Engineering of recombinant Staphylococcal protein A with cysteine residue (SPA-Cys) for preparation of affi nity chromatography stationary phase and formation of bioselective element of immunosensor. Methods. DNA sequences encoding IgG-binding region of SPA, His-tag and cysteine were genetically fused and expressed in E. coli. SPA-Cys was immobilized on maleimide-functionalized silica beads for affi nity chromatography stationary phase preparation and on a gold sensor surface as a bioselective element of immunosensor. Results. SPA-Cys was expressed at a high-level in a soluble form. The target protein was purifi ed and showed a high IgG-binding activity. The capacity of the obtained SPA-Cys-based affi nity chromatography stationary phase was 10-12 mg of IgG /ml. The purity of eluted IgG was more than 95 % in one-step purifi cation procedure. The developed SPA-Cys-based bioselective element of immunosensor selectively interacted with human IgG and did not interact with the control proteins. Conclusions. The recombinant Staphylococcal protein A with cysteine residue was successfully used for the preparation of affi nity chromatography stationary phase and formation of the bioselective element of immunosensor. K e y w o r d s: antibodies, recombinant Staphylococcal protein A, protein immobilization, affi nity chromatography, immunosensor, surface plasmon resonance.
Protein splicing is a post-translational autocatalystic excision of internal protein sequence (intein) with the subsequent ligation of the flanking polypeptides (exteins). The high specificity of excision ensured by intein makes it possible to use a phenomenon of protein splicing for the biotechnology purposes. The aim of this work was optimization of obtaining and purification of the recombinant human growth hormone using the protein splicing. It was experimentally demonstrated that the use of modified intein as auto-removal affine marker makes it possible to perform the rapid and cheap isolation of the recombinant protein Hgh.Furthermore, this approach allows to obtain the human growth hormone with native Nterminus, without formyl-metionine.Protein splicing is a post-translational autocatalystic excision of internal protein sequence (intein) with the subsequent ligation of the flanking polypeptides (exteins). The high
The article raises the problems of intracellular spatial and temporal organization of metabolism, signaling, and energy supply of these processes. To provide cell functions, the enzymes of metabolic chains, molecules of signaling pathways, and macroergs (as units of molecular interactions, accompanied by energy consumption) should find their partners and get their precise spatial relationship. The current views are based on ideas of compartmentalization of all processes as local sites of cellular matrix membrane, where specific stages of different metabolic cycles take place. The assembly of complexes of macromolecules in the number and combinations, required for their adequate functioning in the space of a cell, is generally described as intracellular transport of vesicles, implemented by mobile elements of cytoske-leton. Inside the vesicle there is «effective load»-macromolecules. The membranes of these vesicles fuse with specific sites of the matrix membranes and therefore relocate macromolecules. Neither calculations nor assumptions allow explaining precise formation of enzymatic chains, their interaction, signaling, etc. on this basis. Such transport of macromolecules (inside vesicles) enables solving other tasks. The concept of search-and-address systems in the form of space-scanning micro vesicles is proposed and well-grounded for purposes of searching for partners, forming chains and complexes, and building compartments. The micro vesicles collect corresponding chains of enzymes, signaling, and ensure the interactions on their surface. These micro vesicles are exactly those compartments, which provide for both precision of processes and their relationship.
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