(=1)

(=1). Under first-order conditions, the kinetic dissociation constant kdis directly related to the half-life of the bimolecular complex (t1/2) in irreversible dissociation conditions through the equation: t1/2=ln 0.5/kd, establishing an intuitive and direct Metoclopramide HCl relation between kdand life-time of the complex[48]. readily produced in large amounts (>18 mg/L) in the cytoplasm ofE. coli,and bound with a subpicomolar affinity (Kd0.54 pM) to its target, the HIV-1 Nef protein. When expressed in human cells Neffin could potently inhibit Nef function. Comparable VHH-SH3 fusion proteins could be targeted against many other proteins of interest and could have widespread use in diverse medical and biotechnology applications where biochemical robustness and strong binding affinity are required. == Introduction == Specific recognition and strong binding to chosen target molecules is the cornerstone of modern therapeutic and diagnostic practices. Monoclonal antibody technology pioneered by Khler and Milstein in the 1970s revolutionized medical and other fields of immunodiagnostic development[1], and currently accounts for a significant portion of new drugs approved for treatment of major human diseases, such as malignancy and autoimmune disorders[2],[3]. Subsequent progress in molecular biology has made it possible to generate recombinant antibodies with rationally altered binding properties and multifunctional fusion partners[4],[5]. Recombinant antibodies made up of only the Fab fragment and single-chain antibodies (scFv) comprised only of the variable domains of heavy and light chains joined by a flexible linker peptide represent simpler and smaller alternatives to complete immunoglobulins. Fab and scFv proteins can be easily manipulated and often produced in relatively large amounts in prokaryotic expression systems. The possibility to select recombinant antibodies Metoclopramide HCl from synthetic libraries and to optimize their properties by random and targeted mutagenesis combined with powerfulin vitroaffinity selection schemes have been fruitfully exploited in various biotechnology applications. These approaches enable rational targeting of antibody binding, including target epitopes that might be poorly immunogenic, as well as overcoming the affinity ceiling of monoclonal antibodies. While most natural antibodies haveKdvalues in the range of 108to 1011M[6],[7], orders of magnitude tighter binding has been reported for optimized recombinant antibodies[8]. Despite these advantages, problems and limitations related to recombinant antibodies exist, which have hindered their widespread use. Metoclopramide HCl Due to the complex structure recombinant antibodies show challenging biophysical properties, and are lacking the robustness of ideal recombinant protein reagents[9],[10],[11],[12]. Metoclopramide HCl Accordingly, recombinant antibodies have poor stability under reducing conditions, such as the intracellular environment. Moreover, their antigen recognition can be sensitive for context-specific steric effects, thus limiting the freedom to create multifunctional fusion protein derivatives. Therefore, several investigators have considered the use of non-Ig proteins as sources (scaffold proteins) for novel high affinity ligand binders via applying the same principles of sequence diversification and affinity selection successfully applied in recombinant antibody engineering. A growing number of proteins and protein domains, with normal functions either related or unrelated to protein interactions, have been established as suitable backbones for engineering of artificial proteins with useful binding specificities (for reviews, see[13],[14],[15]). Among the Metoclopramide HCl best validated examples of these are affibodies based on the Z-domain of staphylococcal protein A[16], monobodies based on the 10th extracellular domain name of human fibronectin III[17], and DARPins (designed ankyrin repeat domains) comprised of an optimized target binding interface built from four to six ankyrin repeat modules with designed binding properties[18]. Another attractive non-Ig scaffold is the SH3 domain name[19],[20], representing a small (5560 aa) protein module with a compact beta-sandwich fold lacking disulfide bridges, which can be easily expressed in large amounts and in soluble form inE. coli.By randomizing the non-conserved flexible loops of SH3 domains they have been successfully targeted for binding to diverse ligand proteins with low nanomolar affinities[21],[22],[23]. An alternative approach to address the challenges related to the biochemical properties of recombinant antibodies has been to exploit the ability of certain immunoglobulin variable Rabbit Polyclonal to IkappaB-alpha domains to bind target antigens as impartial monomeric models[24]. In particular, camelids and sharks naturally.