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Purification of recombinant proteins by affinity chromatography
Zemek, Ondřej ; Mácha, Jaroslav (advisor) ; Hrdý, Ivan (referee)
The isolation and purification of recombinant proteins is essential for further study of their structural and functional properties. The affinity chromatography is usually the method of choice for this task. In this paper the most used affinity tags are reviewed for their properties and experience with their application. The tags covered here include CBP, MBP, GST, polyhistidine and polyarginine tags, FLAG-tag, Strep-tag II and SpA. The origin and properties of the tags, their influence on form and localization of fusion protein as well as binding, elution and removal are discussed. Keywords: affinity chromatography, recombinant protein, purification, affinity tag
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Characterization of recombinant fragment of an antibody against CD3 marker.
Písačková, Jana ; Maloy Řezáčová, Pavlína (advisor) ; Obšil, Tomáš (referee)
Monoclonal antibody MEM-57 recognizes CD3 antigen expressed on peripheral blood T-lymphocytes. CD3 surface glycoprotein complex associates with T-cell receptor and is responsible for the transduction of activation signal. Antibody MEM-57 has, therefore, a large diagnostic and therapeutic potential. It could be used in autoimmune diseases diagnostics, for classification of T-cell leukemias and, as an immunosuppressant, in transplantation. The most promising therapeutic use of MEM-57 antibody would be the construction of a "Bispecific T-cell Engager" (BiTE) antibody format with potential application in cancer therapy. In this format, single-chain variable fragment (scFv) of MEM-57 would be fused with an anti-tumor antigen scFv. The thesis is focused on biochemical and biophysical characterization of MEM-57 antibody scFv fragment. Recombinant antibody fragment scFv MEM-57, equipped with the pelB leader sequence, c-myc tag and His5 tag, was produced from a pET22b(+) vector into the periplasmic space of E. coli BL21 (DE3). Two-step purification protocol, employing nickel chelation affinity chromatography and ion-exchange chromatography, was developed to obtain high yield of pure protein. The antigen binding activity of scFv MEM-57 was confirmed by flow cytometry. Structural information on scFv MEM-57...
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Preparation of the 14-3-3 Protein Binding Partners for Structural Studies.
Kopecká, Miroslava ; Obšil, Tomáš (advisor) ; Teisinger, Jan (referee)
Tyrosine hydroxylase belongs to the group of hydroxylases of aromatic acids and catalyzes a key step in the biosynthesis of catecholamine neurotransmitters. The tyrosine hydroxylase possesses the homotetrameric structure and contains three structural domains: the N-terminal regulatory domain, the catalytic domain and the C-terminal tetramerization domain. The activity of tyrosine hydroxylase is regulated by phosphorylation and through the regulation of its expression. Phosphorylation at Ser-19 induces binding of the 14-3-3 protein, which affects the structure of the regulatory domain and protects it against both dephosphorylation and degradation. Since the structure of the regulatory domain is still unknown, we decided to perform its structural characterization using NMR techniques. First, the expression and purification protocol of the regulatory domain of tyrosine hydroxylase was optimized. The protein was expressed as a His-tag fusion protein and its purification is composed from two steps: the chelating chromatography and the size-exclusion chromatography. The dynamic light scattering and the 1 H nuclear magnetic resonance were used to verify its monodispersity, and hence its suitability for further experiments.
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Heterologous expression of NADPH:cytochrome P450 reductase
Stráňava, Martin ; Černá, Věra (advisor) ; Martínek, Václav (referee)
NADPH:cytochrome P450 reductase (CPR) is a 78 kDa flavoprotein, which is together with cytochrome P450 component of monooxygenase system bound in the membrane of the endoplasmic reticulum. Monooxygenase system is involved in the metabolism of a wide range of organic substances, including drugs or various pollutants present in the environment (polycyclic aromatic hydrocarbons, aromatic amines, etc.). CPR works as a transporter of reducing equivalents from NADPH to the cytochromes P450. For proper interaction with cytochromes P450, intact N-terminal hydrophobic domain anchoring protein in the membrane is needed. Removing this domain, e.g. during trypsin proteolysis, gives rise a soluble CPR (72 kDa) and cause loss of catalytic activity towards cytochrome P450. During heterologous expression in E. coli proteolytically sensitive site of CPR (Lys56 - Ile57) is cleaved by intracellular trypsin-like proteases, that may negatively affect the yields of native 78 kDa protein. This thesis describes the heterologous expression, purification and characterization of two forms of rat CPR. WtCPR is a protein naturally occurring in rats (Wistar strain), while mCPR contains one amino acid substitution (K56Q) in the site of proteolytic degradation. The result of that substitution is proteolytically stable CPR,...
