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Current methods in protein-protein interactions research
Křivková, Jana ; Hrdý, Ivan (advisor) ; Kučerová, Jitka (referee)
Protein-protein interactions (PPI) have a crucial role in all processes in living cells. Understanding the interactions between proteins allows us to describe cell processes in more detail and their study opens new possibilities for drug design. The importance of the question of studying PPI is shown in the recent development of various methods for their identification and description. The aim of this thesis is to give an overview of new and improved experimental methods of identification and characterization of protein-protein interactions. Methods described in this thesis are divided in four chapters - proximity-dependent labelling methods (BioID, BioID2, APEX, TurboID, MiniTurbo, PUP-IT, AirID, SPPLAT, EMARS), cross-linking methods (XS-MS), fluorescence methods for identification and visualization (BiFC, FRET, BRET) and biophysical methods for description of kinetics and thermodynamics parameters of interaction (SPR, ITC, MT).
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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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Determination of pepsinogens using IgY and IgG
Kulhavá, Lucie ; Pacáková, Věra (advisor) ; Tichá, Marie (referee)
A decreased concentration of pepsinogen A in serum was found to be a marker of gastric cancer, similarly as a low ratio of pepsinogen A to pepsinogen C. In the present study we have compared properties of immunoglobulin fraction isolated from the egg yolks after immunization of laying hens with pepsinogen isolated from porcine gastric mucosa with those of present in rabbit antiserum obtained after the animal immunization with the same antigen. The characteristics of chicken antibodies against porcine pepsinogen and the comparison with rabbit antibodies raised against the same antigen was carried out using the following methods: ELISA, affinity chromatography on immobilzed antigen and bovine serum albumin, SDS and native electrophoresis and MALDI-TOF MS/MS. While the rabbit specific antibodies interacted with the used antigen and only slightly with bovine serum albumin and there was a diference between pre-immune IgG and specific IgG, in the case of chicken antibodies IgY it did not work. No diference was observed between ELISA tests performed with pre-immune serum and the serum after immunization with porcine pepsinogen and a high interaction of IgY with bovine serum albumin in pre-immune serum and specific IgY after the immunization were detected. Similar results were obtained in experiments with...
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Determination of pepsinogens using IgY and IgG
Kulhavá, Lucie ; Pacáková, Věra (advisor) ; Tichá, Marie (referee)
A decreased concentration of pepsinogen A in serum was found to be a marker of gastric cancer, similarly as a low ratio of pepsinogen A to pepsinogen C. In the present study we have compared properties of immunoglobulin fraction isolated from the egg yolks after immunization of laying hens with pepsinogen isolated from porcine gastric mucosa with those of present in rabbit antiserum obtained after the animal immunization with the same antigen. The characteristics of chicken antibodies against porcine pepsinogen and the comparison with rabbit antibodies raised against the same antigen was carried out using the following methods: ELISA, affinity chromatography on immobilzed antigen and bovine serum albumin, SDS and native electrophoresis and MALDI-TOF MS/MS. While the rabbit specific antibodies interacted with the used antigen and only slightly with bovine serum albumin and there was a diference between pre-immune IgG and specific IgG, in the case of chicken antibodies IgY it did not work. No diference was observed between ELISA tests performed with pre-immune serum and the serum after immunization with porcine pepsinogen and a high interaction of IgY with bovine serum albumin in pre-immune serum and specific IgY after the immunization were detected. Similar results were obtained in experiments with...
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Preparation and testing of polyclonal antibodies against Cbf11 and Cbf12, the fission yeast CSL transcription factors
Tvarůžková, Jarmila ; Půta, František (advisor) ; Moserová, Michaela (referee)
CSL (CBF1/RBP-Jκ, Suppressor of Hairless, Lag-1) protein family members are transcription factors critical for metazoan development as the effectors of Notch signaling pathway as well as in a Notch-independent manner. CSL homologues have been identified in fungal organisms lacking the Notch signaling pathway. Cbf11 and Cbf12 are antagonistic paralogous proteins that are important for proper coordination of cell and nuclear division, regulation of cell adhesion and chromosome integrity in the fission yeast Schizosaccharomyces pombe. The activities of Cbf11 and Cbf12 proteins need to be finely balanced for their proper biological function, however, chromosomally tagged alleles of these proteins exhibit properties different from wild type. Therefore, the availability of specific antibodies would greatly enhance the study of CSL proteins in the fission yeast. In this bachelor's thesis, design and preparation of imunogen for commercial antibody production followed by antibody testing is presented. Using bioinformatics, suitable immunogenic Cbf11 and Cbf12 protein fragments were selected and the corresponding DNA sequences were cloned into an expression vector. His-tagged expression was optimized in a bacterial expression system and the native protein was purified using immobilized metal affinity...
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Isolation of proteins from sow oviductal fluid by affinity chromatography on DNA cellulose
Černá, Tereza ; Liberda, Jiří (advisor) ; Ryšlavá, Helena (referee)
Oviductal fluid was isolated from sow oviduct and then frozen and lyophilized. The obtained material was separated by affinity chromatography on DNA-cellulose and used as well for determination of antimicrobial and glycosidases activity. Two fractions - non-binding and bound - were obtained by affinity chromatography on DNA-cellulose. Four protein zones were detected on the SDS-electrophoreogram in the binding fraction of chromatographic separation - three zones with relative molecular weight 15 000 - 21 000. These zones correspond by their relative molecular weights to relative molecular weight of histones. The last zone was corresponding to ~ 34 000. All of these zones were sent for identification using MALDI-TOF analysis. The antimicrobial activity was detected in the non-binding fraction and in oviductal fluid. Contrary to this, the antimicrobial activity was not present in the bound fraction, due to the fact that in this case only an insignificant amount of proteins was found in the bound fraction. The presence of the glycosidases activity was shown in oviductal fluid. (In Czech)
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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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Analyses of glycoproteins from the salivary glands of the tick \kur{Ixodes ricinus}
BUČINSKÁ, Lenka
I characterized several potential glycoproteins in salivary gland extracts from unfed and partially fed females of ticks Ixodes ricinus using enzyme deglycosylation and lectin labeling. Affinity-based (chromatografic) analysis was applied for isolations of glycoproteins with specificity for GNA (mannose), HPA (N-acetylgalactosamine) and MAA II (sialic acid) lectins. GNA specific 120 kDa glycoprotein was isolated from partially fed females and is modified with N-linked glycans containing {$\alpha$}1,3-mannose. Mass spectrometry analyses confirmed the presence carboxypeptidase M in elution fraction gain with GNA affinity chromatography. GNA specific proteins were purified from unfed female salivary gland extracts. MS analyses identified them as proteins similar to arylsulfatase B and cytoskeletal Sojo protein. Proteins (85 and 56 kDa) isolated with HPA affinity chromatography were characterized as Trappin 12, which is a host protein. MAA II lectin was used for labelling and isolation of 100 kDa protein. N-terminal sequence of the MAA II specific protein predicted similarity with a host protein, Siglec 1. Fucose in salivary gland extract was detected with the labelling of AAA, AAL, UEA I and LTL lectins. Results showed that salivary gland extracts contain {$\alpha$}1,2-; {$\alpha$}1,3- and {$\alpha$}1,6- N-linked fucose and O-linked fucose probably as well. GNA specific proteins were detected in partially fed salivary glands acini type II and III using electron transmission microscopy. Fucose was detected on gut and salivary gland structures using fucose-specific lectin AAL.
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