• MeSH
• biotransformace MeSH
• proteinové inženýrství MeSH
• protilátky terapeutické užití   genetika   MeSH
• Publikační typ
• laboratorní příručky MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biologie

Cardio- and cerebrovascular diseases are leading causes of death and disability, resulting in one of the highest socio-economic burdens of any disease type. The discovery of bacterial and human plasminogen activators and their use as thrombolytic drugs have revolutionized treatment of these pathologies. Fibrin-specific agents have an advantage over non-specific factors because of lower rates of deleterious side effects. Specifically, staphylokinase (SAK) is a pharmacologically attractive indirect plasminogen activator protein of bacterial origin that forms stoichiometric noncovalent complexes with plasmin, promoting the conversion of plasminogen into plasmin. Here we report a computer-assisted re-design of the molecular surface of SAK to increase its affinity for plasmin. A set of computationally designed SAK mutants was produced recombinantly and biochemically characterized. Screening revealed a pharmacologically interesting SAK mutant with ∼7-fold enhanced affinity toward plasmin, ∼10-fold improved plasmin selectivity and moderately higher plasmin-generating efficiency in vitro. Collectively, the results obtained provide a framework for SAK engineering using computational affinity-design that could pave the way to next-generation of effective, highly selective, and less toxic thrombolytics.

• Publikační typ
• časopisecké články MeSH

BACKGROUND: Lectins are proteins of non-immune origin capable of binding saccharide structures with high specificity and affinity. Considering the high encoding capacity of oligosaccharides, this makes lectins important for adhesion and recognition. The present study is devoted to the PA-IIL lectin from Pseudomonas aeruginosa, an opportunistic human pathogen capable of causing lethal complications in cystic fibrosis patients. The lectin may play an important role in the process of virulence, recognizing specific saccharide structures and subsequently allowing the bacteria to adhere to the host cells. It displays high values of affinity towards monosaccharides, especially fucose--a feature caused by unusual binding mode, where two calcium ions participate in the interaction with saccharide. Investigating and understanding the nature of lectin-saccharide interactions holds a great potential of use in the field of drug design, namely the targeting and delivery of active compounds to the proper site of action. RESULTS: In vitro site-directed mutagenesis of the PA-IIL lectin yielded three single point mutants that were investigated both structurally (by X-ray crystallography) and functionally (by isothermal titration calorimetry). The mutated amino acids (22-23-24 triad) belong to the so-called specificity binding loop responsible for the monosaccharide specificity of the lectin. The mutation of the amino acids resulted in changes to the thermodynamic behaviour of the mutants and subsequently in their relative preference towards monosaccharides. Correlation of the measured data with X-ray structures provided the molecular basis for rationalizing the affinity changes. The mutations either prevent certain interactions to be formed or allow formation of new interactions--both of afore mentioned have strong effects on the saccharide preferences. CONCLUSION: Mutagenesis of amino acids forming the specificity binding loop allowed identification of one amino acid that is crucial for definition of the lectin sugar preference. Altering specificity loop amino acids causes changes in saccharide-binding preferences of lectins derived from PA-IIL, via creation or blocking possible binding interactions. This finding opens a gate towards protein engineering and subsequent protein design to refine the desired binding properties and preferences, an approach that could have strong potential for drug design.

• MeSH
• bakteriální adheziny genetika   chemie   MeSH
• chromatografie afinitní MeSH
• financování organizované MeSH
• jednonukleotidový polymorfismus MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• lektiny genetika   chemie   MeSH
• molekulární modely MeSH
• monosacharidy chemie   MeSH
• mutageneze cílená MeSH
• proteinové inženýrství MeSH
• Pseudomonas aeruginosa genetika   MeSH
• Ralstonia solanacearum chemie   MeSH
• rekombinantní proteiny genetika   izolace a purifikace   MeSH
• rostlinné lektiny chemie   MeSH
• substituce aminokyselin MeSH
• vazebná místa MeSH

