) is a member of the interleukin-6 (IL-6) family of cytokines. IL-11 is a regulator of multiple events in hematopoiesis, and IL-11-mediated signaling is implicated in inflammatory disease, cancer, and fibrosis. All IL-6 family cytokines signal through the signal-transducing receptor, glycoprotein 130 (gp130), but these cytokines have distinct as well as overlapping biological functions. To understand IL-11 signaling at the molecular level, we performed a comprehensive interaction analysis of the IL-11 signaling complex, comparing it with the IL-6 complex, one of the best-characterized cytokine complexes. Our thermodynamic analysis revealed a clear difference between IL-11 and IL-6. Surface plasmon resonance analysis showed that the interaction between IL-11 and IL-11 receptor α (IL-11Rα) is entropy driven, whereas that between IL-6 and IL-6 receptor α (IL-6Rα) is enthalpy driven. Our analysis using isothermal titration calorimetry revealed that the binding of gp130 to the IL-11/IL-11Rα complex results in entropy loss, but that the interaction of gp130 with the IL-6/IL-6Rα complex results in entropy gain. Our hydrogen-deuterium exchange mass spectrometry experiments suggested that the D2 domain of gp130 was not involved in IL-6-like interactions in the IL-11/IL-11Rα complex. It has been reported that IL-6 interaction with gp130 in the signaling complex was characterized through the hydrophobic interface located in its D2 domain of gp130. Our findings suggest that unique interactions of the IL-11 signaling complex with gp130 are responsible for the distinct biological activities of IL-11 compared to IL-6.

Addition of arginine hydrochloride and proline to the culture medium enhances recombinant protein expression in Brevibacillus choshinensis: The case of RBD of SARS-CoV-2 spike protein and its antibody.

Brevibacillus choshinensis is a gram-positive bacterium that is known to efficiently secrete recombinant proteins. However, the expression of these proteins is often difficult depending upon the expressed protein. In this study, we demonstrated that the addition of arginine hydrochloride and proline to the culture medium dramatically increased protein expression. By culturing bacterial cells in 96-well plates, we were able to rapidly examine the expression conditions and easily scale up to 96 mL of culture for production. Although functional expression of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein without any solubility-enhancing tag in bacterial strains (including Escherichia coli) has not been reported to date, we succeeded in efficiently producing RBD which showed a similar CD spectrum to that of RBD produced by eukaryotic cell expression systems. Furthermore, RBD from the omicron variant (B.1.1.529) was also produced. Physicochemical analyses indicated that omicron RBD exhibited markedly increased instability compared to the wild-type. We also revealed that the Fab format of the anti-SARS-CoV-2 antibody C121 can be produced in large quantities using the same expression system. The obtained C121 Fab bound to wild-type RBD but not to omicron RBD. These results strongly suggest that the Brevibacillus expression system is useful for facilitating the efficient expression of proteins that are difficult to fold and will thus contribute to the rapid physicochemical evaluation of functional proteins.

Hyperthin nanochains composed of self-polymerizing protein shackles.

Protein fibrils are expected to have applications as functional nanomaterials because of their sophisticated structures; however, nanoscale ordering of the functional units of protein fibrils remains challenging. Here we design a series of self-polymerizing protein monomers, referred to as protein shackles, derived from modified recombinant subunits of pili from Streptococcus pyogenes. The monomers polymerize into nanochains through spontaneous irreversible covalent bond formation. We design the protein shackles so that their reactions can be controlled by altering redox conditions, which affect disulphide bond formation between engineered cysteine residues. The interaction between the monomers improves their polymerization reactivity and determines morphologies of the polymers. In addition, green fluorescent protein-tagged protein shackles can polymerize, indicating proteins can be stably attached to the nanochains with its functionality preserved. Furthermore we demonstrate that a molecular-recognizable nanochain binds to its partner with an enhanced binding ability in solution. These characteristics are expected to be applied for novel protein nanomaterials.