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Preparation of expression vectors for the production of ZIKA and Dengue NS3 helicase
Daňhelová, Kateřina ; Konvalinka, Jan (advisor) ; Heidingsfeld, Olga (referee)
Zika and Dengue viruses have spread, due to globalisation, to all continents which lie at least in part in the subtropic and tropic climatic zones. This spread of these viruses is a reason of an increasing number of severe diseases caused by them. New drugs, which would be effective against these infections, could be an answer to this challenge. Various viral proteins, among them also viral helicase, which is the topic of this bachelor thesis, can be a suitable drug target. The task was to prepare expression constructs for production of recombinant helicases of Zika and Dengue viruses via the suitable bacterial strain Escherichia coli. Several constructs derived from plasmid pET-16b were prepared with inserted helicase of Zika and Dengue viruses. One of them was used for the preparation of recombinant purified helicase of Zika virus, that will be used for further research. [IN CZECH] Keywords: Flavivirus, Zika virus, Dengue virus, helicase, expression, purification, enzyme activity [IN CZECH]
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Expression and purification of recombinant proteins of human Aichi virus and other kobuviruses
Ludvík, Tomáš ; Bouřa, Evžen (advisor) ; Petrvalská, Olívia (referee)
Viral 2Apro plays many important functions in the life cycle of virus. It is protease that is involved in the cleavage of the viral polyprotein in the VP1/2A region. The structure of protein contains active site, thanks to this the protein is able to bind various substrates in the host cell, which is essential for viral infection. With the help of 2Apro , the virus can bypass the host immune response and stop the production of key host proteins. Therefore, 2Apro is suitable target for antiviral intervention. In this work were prepared two recombinant proteins from Aichi virus and Coxsackievirus B3. These are viruses of the family Picornaviridae, which belong among the human pathogens that in some cases cause serious diseases. Aichi virus is the cause of gastroenteritis and Coxsackievirus B3 in the worst cases causes dilated cardiomyopathy. Cardiomyopathy leads to poor heart function due to it is enlargement and can lead to heart failure. (In Czech) Key words: recombinant proteins, 2Apro , Aichi virus, Coxsackievirus B3
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Ergothioneine and mycothiol in the biosynthesis of lincosamides
Seidlová, Bára ; Kameník, Zdeněk (advisor) ; Kopecký, Jan (referee)
Specialized microbial metabolites are described as low-molecular-weight bioactive compounds, which are dispensable for the growth, evolution, or reproduction of its producer. This group of substances includes the lincosamides, which are produced mainly by the bacteria of the Streptomyces genera. Apart from other precursors, two low-molecular-weight thiols, ergothioneine and mycothiol, are essential participants of the lincosamide biosynthesis. Mycothiol (MSH) serves in this pathway as a source of sulphur, on the other hand, ergothioneine (ESH) constitutes a conjugate with the aminosugar moiety of lincosamide structure. The conjugate is condensed with an activated amino acid, which is catalyzed by an unusual enzyme to form a core of the lincosamide molecule. The objective of this diploma thesis is to isolate the conjugate of ESH and aminooctose, which serves as a substrate of the LmbD biosynthetic protein. Another aim is to study the links between the thiol metabolism and the biosynthesis of three lincosamides, lincomycin, celesticetin, and intervencin, which are produced by different bacterial strains. Bacterial strains were cultivated under laboratory conditions and methods of liquid chromatography with UV and MS detection were used for the analysis. The parameters of the methods were developed...
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Focused eletron beam induced deposition
Juřík, Karel ; Zmeškal, Oldřich (referee) ; Pospíšil, Jan (advisor)
Within this work, a set of depositions induced by focused electron beam was prepared. The depositions were prepared in the presence of water vapours from trimethyl(methylcyclopentadienyl)platinum(IV) precursor. The dependence of prepared materials purity on beam accelerating voltage and water vapour pressure was measured. The best platinum content was achieved at (27,2 ± 0,4) at. %, with beam accelerating voltage 5 kV, beam current 1600 pA and water vapour pressure 100 Pa. Due to subsequent long-term exposure to light, air humidity and air oxygen, the platinum content was increased to (39,2 ± 2,1) at. %.
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Constant Current Chronopotentiometry as a Method for Detection of Singlet Oxygen Protein Damage
Ostatná, Veronika ; Vargová, Veronika ; Černocká, Hana ; Paleček, Emil
Proteins are one of the major targets for oxidative damage in the cell.Indirect non-radical oxidation of the protein via formation and subsequent reaction with single oxygen (O-1(2))is one of the major processes.In this work we studied the single oxygen(O-1(2))-mediated oxidation of bovine serum albumin (BSA) by constant current chronopotentiometry in combination with mercury electrode.Our chronopotentiometric data show that photo-oxidized BSA was more susceptible to the electric field-induced denaturation than non-oxidized native BSA. Our method utilizing intrinsic electrochemical signal of proteins provides a sensitive detection methods for minor damages in various proteins.
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