Stabilizing proteins through saturation suppressor mutagenesis

Abstract

While there have been recent, transformative advances in the area of protein structure prediction, prediction of point mutations that improve protein stability remains challenging. It is possible to construct and screen large mutant libraries for improved activity or ligand binding, however reliable screens for mutants that improve protein stability do not exist, especially for proteins that are well folded and relatively stable. We demonstrate that incorporation of a single, specific destabilizing, (parent inactive) mutation into each member of a single-site saturation mutagenesis library followed by screening for suppressors, allows for robust and accurate identification of stabilizing mutations. When coupled to FACS sorting of a yeast surface display library of the bacterial toxin CcdB, followed by deep sequencing of sorted populations, multiple stabilizing mutations could be identified after a single round of sorting. Multiple libraries with different parent inactive mutations could be pooled and simultaneously screened to further enhance the accuracy of identification of stabilizing mutations. Individual stabilizing mutations could be combined to result in a multimutant with increase in thermal melting temperature of about 20 degrees Celsius and enhanced tolerance to high temperature exposure. The method employs small library sizes and can be readily extended to other display and screening formats to rapidly isolate stabilized protein mutants.

A Stabilized, Monomeric, Receptor Binding Domain Elicits High-Titer Neutralizing Antibodies Against All SARS-CoV-2 Variants of Concern

“…and Ahmed et al. ( 32 , 33 ). We generated an SSSM library of RBD containing the V512A PIM (parent inactive mutation) and displayed it on the yeast cell surface.…”

“…It has previously been shown in the context of HIV-1 antigens that stabilized mutants of a protein can enhance immunogenicity (29)(30)(31)46). We recently described a workflow employing second-site saturation-suppressor mutagenesis (SSSM) coupled to yeast surface display to rapidly identify stabilizing mutations (33). Recent work describes related approaches to identify stabilizing point mutants of GFP and CI2 from multi-mutant libraries in E. coli (47)(48)(49).…”

“…A schematic of the principle of second-site saturation suppressor mutagenesis (SSSM) to isolate stabilizing mutations is shown in Figures 1A-D; more detailed descriptions can be found in Sahoo et al and Ahmed et al (32,33). We generated an SSSM library of RBD containing the V512A PIM (parent inactive mutation) and displayed it on the yeast cell surface.…”

“…Earlier studies in other systems have shown that improving thermostability can enhance immunogenicity ( 29 – 31 ). 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.…”

“…However, once the stability crosses a certain threshold, further stability increases are not accompanied by increased binding on the yeast surface; hence, it is challenging to isolate mutants 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 1A–D ). A significant fraction of these suppressors is found to be stabilizing even in the wild-type background.…”