Biomass feedstock is an efficient and harmless source of energy. There are various sources of feedstock, such as plant, microbial, macro, and microalgae, and agricultural waste. The major component in biomass feedstock material is a polysaccharide, such as cellulose, cellobiose, starch, and alginate. Alginate is mainly found in macroalgae as one of the significant polysaccharide components. It is made up of β-d-mannuronate (M) and α-l-guluronate (G) blocks. Alginate lyase is an enzyme that degrades alginate by breaking the glycosidic linkage between the poly M and G blocks to liberate oligosaccharides. Several organisms, including bacteria, fungi, viruses, and algae can produce alginate lyases. The species of bacteria, such as Bacillus, Vibrio, Pseudomonas, and Microbulbifer, are some of the important sources of alginate lyases. They are industrially essential enzymes used in food, biofuel, and biomedical industries. There are various assays available to determine the alginate lyase activity qualitatively as well as quantitatively. Qualitatively, different dyes like Gram's iodine, cetyl pyridinium chloride, and rutanium red can be used to visualize the zone formed due to the alginate lyase activity. DNS assay, UV absorption, and the Somogyi-Nelson method help to determine the alginate lyase activity quantitatively. Since the alginate lyase production in the native organisms is relatively lower, the genes encoding alginate lyases are heterologously cloned and expressed in E. coli to maximize the production and to characterize the enzyme. Different chromatographic techniques like size exclusion, affinity, gel permeation, and ion-exchange chromatography are used to purify the protein. In this paper, the source of alginate and alginate lyases, the mechanism of action of the enzyme, the engineering approaches to enhance the enzyme production, its purification strategy, and the potential applications of alginate lyases has been discussed.

• MeSH
• algináty chemie   MeSH
• Bacteria metabolismus   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• biotechnologie * MeSH
• genetické inženýrství * MeSH
• houby metabolismus   MeSH
• metagenom MeSH
• mořské řasy metabolismus   MeSH
• polysacharid-lyasy chemie   genetika   metabolismus   MeSH
• substrátová specifita MeSH
• viry MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Počítačová předpověď proteinových mutací vedoucí ke zvýšení jejich afinity k cílovému proteinu není běžně používaný, ale užitečný nástroj proteinového inženýrství. Zde ukazujeme, že dostupnými výpočetními prostředky je možné předpovědět zvýšení afinity receptoru 1 k interferonu-γ. U dvou, ze čtrnácti předpovězených mutant, došlo k pětinásobnému zvýšení disociační rovnovážné konstanty Kd jak bylo ukázáno měřením povrchovou plasmonovou resonancí, SPR.

Computer design of mutations increasing affinity to the target protein is not a standard but useful tool of protein engineering. Here we show that it is possible to apply relatively simple and accessible computer techniques based on empirical potential to predict mutants of receptor 1 to interferon-γ. Two out of fourteen designed mutants showed about five-fold increase of dissociation equilibrium constant K d as measured by surface plasmon resonance, SPR.

• MeSH
• financování organizované MeSH
• interferon gama MeSH
• mutace MeSH
• počítačová simulace využití   MeSH
• proteinové inženýrství metody   využití   MeSH
• receptory interferonů genetika   imunologie   MeSH
• terciární struktura proteinů genetika   MeSH

N-Acetylhexosamine oligosaccharides terminated with GalNAc act as selective ligands of galectin-3, a biomedically important human lectin. Their synthesis can be accomplished by β-N-acetylhexosaminidases (EC 3.2.1.52). Advantageously, these enzymes tolerate the presence of functional groups in the substrate molecule, such as the thiourea linker useful for covalent conjugation of glycans to a multivalent carrier, affording glyconjugates. β-N-Acetylhexosaminidases exhibit activity towards both N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) moieties. A point mutation of active-site amino acid Tyr into other amino acid residues, especially Phe, His, and Asn, has previously been shown to strongly suppress the hydrolytic activity of β-N-acetylhexosaminidases, creating enzymatic synthetic engines. In the present work, we demonstrate that Tyr470 is an important mutation hotspot for altering the ratio of GlcNAcase/GalNAcase activity, resulting in mutant enzymes with varying affinity to GlcNAc/GalNAc substrates. The enzyme selectivity may additionally be manipulated by altering the reaction medium upon changing pH or adding selected organic co-solvents. As a result, we are able to fine-tune the β-N-acetylhexosaminidase affinity and selectivity, resulting in a high-yield production of the functionalized GalNAcβ4GlcNAc disaccharide, a selective ligand of galectin-3.

