Experimental Protocols for Generating Focused Mutant Libraries and Screening for Thermostable Proteins (original) (raw)
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Engineering highly functional thermostable proteins using ancestral sequence reconstruction
Nature Catalysis, 2018
Commercial enzymatic processes require robust catalysts able to withstand elevated temperatures and long incubations, conditions under which most native enzymes perform poorly. Incremental increases in thermostability can be achieved by repeated rounds of mutagenesis and screening, but general strategies are needed for designing highly thermostable enzymes a priori. Here we show that enzymes can be created that can withstand temperatures ~ 30 °C higher and incubations ≥ 100 times longer than extant forms in a single step using ancestral reconstruction. We exemplify the approach with the first ancestral resurrections of two unrelated enzyme families: cytochrome P450 monooxygenases, that stereo-and regioselectively functionalize un-activated C-H bonds in pharmaceutical, flavour, fragrance and other fine chemical syntheses; and ketol acid reductoisomerases, used to make butanol-based biofuels. This shows thermostability can be designed into proteins using sequence data alone, potentially enhancing the economic feasibility of any process or product requiring a highly stable protein.
High-throughput screening for enhanced protein stability
Current Opinion in Biotechnology, 2006
High thermostability of proteins is a prerequisite for their implementation in biocatalytic processes and in the evolution of new functions. Various protein engineering methods have been applied to the evolution of increased thermostability, including the use of combinatorial design where a diverse library of proteins is generated and screened for variants with increased stability. Current trends are toward the use of data-driven methods that reduce the library size by using available data to choose areas of the protein to target, without specifying the precise changes. For example, the half-lives of subtilisin and a Bacillus subtilis lipase were increased 1500-fold and 300-fold, respectively, using a crystal structure to guide mutagenesis choices. Sequence homology based methods have also produced libraries where 50% of the variants have improved thermostability. Moreover, advances in the high-throughput measurement of denaturation curves and the application of selection methods to thermostability evolution have enabled the screening of larger libraries. The combination of these methods will lead to the rapid improvement of protein stability for biotechnological purposes.
Protein thermostability engineering
RSC Advances, 2016
The use of enzymes for industrial and biomedical applications is limited to their function at elevated temperatures. The principles of thermostability engineering need to be implemented for proteins with low thermal stability to broaden their applications. Therefore, understanding the thermal stability modulating factors of proteins is necessary for engineering their thermostability. In this review, first different thermostability enhancing strategies in both the sequence and structure levels, discovered by studying the natural proteins adapted to different conditions, are introduced. Next, the progress in the development of various computational methods to engineer thermostability of proteins by learning from nature and introducing several popular tools and algorithms for protein thermostability engineering is highlighted. Further discussion includes the challenges in the field of protein thermostability engineering such as the protein stability-activity trade-off. Finally, how thermostability engineering could be instrumental for the design of protein drugs for biomedical applications is demonstrated.
Journal of Biological Chemistry, 2018
Abstract Aldehyde-deformylating oxygenase (ADO) is an essential enzyme for production of long-chain alkanes as drop-in biofuels, which are compatible with existing fuel systems. The most active ADOs are present in mesophilic cyanobacteria, especially Nostoc punctiforme. Given the potential applications of thermostable enzymes in biorefineries, here we generated a thermostable (Cts)-ADO based on a consensus of ADO sequences from several thermophilic cyanobacterial strains. Using an in silico design pipeline and a metagenome library containing 41 hot-spring microbial communities, we created Cts-ADO. Cts-ADO displayed a 3.8-fold increase in pentadecane production on raising the temperature from 30°C to 42°C, whereas ADO from N. punctiforme (Np-ADO) exhibited a 1.7-fold decline. 3D structure modeling and molecular dynamics simulations of Cts- and Np-ADO at different temperatures revealed differences between the two enzymes in residues clustered on exposed loops of these variants, which affected the conformation of helices involved in forming the ADO catalytic core. In Cts-ADO, this conformational change promoted ligand binding to its preferred iron, Fe2, in the di-iron cluster at higher temperature, but the reverse was observed in Np-ADO. Detailed mapping of residues conferring Cts-ADO thermostability identified four amino acids, which we substituted individually and together in Np-ADO. Among these substitution variants, A161E was remarkably similar to Cts-ADO in terms of activity optima, kinetic parameters, and structure at higher temperature. A161E was located in loop L6, which connects helices H5 and H6, and supported ligand binding to Fe2 at higher temperatures, thereby promoting optimal activity at these temperatures and explaining the increased thermostability of Cts-ADO.
FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants
There is great interest in increasing proteins’ stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt’s reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.
Enzyme stabilization by domain insertion into a thermophilic protein
Protein Engineering Design and Selection, 2009
Insufficient kinetic stability of exoinulinase (EI) restricts its application in many areas including enzymatic transformation of inulin for production of ultra-high fructose syrup and oligofructan, as well as fermentation of inulin into bioethanol. The conventional method for enzyme stabilization involves mutagenesis and therefore risks alteration of an enzyme's desired properties, such as activity. Here, we report a novel method for stabilization of EI without any modification of its primary sequence. Our method employs domain insertion of an entire EI domain into a thermophilic scaffold protein. Insertion of EI into a loop of a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) resulted in improvement of kinetic stability (the duration over which an enzyme remains active) at 378 8 8 8 8C without any compromise in EI activity. Our analysis suggests that the improved kinetic stability at 378 8 8 8 8C might originate from a raised kinetic barrier for irreversible conversion of unfolded intermediates to completely inactivated species, rather than an increased energy difference between the folded and unfolded forms.
Design of Temperature-Sensitive Mutants Solely From Amino Acid Sequence
Proceedings of the …, 2004
Temperature-sensitive (Ts) mutants are a powerful tool with which to study gene function in vivo. Ts mutants are typically generated by random mutagenesis followed by laborious screening procedures. By using the Escherichia coli cytotoxin CcdB as a model system, simple ...
Engineering an enzyme to resist boiling
Proceedings of the National Academy of Sciences, 1998
In recent years, many efforts have been made to isolate enzymes from extremophilic organisms in the hope to unravel the structural basis for hyperstability and to obtain hyperstable biocatalysts. Here we show how a moderately stable enzyme (a thermolysin-like protease from Bacillus stearothermophilus, TLP-ste) can be made hyperstable by a limited number of mutations. The mutational strategy included replacing residues in TLP-ste by residues found at equivalent positions in naturally occurring, more thermostable variants, as well as rationally designed mutations. Thus, an extremely stable 8-fold mutant enzyme was obtained that was able to function at 100°C and in the presence of denaturing agents. This 8-fold mutant contained a relatively large number of mutations whose stabilizing effect is generally considered to result from a reduction of the entropy of the unfolded state (''rigidifying'' mutations such as Gly 3 Ala, Ala 3 Pro, and the introduction of a disulfide bridge). Remarkably, whereas hyperstable enzymes isolated from natural sources often have reduced activity at low temperatures, the 8-fold mutant displayed wild-type-like activity at 37°C.