Directed evolution
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Engineered small non-antibody protein scaffolds are a promising alternative to antibodies and are especially attractive for use in protein therapeutics and diagnostics. The advantages include smaller size and a more robust, single-domain structural framework with a defined binding surface amenable to mutation. This calls for a more systematic approach in designing new scaffolds suitable for use in one or more methods of directed evolution. We hereby describe a process based on an analysis of protein structures from the Protein Data Bank and their experimental examination. The candidate protein scaffolds were subjected to a thorough screening including computational evaluation of the mutability, and experimental determination of their expression yield in E. coli, solubility, and thermostability. In the next step, we examined several variants of the candidate scaffolds including their wild types and alanine mutants. We proved the applicability of this systematic procedure by selecting a monomeric single-domain human protein with a fold different from previously known scaffolds. The newly developed scaffold, called ProBi (Protein Binder), contains two independently mutable surface patches. We demonstrated its functionality by training it as a binder against human interleukin-10, a medically important cytokine. The procedure yielded scaffold-related variants with nanomolar affinity.
- MeSH
- databáze proteinů MeSH
- interleukin-10 metabolismus MeSH
- konformace proteinů MeSH
- počítačová simulace MeSH
- proteinové inženýrství MeSH
- proteiny chemie genetika metabolismus MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- ribozomy metabolismus MeSH
- řízená evoluce molekul metody MeSH
- sekvence aminokyselin MeSH
- stabilita proteinů MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Here we present an optimized procedure to generate amino acid variations at specific site(s) of proteins, followed by a simple one-step screen for mutants with the desired β-glucosidase activity. The procedure was evaluated by introducing sequence variation into a codon specifying a non-functional variant of the catalytic nucleophile (E401) of the maize β-glucosidase Zm-p60.1. Observed and theoretically expected frequencies of the four possible variants of the codon and the two possible phenotypes (functional and non-functional) were investigated. Deviations in codon and phenotype frequencies were expressed as a coefficient. This coefficient was then used to estimate the extent of oversampling, of the mutant library, which would be necessary to compensate for the underrepresentation of some sequences. This evaluation of the overall performance of the method allows experimentally derived parameters to be incorporated into mutant library design. This method combines the application of a well-defined distribution of variability with a reliable screening process. Thus, it facilitates the production of novel functional variants of β-glucosidases for either fundamental studies or potential biotechnological applications.
Freshwater environments teem with microbes that do not have counterparts in culture collections or genetic data available in genomic repositories. Currently, our apprehension of evolutionary ecology of freshwater bacteria is hampered by the difficulty to establish organism models for the most representative clades. To circumvent the bottlenecks inherent to the cultivation-based techniques, we applied ecogenomics approaches in order to unravel the evolutionary history and the processes that drive genome architecture in hallmark freshwater lineages from the phylum Planctomycetes. The evolutionary history inferences showed that sediment/soil Planctomycetes transitioned to aquatic environments, where they gave rise to new freshwater-specific clades. The most abundant lineage was found to have the most specialised lifestyle (increased regulatory genetic circuits, metabolism tuned for mineralization of proteinaceous sinking aggregates, psychrotrophic behaviour) within the analysed clades and to harbour the smallest freshwater Planctomycetes genomes, highlighting a genomic architecture shaped by niche-directed evolution (through loss of functions and pathways not needed in the newly acquired freshwater niche).
... The Structure of Adaptive Landscapes Underlying Protein Evolution, 121 -- Adaptive Maturation of the ... ... Immune Response, 122 Evolution of Novel Catalytic Functions, 142 -- Applied Molecular Evolution: Direct ... ... - PART III ORDER AND ONTOGENY, 407 -- 11 The Architecture of Genetic Regulatory Circuits and Its Evolution ... ... , 411 -- Independence of the Molecular Evolutionary Clock and Morphological Evolution, 412 -- CONTENTS ... ... Genetic Regulatory Systems of Prokaryotes and Eukaryotes, 412 -- An Ensemble Theory Based on Random Directed ...
1st ed. 709 s. : il.
- Klíčová slova
- Biologie, Evoluce, Fylogeneze,
- MeSH
- biologická evoluce MeSH
- biologie MeSH
- fylogeneze MeSH
- molekulární evoluce MeSH
- původ života MeSH
Adenylation domains CcbC and LmbC control the specific incorporation of amino acid precursors in the biosynthesis of lincosamide antibiotics celesticetin and lincomycin. Both proteins originate from a common L-proline-specific ancestor, but LmbC was evolutionary adapted to use an unusual substrate, (2S,4R)-4-propyl-proline (PPL). Using site-directed mutagenesis of the LmbC substrate binding pocket and an ATP-[32P]PPi exchange assay, three residues, G308, A207 and L246, were identified as crucial for the PPL activation, presumably forming together a channel of a proper size, shape and hydrophobicity to accommodate the propyl side chain of PPL. Subsequently, we experimentally simulated the molecular evolution leading from L-proline-specific substrate binding pocket to the PPL-specific LmbC. The mere change of three amino acid residues in originally strictly L-proline-specific CcbC switched its substrate specificity to prefer PPL and even synthetic alkyl-L-proline derivatives with prolonged side chain. This is the first time that such a comparative study provided an evidence of the evolutionary relevant adaptation of the adenylation domain substrate binding pocket to a new sterically different substrate by a few point mutations. The herein experimentally simulated rearrangement of the substrate binding pocket seems to be the general principle of the de novo genesis of adenylation domains' unusual substrate specificities. However, to keep the overall natural catalytic efficiency of the enzyme, a more comprehensive rearrangement of the whole protein would probably be employed within natural evolution process.
Thrombosis and haemostasis, ISSN 0340-6245 Volume 90, 2003, příloha
22 stran : ilustrace ; 28 cm
- MeSH
- fondaparinux terapeutické užití MeSH
- inhibitory faktoru Xa terapeutické užití MeSH
- ortopedické výkony MeSH
- tromboembolie farmakoterapie MeSH
- trombóza farmakoterapie MeSH
- Publikační typ
- souborné dílo MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- hematologie a transfuzní lékařství
- angiologie
Traditional directed evolution experiments are often time-, labor- and cost-intensive because they involve repeated rounds of random mutagenesis and the selection or screening of large mutant libraries. The efficiency of directed evolution experiments can be significantly improved by targeting mutagenesis to a limited number of hot-spot positions and/or selecting a limited set of substitutions. The design of such "smart" libraries can be greatly facilitated by in silico analyses and predictions. Here we provide an overview of computational tools applicable for (a) the identification of hot-spots for engineering enzyme properties, and (b) the evaluation of predicted hot-spots and selection of suitable amino acids for substitutions. The selected tools do not require any specific expertise and can easily be implemented by the wider scientific community.
