Plates were washed four times with 200 l of PBST/well. clinical use or clinical trials MLT-748 are derived from the wild-type (WT) sequence (21) and employ the full-length viral spike as the primary antigen. In recent months, several viral variants of concern (VOC) have emerged. Current VOC are B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), and B.1.617.2 (delta). Both B.1.351 and B.1.617.2 show substantially decreased neutralization by many existing monoclonal antibodies and by convalescent as well as vaccine sera (22C24). The B.1.617.2 variant is currently driving a worldwide resurgence in infections (25C27). Thus, there is still a need for inexpensive, rapidly producible, and highly efficacious vaccines against VOC, which preferably do not require low-temperature storage. We recently showed that both monomeric and intermolecular disulfide-linked, trimeric RBD derivatives were highly thermotolerant with the latter showing improved immunogenicity (28), albeit with significant antibody titers against the trimerization domain. Earlier studies in other systems have shown that improving thermostability can enhance immunogenicity (29C31). In the present work, we use second-site, saturation suppressor mutagenesis (SSSM) (32, 33) to isolate multiple single-site and multisite stabilized RBD derivatives that were expressed in high yield in mammalian cell culture. The principle of SSSM is as follows. We have previously shown in MLT-748 the context of unstable proteins displayed on the yeast surface that the relative amount of properly folded mutant proteins displayed on the yeast surface correlates with the thermal stability of the corresponding purified mutant measured (34). However, once the stability crosses a certain threshold, further PRKD3 stability increases are not accompanied by increased binding on the yeast surface; hence, it is challenging to isolate mutants MLT-748 with higher stability than the wild type from single-site saturation mutagenesis (SSM) libraries using this approach. To overcome this, a destabilizing mutant (termed as parent inactive mutant or PIM) can be introduced into all members of the mutant library (32, 33) and suppressors isolated ( Figures?1ACD ). A significant fraction of these suppressors is found to be stabilizing even in the wild-type background. Open in a separate window Figure?1 Stabilized mutant identification using second-site saturation suppressor mutagenesis (SSSM). Schematic representation of second-site saturation suppressor mutagenesis. Proteins exist in an equilibrium between folded and unfolded states. (A) Generally, in the case of WT proteins, equilibrium is shifted toward the folded conformation. Such proteins show high levels of folded expressed protein when expressed on the yeast cell surface and bound to their cognate partner. However, upon introduction of a (B) parent inactive mutation (PIM), the equilibrium is shifted toward the unfolded state and the extent of equilibrium shift will be determined by the destabilizing effect of the PIM. Such PIMs show a lower level of expressed as well as folded proteins on the yeast cell surface. (C) The equilibrium between folded and unfolded states of the PIM can be shifted toward the folded state if a suppressor mutation is introduced. Such double mutants show higher levels of folded expressed proteins on yeast cell surface compared to the PIM alone. (D) Such suppressor mutants generally have a higher amount of folded fraction at the equilibrium than the WT protein. However, the amount of expressed protein on the yeast cell surface will be similar to WT. Normalized MFI of suppressor mutant (E) expression and (F) binding of individually analyzed putative suppressors in the background of the PIM. The MFI of double mutants was normalized with the MFI of PIM, and a twofold cutoff was used to differentiate between putative suppressors and non-suppressor mutations. (G) Tm of purified single mutants identified from suppressor analysis, measured by nano-DSF. The mutants were categorized into stabilized, WT-like, and destabilized mutants indicated by light gray, dark gray, and back bars, respectively. Binding MFI of double mutants relative to PIM (E) robustly identifies stabilizing suppressors. (H) Tm of multi-mutants which were.