scholarly journals Stabilizing proteins through saturation suppressor mutagenesis.

2021 ◽  
Author(s):  
Shahbaz Ahmed ◽  
Kavyashree Manjunath ◽  
Raghavan Varadarajan
Keyword(s):  
Protein Stability ◽  
Structure Prediction ◽  
Surface Display ◽  
Point Mutations ◽  
Yeast Surface Display ◽  
Saturation Mutagenesis ◽  
Bacterial Toxin ◽  
Accurate Identification ◽  
Protein Mutants ◽  
Temperature Exposure

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.

scholarly journals Prediction of residue-specific contributions to binding and thermal stability using yeast surface display

2021 ◽  
Author(s):  
shahbaz ahmed ◽  
Kavyashree Manjunath ◽  
Raghavan Varadarajan
Keyword(s):  
Thermal Stability ◽  
Active Site ◽  
Surface Display ◽  
Protein Stabilization ◽  
Yeast Surface Display ◽  
Saturation Mutagenesis ◽  
Quantitative Prediction ◽  
Bacterial Toxin ◽  
Fluorescent Intensity ◽  
Yeast Surface

Quantitative prediction of residue-specific contributions to protein stability and activity is challenging, especially in the absence of experimental structural information. This is important for prediction and understanding of disease causing mutations, and for protein stabilization and design. Using yeast surface display of a saturation mutagenesis library of the bacterial toxin CcdB, we probe the relationship between ligand binding and expression level of displayed protein, with in vivo solubility in E.coli and in vitro thermal stability. We find that both the stability and solubility correlate well with the total amount of active protein on the yeast cell surface but not with total amount of expressed protein. We coupled FACS and deep sequencing to reconstruct the binding and expression mean fluorescent intensity of each mutant. The reconstructed mean fluorescence intensity (MFIseq) was used to differentiate between buried site, exposed non active-site and exposed active-site positions with high accuracy. The MFIseq was also used as a criterion to identify destabilized as well as stabilized mutants in the library, and to predict the melting temperatures of destabilized mutants. These predictions were experimentally validated and were more accurate than those of various computational predictors. The approach was extended to successfully identify buried and active-site residues in the receptor binding domain of the spike protein of SARS-CoV-2, suggesting it has general applicability.

scholarly journals Validation and Stabilization of a Prophage Lysin of Clostridium perfringens by Using Yeast Surface Display and Coevolutionary Models

2019 ◽  
Vol 85 (10) ◽  
Author(s):  
Seth C. Ritter ◽  
Benjamin J. Hackel
Keyword(s):  
Protein Stability ◽  
Clostridium Perfringens ◽  
High Throughput Sequencing ◽  
Surface Display ◽  
High Specificity ◽  
Classification Performance ◽  
Yeast Surface Display ◽  
Content Type ◽  
Cell Wall Binding ◽  
Yeast Surface

ABSTRACT Bacteriophage lysins are compelling antimicrobial proteins whose biotechnological utility and evolvability would be aided by elevated stability. Lysin catalytic domains, which evolved as modular entities distinct from cell wall binding domains, can be classified into one of several families with highly conserved structure and function, many of which contain thousands of annotated homologous sequences. Motivated by the quality of these evolutionary data, the performance of generative protein models incorporating coevolutionary information was analyzed to predict the stability of variants in a collection of 9,749 multimutants across 10 libraries diversified at different regions of a putative lysin from a prophage region of a Clostridium perfringens genome. Protein stability was assessed via a yeast surface display assay with accompanying high-throughput sequencing. Statistical fitness of mutant sequences, derived from second-order Potts models inferred with different levels of sequence homolog information, was predictive of experimental stability with areas under the curve (AUCs) ranging from 0.78 to 0.85. To extract an experimentally derived model of stability, a logistic model with site-wise score contributions was regressed on the collection of multimutants. This achieved a cross-validated classification performance of 0.95. Using this experimentally derived model, 5 designs incorporating 5 or 6 mutations from multiple libraries were constructed. All designs retained enzymatic activity, with 4 of 5 increasing the melting temperature and with the highest-performing design achieving an improvement of +4°C. IMPORTANCE Bacteriophage lysins exhibit high specificity and activity toward host bacteria with which the phage coevolved. These properties of lysins make them attractive for use as antimicrobials. Although there has been significant effort to develop platforms for rapid lysin engineering, there have been numerous shortcomings when pursuing the ultrahigh throughput necessary for the discovery of rare combinations of mutations to improve performance. In addition to validation of a putative lysin and stabilization thereof, the experimental and computational methods presented here offer a new avenue for improving protein stability and are easily scalable to analysis of tens of millions of mutations in single experiments.

