Production of Third Type Domain of Fibronectin Fragments in E.coli Expression System

Gyvų ląstelių analizė ir manipuliacijos jomis yra tarpdisciplininė tyrimų sritis, kuriai daug sunkumų kelia audinių natyvios aplinkos efektyvus atkartojimas. Fibronektinas (FN), naudojamas ląstelių adhezijai ant polimerinių struktūrų, yra 230-250 kDa baltymas sudarytas iš trijų modulių tipų – I, II ir III tipo domenų. FNIII9-10 domenai atsakingi už ląstelės prisijungimą prie FN baltymo. Arg-Gly-Asp (RGD) seka yra III10 domene ir atsakinga už ląstelės prisijungimą prie paviršiaus per α5β1 ir αVβ3 integrinus. Papildomai, III9 domene yra sinerginė zona būtina fibronektino sąveikai su α5β1 integrinais. Heterologinė didelių eukariotinių baltymų, tokių kaip FN, raiška bakterinėse raiškos sistemose yra problematiška. Todėl, šio tyrimo tikslas buvo bakterinės rekombinantinių baltymų gamybos systemos tinkamos FN baltymo fragmentų gaminimui sukūrimas. Fragmentai apimantys III6-10 ir III9-10 žmogaus fibronektino baltymo domenus buvo klonuoti naudojant klonavimą nenaudojant ligazės. Konstruktai sukurti įterpiant 6xHis-tag žymę rekombinantinių baltymų gryninimui. Ląstelių auginimo ir baltymo indukcijos sąlygos buvo optimizuotos siekiant maksimalios baltymo gamybos išeigos. Baltymų fragmentai buvo gryninti naudojant optimizuotą IMAC metodą, o jų funkcionalumas patvirtintas naudojant žmogaus fibroblastų ląsteles.Analysis and manipulation of living cells is a multidisciplinary area of research chalanged by efficient mimetics of the native environment of the tissues that has to be reproduced. Fibronectin (FN), used for cell adhesion to polymer structures, is a 230–250 kDa protein that contains three types of modules: type I, II, and III. The modules III9–10 correspond to the "cell-binding domain" of FN protein. The Arg–Gly–Asp (RGD) sequence is located in module III10 and is the site of cell attachment via α5β1 and αVβ3 integrins on the cell surface. In addition the "synergy site" located in III9 is important for association of fibronectin with α5β1 integrins. Heterologous expression of large eucaryotic proteins, such as FN, in bacterial expression system is problematical. Therefore aim of this study was to develop a bacterial recombinant protein production system for fragments of FN peptide suitable for microfabrication and integrin-mediated imobilization of cells to synthetic scaffolds. Fragments encompasing modules III6-10 and III9-10 of human FN protein has been cloned using ligation independent cloning system. The construct was build to include N-terminal 6xHis-tag for the recombinant peptide purification. Cell growth and protein induction conditions were optimized to ensure maximum yields of the protein production. Protein fragments were purified using optimiezed IMAC method and their functionality proved using fibroblast cells.Gamtos mokslų fakultetasVytauto Didžiojo universiteta

Cloning and expression of transmembrane domain segments of Arabidopsis thaliana RBOH D enzyme

Production of reactive oxygen species (ROS) is important cell signaling component involved in plant response to stress. NADPH-oxidase, also known as respiratory burst oxidase homologue (RBOH) in plants, is a superoxide producing NOX family protein that is located in plasma membrane and contributes to ROS generation outside the plasma membrane in the apoplast. A. thaliana has ten RBOH genes (RBOH A-J). RBOH D is expressed in all plant tissues and takes part in regulation of response to pathogen and abiotic stress. Plant RBOH proteins show unique structural and regulation features that include calcium binding and different cytosolic component role in regulation of the enzyme activity. Meanwhile, structure of small N-terminal cytosolic domain of plant RBOH was studied so far, further structural and functional studies of plant RBOH homologues could help to understand the role of these enzymes in cell signaling. Heterologous expression and purification of transmembrane proteins is challenging due to their hydrophobic membrane domain. However, valuable information about transmembrane protein structure and function may be obtained by expression and characterization of separate membrane domain fragments. Aim of this study was to identify, clone and optimize the production of segments of A. thaliana RBOH D protein membrane domain in E. coli expression system. To identify transmembrane domain of RBOH D, bioinformatics analysis of amino acid sequence hydrophobicity profile, transmembrane protein domain modeling and assessment of previously predicted structures of other NOX family proteins, such as NOX2, was used. Six transmembrane helices were identified and the third and fifth helices each contained two conserved His residues, which are considered to coordinate the two hemes involved in superoxide production. [...]Gamtos mokslų fakultetasLietuvos agrarinių ir miškų mokslų centro filialas Sodininkystės ir daržininkystės institutasVytauto Didžiojo universiteta

Production of recombinant Fibronectin tipe III 9-10 domain in bacterial expression system

Fibronectin (Fn) is a glycoprotein that plays important roles in cell adhesion, growth, differentiation and migration by mediating a wide variety of cellular interactions with the extracellular matrix (ECM). Fn usually exists as a dimer composed of two nearly identical 220~250 kDa subunits where each monomer is composed of homologous repeats of three prototypical domains: type I, type II and type III. Fn interacts with many integrins such as α3β1, α5β1, α8β1, αvβ1, αIIββ3, αvβ3, αvβ5, and αvβ6. In previous studies, the specific integrin-recognition sequences involved in cell adhesion have been identified. The best known of these – Arg-Gly-Asp (RGD) sequence – is located in the central cell-binding domain - FnIII10. It is the most important recognition site that can interact with about half of all known integrins. Another important sequence which acts in synergy with the RGD site is - Pro-His-Ser-Arg-Asn (the ‘synergy site’ PHSRN) found in Fn repeat III9, that promotes specific α5β1 integrin binding. Because of the ECM binding properties Fn is a perfect protein for nanofabrication and integrin-mediated immobilization of cells into synthetic scaffolds. Heterologous expressions of large eucaryotic proteins, such as Fn, in bacterial expression system is complicated. Therefore the aim of this study was to establish a recombinant protein production system for the "cell-binding domain" of Fn protein - including FnIII9-10 fragment and to assess the effect of His-tag position on the recombinant peptide purification efficiency. For this purpose, a sequence of the FnIII9-10 fragment was cloned to pLATE bacterial expression vector using a ligation independent cloning system. This vector includes bacteriophage T7 promoter that ensures high yields of expressed proteins. Two constructs including either amino- or carboxy- terminal 6xHis-tag were developed.[...]Biochemijos katedraGamtos mokslų fakultetasLietuvos agrarinių ir miškų mokslų centro Sodininkystės ir daržininkystės institutasVilniaus universitetasVytauto Didžiojo universiteta