Lehmann, M. & Wyss, M. Engineering proteins for thermostability: the use of sequence alignments versus rational design and directed evolution. Curr. Opin. Biotechnol. 12, 371-375

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Current Opinion in Biotechnology (Impact Factor: 7.12). 09/2001; 12(4):371-5. DOI: 10.1016/S0958-1669(00)00229-9
Source: PubMed

ABSTRACT With the advent of directed evolution techniques, protein engineering has received a fresh impetus. Engineering proteins for thermostability is a particularly exciting and challenging field, as it is crucial for broadening the industrial use of recombinant proteins. In addition to directed evolution, a variety of partially successful rational concepts for engineering thermostability have been developed in the past. Recent results suggest that amino acid sequence comparisons of mesophilic proteins alone can be used efficiently to engineer thermostable proteins. The potential benefits of the underlying, semirational 'consensus concept' are compared with those of rational design and directed evolution approaches.

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Available from: Martin Lehmann, Sep 28, 2015
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    • "Protein modification is a covalent reaction between the functional groups on the surface of enzyme molecule and chemicals, e.g., polymers in order to change the chemical feature of enzyme surface, which can lead to greater stability of the pretreated enzyme [4] [5] [6] [7] [8] [9]. Protein stabilization techniques, such as protein engineering [10] [11] [12] [13], reaction medium engineering [7] [14] [15] and protein immobilization are often applied methods in order to stabilize enzyme catalytic activity, i.e., to increase its functional time [6], though immobilized enzymes often have lesser activity than the native ones [16] [17]. Protein or enzyme immobilization means an attachment of protein to the surface or onto the inner cavities of greater structures with adsorption or covalent linkage [4] [5]. "
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    ABSTRACT: The drug carrier function of single protein nanoparticles, i.e., each individual protein molecule covered by a very thin, porous and few nanometer thick polymer layer, has been investigated. This layer around protein molecule is very thin, about 3-5 nm thick and highly porous, thus it does not reduce seriously the enzymatic function of protein molecule. The spatial structure of encapsulated protein molecule, which is essential in its function, can stabilize by thispolymer layer. Bovine serum albumin was used as protein drug molecule and it was encapsulated with acrylamide-bisarylamide random copolymer. The polymerization, starting from the modified sites of the surface of bovine serum albumin molecules was initiated by TEMED (tetramethylethylenediamine). These single albumin nanoparticles were painted with fluorescein isothiocyanate. This material was then injected into the inferior vena cava of rats. The treated rats were decapitated after 1 to 10 minutes and its brain was investigated by fluorescent microscopy. It was proved that bovine serum albumin molecules as drugs encapsulated in polymer nano-layer with a reduced size (about 10 nm) can pass through the blood brain barrier. The results suggest that this method is capable of transformation of biomacromolecules to access the brain tissue via the blood.
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    • "A serious shortcoming of such a random approach is that it can only be applied to enzymes for which high-throughput expression and activity screens are available. Other methods to stabilize enzymes are consensus design (Lehmann and Wyss, 2001; Bommarius et al., 2006), rational protein engineering (Eijsink et al., 2004), the creation of chimeric enzymes (Romero et al., 2013) and computational design (Korkegian et al., 2005; Gribenko et al., 2009; Joo et al., 2011). The number of stabilizing mutations that are introduced is usually rather low and currently none of these methods work well enough to reliably achieve a large stability increase of a target enzyme. "
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    ABSTRACT: The ability to engineer enzymes and other proteins to any desired stability would have wide-ranging applications. Here, we demonstrate that computational design of a library with chemically diverse stabilizing mutations allows the engineering of drastically stabilized and fully functional variants of the mesostable enzyme limonene epoxide hydrolase. First, point mutations were selected if they significantly improved the predicted free energy of protein folding. Disulfide bonds were designed using sampling of backbone conformational space, which tripled the number of experimentally stabilizing disulfide bridges. Next, orthogonal in silico screening steps were used to remove chemically unreasonable mutations and mutations that are predicted to increase protein flexibility. The resulting library of 64 variants was experimentally screened, which revealed 21 (pairs of) stabilizing mutations located both in relatively rigid and in flexible areas of the enzyme. Finally, combining 10-12 of these confirmed mutations resulted in multi-site mutants with an increase in apparent melting temperature from 50 to 85°C, enhanced catalytic activity, preserved regioselectivity and a >250-fold longer half-life. The developed Framework for Rapid Enzyme Stabilization by Computational libraries (FRESCO) requires far less screening than conventional directed evolution.
    Protein Engineering Design and Selection 01/2014; 27(2). DOI:10.1093/protein/gzt061 · 2.54 Impact Factor
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    • "modeling tool [13] to design stabilized MetA mutants. The consensus concept approach for engineering thermally stable proteins is based on an idea that by multiple sequence alignment of the homologous counterparts from mesophiles and thermophiles, the nonconsensus amino acid might be determined and substituted with the respective consensus amino acid, contributing to the protein stability [12]. I-Mutant2.0 is a support vector machine-based web server for the automatic prediction of protein stability changes with single-site mutations ( "
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    ABSTRACT: The growth of Escherichia coli at elevated temperatures is limited due to the inherent instability of homoserine o-succinyltransferase, MetA, which is the first enzyme in the methionine biosynthesis pathway. MetA is also unstable under other stressful conditions, such as weak organic acids and oxidative stress. The MetA protein unfolds, even at 25[degree sign]C, forms considerable aggregates at 37[degree sign]C and completely aggregates at 44[degree sign]C. We extended the MetA mutation studies using a consensus concept based on statistics and sequence database analysis to predict the point mutations resulting in increased MetA stability. In this study, four single amino acid substitutions (Q96K, I124L, I229Y and F247Y) in MetA designed according to the consensus concept and using the I-mutant2.0 modeling tool conferred accelerated growth on the E. coli strain WE at 44[degree sign]C. MetA mutants that enabled E. coli growth at higher temperatures did not display increased melting temperatures (Tm) or enhanced catalytic activity but did show improved in vivo stability at mild (37[degree sign]C) and elevated (44[degree sign]C) temperatures. Notably, we observed that the stabilized MetA mutants partially recovered the growth defects of E. coli mutants in which ATP-dependent proteases or the DnaK chaperone was deleted. These results suggest that the impaired growth of these E. coli mutants primarily reflect the inherent instability of MetA and, thus, the methionine supply. As further evidence, the addition of methionine recovered most of the growth defects in mutants lacking either ATP-dependent proteases or the DnaK chaperone. A collection of stable single-residue mutated MetA enzymes were constructed and investigated as background for engineering the stabilized mutants. In summary, the mutations in a single gene, metA, reframe the window of growth temperature in both normal and mutant E. coli strains.
    BMC Microbiology 07/2013; 13(1):179. DOI:10.1186/1471-2180-13-179 · 2.73 Impact Factor
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