• MeSH
• aktivace enzymů MeSH
• beta-N-acetylhexosaminidasy chemie   genetika   metabolismus   MeSH
• hydrolýza MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• mutace MeSH
• polysacharidy biosyntéza   chemie   farmakologie   MeSH
• proteinové inženýrství MeSH
• vodíková vazba MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Metody exprese a purifikace rekombinantních proteinů umožňují produkci a detailní charakterizaci proteinů v základním výzkumu během in vitro experimentů, ale také přípravu proteinů s terapeutickým využitím. Publikace shrnuje základní postupy od přípravy expresních vektorů až po techniku afinitní purifikace. Dále pojednává o vlastnostech různých prokaryotických a eukaryotických expresních systémů a možnostech jejich využití. Molekulární klonování, které slouží k přípravě expresních vektorů pro rekombinantní proteiny, umožňuje cíleně modifikovat vlastnosti těchto proteinů tak, aby byla usnadněna jejich purifikace a také pozměněna jejich stabilita, aktivita nebo funkce. V současné době je k dispozici široká škála metodických přístupů, jež umožňují rychlou a efektivní přípravu expresních vektorů. Zvolený produkční organizmus a způsob purifikace rekombinantního proteinu určují výběr expresního vektoru. První volbou často bývá expresní systém využívající bakterii Escherichia coli, jehož přednostmi jsou zejména technická, časová i finanční nenáročnost. Tento expresní systém není příliš vhodný pro produkci komplexních savčích proteinů, pro které jsou optimální expresní systémy založené na využití eukaryotických organizmů (kvasinky, hmyzí buňky nebo savčí buňky). Kultivace hmyzích a savčích buněk je však technicky i finančně náročná. Rekombinantní proteiny jsou purifikovány nejčastěji metodou afinitní chromatografie využívající specifickou interakci peptidu nebo proteinu s afinitní matricí. Tyto peptidy či proteiny jsou fúzovány s N‑ nebo C‑koncem purifikovaného proteinu. Purifikace probíhá ve třech krocích, kdy je rekombinantní protein prostřednictvím afinitních značek specificky zachycen na matrici chromatografické kolony, dále následuje promývací krok, po kterém je uvolněn z kolony čistý protein.

Production of recombinant proteins is essential for many applications in both basic research and also in medicine, where recombinant proteins are used as pharmaceuticals. This review sum­marizes procedures involved in recombinant protein expression and purification, including molecular cloning of target genes into expression vectors, selection of the appropriate expres­sion system, and protein purification techniques. Recombinant DNA technology allows protein engineering to modify protein stability, activity and function or to facilitate protein purification by affinity tag fusions. A wide range of cloning systems enabling fast and effective design of expression vectors is currently available. A first choice of protein expression system is usually the bacteria Escherichia coli. The main advantages of this prokaryotic expression system are low cost and simplicity; on the other hand this system is often unsuitable for production of complex mam­malian proteins. Protein expression mediated by eukaryotic cells (yeast, insect and mammalian cells) usually produces properly folded and posttranslationally modified proteins. How­ever, cultivation of insect and, especially, mammalian cells is time consuming and expensive. Affinity tagged recombinant proteins are purified efficiently using affinity chromatography. An affinity tag is a protein or peptide that mediates specific binding to a chromatography column, unbound proteins are removed during a washing step and pure protein is subsequently eluted. Key words: recombinant protein – molecular cloning – purification – expression system This work was supported by the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101) and by MH CZ – DRO (MMCI, 00209805). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for bio­medical papers. Submitted: 22. 1. 2014 Accepted: 20. 3. 2014

• Klíčová slova
• purifikace, expresní systém, expresní vektor,
• MeSH
• chromatografie afinitní MeSH
• Escherichia coli genetika   MeSH
• eukaryotické buňky MeSH
• exprese genu * MeSH
• genetická transkripce MeSH
• genetické vektory * MeSH
• klonování DNA * MeSH
• kultivační média MeSH
• kvasinky genetika   MeSH
• prokaryotické buňky MeSH
• proteinové inženýrství MeSH
• rekombinantní proteiny * genetika   chemická syntéza   MeSH
• virové proteiny genetika   MeSH
• Publikační typ
• práce podpořená grantem MeSH