scholarly journals Generating quantitative binding landscapes through fractional binding selections, deep sequencing and data normalization

2019 ◽  
Author(s):  
Michael Heyne ◽  
Niv Papo ◽  
Julia Shifman
Keyword(s):  
Free Energy ◽  
Protein Interactions ◽  
Binding Free Energy ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Protein Protein Interactions ◽  
Protein Mutants ◽  
Display Technology ◽  
Data Points ◽  
Affinity Interaction

AbstractQuantifying the effects of various mutations on binding free energy is crucial for understanding the evolution of protein-protein interactions and would greatly facilitate protein engineering studies. Yet, measuring changes in binding free energy (ΔΔGbind) remains a tedious task that requires expression of each mutant, its purification, and affinity measurements. We developed a new approach that allows us to quantify ΔΔGbindfor thousands of protein mutants in one experiment. Our protocol combines protein randomization, Yeast Surface Display technology, Next Generation Sequencing, and a few experimental ΔΔGbinddata points on purified proteins to generate ΔΔGbindvalues for the remaining numerous mutants of the same protein complex. Using this methodology, we comprehensively map the single-mutant binding landscape of one of the highest-affinity interaction between BPTI and Bovine Trypsin. We show that ΔΔGbindfor this interaction could be quantified with high accuracy over the range of 12 kcal/mol displayed by various BPTI single mutants.

scholarly journals Development and Characterization of Two Types of Surface Displayed Levansucrases for Levan Biosynthesis

Catalysts ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 757
Author(s):  
Huiyi Shang ◽  
Danni Yang ◽  
Dairong Qiao ◽  
Hui Xu ◽  
Yi Cao
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Large Scale ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Fusion Partner ◽  
Whole Cell ◽  
Food Industries ◽  
Yeast Surface ◽  
The Stability

Levan has wide applications in chemical, cosmetic, pharmaceutical and food industries. The free levansucrase is usually used in the biosynthesis of levan, but the poor reusability and low stability of free levansucrase have limited its large-scale use. To address this problem, the surface-displayed levansucrase in Saccharomyces cerevisiae were generated and evaluated in this study. The levansucrase from Zymomonas mobilis was displayed on the cell surface of Saccharomyces cerevisiae EBY100 using a various yeast surface display platform. The N-terminal fusion partner is based on a-agglutinin, and the C-terminal one is Flo1p. The yield of levan produced by these two whole-cell biocatalysts reaches 26 g/L and 34 g/L in 24 h, respectively. Meanwhile, the stability of the surface-displayed levansucrases is significantly enhanced. After six reuses, these two biocatalysts retained over 50% and 60% of their initial activities, respectively. Furthermore, the molecular weight and polydispersity test of the products suggested that the whole-cell biocatalyst of levansucrase displayed by Flo1p has more potentials in the production of levan with low molecular weight which is critical in certain applications. In conclusion, our method not only enable the possibility to reuse the enzyme, but also improves the stability of the enzyme.

scholarly journals The Role of Yeast-Surface-Display Techniques in Creating Biocatalysts for Consolidated BioProcessing

Catalysts ◽  
2018 ◽  
Vol 8 (3) ◽  
pp. 94 ◽  
Author(s):  
Ian Dominic Flormata Tabañag ◽  
I-Ming Chu ◽  
Yu-Hong Wei ◽  
Shen-Long Tsai
Keyword(s):  
Climate Change ◽  
Lignocellulosic Biomass ◽  
Surface Engineering ◽  
Consolidated Bioprocessing ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Qualitative Assessment ◽  
The Status ◽  
Yeast Surface

Climate change is directly linked to the rapid depletion of our non-renewable fossil resources and has posed concerns on sustainability. Thus, imploring the need for us to shift from our fossil based economy to a sustainable bioeconomy centered on biomass utilization. The efficient bioconversion of lignocellulosic biomass (an ideal feedstock) to a platform chemical, such as bioethanol, can be achieved via the consolidated bioprocessing technology, termed yeast surface engineering, to produce yeasts that are capable of this feat. This approach has various strategies that involve the display of enzymes on the surface of yeast to degrade the lignocellulosic biomass, then metabolically convert the degraded sugars directly into ethanol, thus elevating the status of yeast from an immobilization material to a whole-cell biocatalyst. The performance of the engineered strains developed from these strategies are presented, visualized, and compared in this article to highlight the role of this technology in moving forward to our quest against climate change. Furthermore, the qualitative assessment synthesized in this work can serve as a reference material on addressing the areas of improvement of the field and on assessing the capability and potential of the different yeast surface display strategies on the efficient degradation, utilization, and ethanol production from lignocellulosic biomass.

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