Directed evolution to improve protein folding in vivo

Sachsenhauser, Veronika; Bardwell, James C.A.

doi:10.1016/j.sbi.2017.12.003

Cited by 34 publications

(21 citation statements)

References 77 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Likewise, the abundant presence of misfolded polypeptides with weak folding mutations disrupts the balance of the protein-folding quality control and can establish aberrant physical and genetic interactions that interfere with the folding of marginally stable proteins (Gidalevitz et al 2006(Gidalevitz et al , 2009. Remarkably, directed evolution experiments have shown that more stable proteins can readily be engineered from marginally stable proteins (Foit et al 2009;Sachsenhauser and Bardwell 2018). This shows that most cellular proteins have not been optimized for maximum but optimum stability and suggests that the stability of the proteome is subject to functional constraints.…”

Section: Protein-intrinsic Factors That Regulate Protein Solubility Amentioning

confidence: 99%

Protein Phase Separation as a Stress Survival Strategy

Franzmann

Alberti

2019

Cold Spring Harb Perspect Biol

133

103

View full text Add to dashboard Cite

Cells under stress must adjust their physiology, metabolism, and architecture to adapt to the new conditions. Most importantly, they must down-regulate general gene expression, but at the same time induce synthesis of stress-protective factors, such as molecular chaperones. Here, we investigate how the process of phase separation is used by cells to ensure adaptation to stress. We summarize recent findings and propose that the solubility of important translation factors is specifically affected by changes in physical-chemical parameters such temperature or pH and modulated by intrinsically disordered prion-like domains. These stresstriggered changes in protein solubility induce phase separation into condensates that regulate the activity of the translation factors and promote cellular fitness. Prion-like domains play important roles in this process as environmentally regulated stress sensors and modifier sequences that determine protein solubility and phase behavior. We propose that protein phase separation is an evolutionary conserved feature of proteins that cells harness to regulate adaptive stress responses and ensure survival in extreme environmental conditions.

show abstract

Section: Protein-intrinsic Factors That Regulate Protein Solubility Amentioning

confidence: 99%

Protein Phase Separation as a Stress Survival Strategy

Franzmann

Alberti

2019

Cold Spring Harb Perspect Biol

133

103

View full text Add to dashboard Cite

show abstract

“…Examples include fusion reporter proteins for assessing protein folding and solubility using fluorescence, enzymatic reactions, antibiotic resistance or ligand binding as reporters for the production of soluble and folded proteins. [16][17][18][19][20] and their promotors may be used to drive stress induced heterologous protein expression 21 . Thus, chaperone promoters can be used to construct reporters for the presence of e.g.…”

Section: Introductionmentioning

confidence: 99%

A dual-reporter system for investigating and optimizing protein translation and folding inE. coli

Zutz

Hamborg

Pedersen

et al. 2020

Preprint

View full text Add to dashboard Cite

Strategies for investigating and optimising the expression and folding of proteins for biotechnological and pharmaceutical purposes are in high demand. Here, we describe a dual-reporter biosensor system that simultaneously assesses in vivo protein translation and protein folding, thereby enabling rapid screening of mutant libraries. We have validated the dual-reporter system on five different proteins and find an excellent correlation between reporter intensity signals and the levels of protein expression and solubility of the proteins. We further demonstrate the applicability of the dual-reporter system as a screening assay for deep mutational scanning experiments. The system enables high throughput selection of protein variants with high expression levels and altered protein stability. Next generation sequencing analysis of the resulting libraries of protein variants show a good correlation between computationally predicted and experimentally determined protein stabilities. We furthermore show that the mutational experimental data obtained using this system may be useful for protein structure calculations.

show abstract

“…Enzyme engineering, for improved catalytic efficiency, substrate and product preferences, and solubility, is traditionally used as a strategy to overcome issues with product synthesis. It can, however, be time‐consuming, even when aided by computational protein engineering tools (e.g., Hassanpour, Ullah, Yousofshahi, Nair, & Hassoun, ; Sachsenhauser & Bardwell, ), especially if several enzymes within a pathway are insoluble. Thus, implementing pathways with the knowledge that the enzymes selected have a high propensity for solubility in the host can reduce the traditional trial‐and‐error approach to enzyme selection.…”

Section: Discussionmentioning

confidence: 99%

“…Enzyme engineering, for improved catalytic efficiency, substrate and product preferences, and solubility, is traditionally used as a strategy to overcome issues with product synthesis. It can, however, be time-consuming, even when aided by computational protein engineering tools (e.g., Hassanpour, Ullah, Yousofshahi, Nair, & Hassoun, 2017;Sachsenhauser & Bardwell, 2018), F I G U R E 6 Three pathways for producing 3-hydroxypropanoic acid (3-HP) in E. coli were identified by ProPASS and proposed by (a) Luo et al (2016), (b) Cheng et al (2016), and (c) Song et al (2016). Two predicted solubility scores are provided for each enzymatic reaction.…”

Section: 3-butanediolmentioning

confidence: 99%

Establishing synthesis pathway‐host compatibility via enzyme solubility

Amin

Gopinarayanan

Nair

et al. 2019

Biotech & Bioengineering

View full text Add to dashboard Cite

Current pathway synthesis tools identify possible pathways that can be added to a host to produce a desired target molecule through the exploration of abstract metabolic and reaction network space. However, not many of these tools do explore gene-level information required to physically realize the identified synthesis pathways, and none explore enzyme-host compatibility. Developing tools that address this disconnect between abstract reactions/metabolic design space and physical genetic sequence design space will enable expedited experimental efforts that avoid exploring unprofitable synthesis pathways. This work describes a workflow, termed Probabilistic Pathway Assembly with Solubility Scores (ProPASS), which links synthesis pathway construction with the exploration of the physical design space as imposed by the availability of enzymes with characterized activities within the host. Predicted protein solubility propensity scores are used as a confidence level to quantify the compatibility of each pathway enzyme with the host (E. coli). This work also presents a database, termed Protein Solubility Database (ProSol DB), which provides solubility confidence scores in E. coli for 240,016 characterized enzymes obtained from UniProtKB/Swiss-Prot. The utility of ProPASS is demonstrated by generating genetic implementations of heterologous synthesis pathways in E. coli that target several commercially useful biomolecules.

show abstract

Directed evolution to improve protein folding in vivo

Cited by 34 publications

References 77 publications

Protein Phase Separation as a Stress Survival Strategy

Protein Phase Separation as a Stress Survival Strategy

A dual-reporter system for investigating and optimizing protein translation and folding inE. coli

Establishing synthesis pathway‐host compatibility via enzyme solubility

Contact Info

Product

Resources

About