The RNA recognition motif (RRM) is the most common RNA binding domain across eukaryotic proteins. It is therefore of great value to engineer its specificity to target RNAs of arbitrary sequence. This was recently achieved for the RRM in Rbfox protein, where four mutations R118D, E147R, N151S, and E152T were designed to target the precursor to the oncogenic miRNA 21. Here, we used a variety of molecular dynamics-based approaches to predict specific interactions at the binding interface. Overall, we have run approximately 50 microseconds of enhanced sampling and plain molecular dynamics simulations on the engineered complex as well as on the wild-type Rbfox·pre-miRNA 20b from which the mutated systems were designed. Comparison with the available NMR data on the wild type molecules (protein, RNA, and their complex) served to establish the accuracy of the calculations. Free energy calculations suggest that further improvements in affinity and selectivity are achieved by the S151T replacement.

• MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• mikro RNA chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• motiv rozpoznávající RNA * genetika   MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• proteinové inženýrství MeSH
• proteiny vázající RNA chemie   genetika   metabolismus   MeSH
• RNA chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• simulace molekulární dynamiky MeSH
• stabilita RNA MeSH
• vazba proteinů MeSH
• vazebná místa genetika   MeSH
• výpočetní biologie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

This article focuses on designing mutations of the PA-IIL lectin from Pseudomonas aeruginosa that lead to change in specificity. Following the previous results revealing the importance of the amino acid triad 22-23-24 (so-called specificity-binding loop), saturation in silico mutagenesis was performed, with the intent of finding mutations that increase the lectin's affinity and modify its specificity. For that purpose, a combination of docking, molecular dynamics and binding free energy calculation was used. The combination of methods revealed mutations that changed the performance of the wild-type lectin and its mutants to their preferred partners. The mutation at position 22 resulted in 85% in inactivation of the binding site, and the mutation at 23 did not have strong effects thanks to the side chain being pointed away from the binding site. Molecular dynamics simulations followed by binding free energy calculation were performed on mutants with promising results from docking, and also at those where the amino acid at position 24 was replaced for bulkier or longer polar chain. The key mutants were also prepared in vitro and their binding properties determined by isothermal titration calorimetry. Combination of the used methods proved to be able to predict changes in the lectin performance and helped in explaining the data observed experimentally.

• MeSH
• bakteriální adheziny chemie   genetika   metabolismus   MeSH
• design s pomocí počítače MeSH
• lektiny chemie   genetika   metabolismus   MeSH
• metabolismus sacharidů MeSH
• mutace MeSH
• mutageneze * MeSH
• počítačová simulace MeSH
• Pseudomonas aeruginosa chemie   genetika   metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• simulace molekulového dockingu MeSH
• termodynamika MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

IL-2 has been used to treat diseases ranging from cancer to autoimmune disorders, but its concurrent immunostimulatory and immunosuppressive effects hinder efficacy. IL-2 orchestrates immune cell function through activation of a high-affinity heterotrimeric receptor (composed of IL-2Rα, IL-2Rβ, and common γ [γc]). IL-2Rα, which is highly expressed on regulatory T (TReg) cells, regulates IL-2 sensitivity. Previous studies have shown that complexation of IL-2 with the JES6-1 Ab preferentially biases cytokine activity toward TReg cells through a unique mechanism whereby IL-2 is exchanged from the Ab to IL-2Rα. However, clinical adoption of a mixed Ab/cytokine complex regimen is limited by stoichiometry and stability concerns. In this study, through structure-guided design, we engineered a single agent fusion of the IL-2 cytokine and JES6-1 Ab that, despite being covalently linked, preserves IL-2 exchange, selectively stimulating TReg expansion and exhibiting superior disease control to the mixed IL-2/JES6-1 complex in a mouse colitis model. These studies provide an engineering blueprint for resolving a major barrier to the implementation of functionally similar IL-2/Ab complexes for treatment of human disease.

• MeSH
• aktivace lymfocytů MeSH
• autoimunitní nemoci imunologie   terapie   MeSH
• cytokiny genetika   imunologie   metabolismus   MeSH
• imunoterapie metody   MeSH
• kolitida imunologie   terapie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• proliferace buněk MeSH
• proteinové inženýrství MeSH
• protilátky genetika   metabolismus   MeSH
• receptory interleukinu-2 imunologie   MeSH
• regulační T-lymfocyty imunologie   MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH