The current level of genetic engineering technology provides the obtaining of necessary amounts of target proteins for research, drug discovery and diagnostics via expression in heterologous systems. Due to its well-characterized genetics, rapid growth and production, E. coli is a preferred choice among other expression systems for production of non-glycosylated proteins. Nearly 30% of currently approved recombinant therapeutic proteins and most immunodiagnostic tools are produced in E. coli. As objects for our investigations were chosen proteins, the use of which is promising for the diagnosis of tuberculosis (MPT-63, MPT-83, MPT-63-83 fusion protein); development of subunit vaccines against swine erysipelas (SPAA(tr); bioligand for chromatographic purification of target antigens (scFv-CBD); bioligands for chromatographic purification of IgG (SPA-CBD, SPA-CBD2, SPA-Cys); genetically engineered immunoconjugate for immunodetection (SPA-BAPmut). To enhance the protein expression was used a modified method of auto-induction in high density shaking cultures. As the result, 8 of 9 investigated proteins were obtained in preparative amounts via expression in E. coli. The MPT-63, MPT-83, MPT-63-83, SPAA(tr), ScFv-CBD were accumulated in the form of bacterial inclusion bodies. The matrix-assistant refolding method was used for obtaining of soluble and purified MPT-63, MPT-83, MPT-63-83 fusion protein, SPAА(tr). Solubilized proteins under denaturing conditions were loaded into a metal affinity column and refolded by decreasing urea gradient using an automated FPLC chromatography system. The purity of recombinant proteins after refolding was more than 90 %. We have shown that polyclonal antibodies induced by recombinant analogs of MPT63 and MPT83, as well as by their fusion, were able to detect the appropriate natural antigens in Western blot analysis. Antigenic specificity of the recombinant SPAА(tr) protein was confirmed by immunoblotting with polyclonal antibodies against Er.rhusiopathiae museum strains, and with specific rabbit antiserum against recombinant SPAА(tr) and Er.rhusiopathiae strains lysates of different serotypes. Also, in experiments on mice we have found that recombinant SPAA(tr), in amount of 10 ?g, demonstrated 100% protective effect. An inexpensive and effective refolding method for ScFv-CBD fusion protein recovery from E. coli inclusion bodies has been developed. The principle of the proposed method is the stepwise dilution of solubilized ScFv-CBD in the presence of 6 M guanidine hydrochloride with refolding buffer containing L-arginine and glutathione. The influences of initial protein concentration and the molar ratio of oxidized and reduced forms of glutathione which were added at appropriate refolding stages have been carefully investigated. The optimal refolding conditions providing highest yield of soluble monomeric ScFv-CBD as well as the highest functional activity of antigen-binding (ScFv) and cellulose-binding (CBD) moieties (~63 % and ~96% respectively) have been proposed. SPA is a cell wall associated protein exposed on the surface of the S. aureus. SPA has high affinity to IgG from various animal species and human. The most applied affinity system for the purification of antibodies is based on SPA. The DNA sequences encoding SPA and two CBD were genetically fused, expressed in E. coli system in a soluble form. The SPA-CBD2 fusion protein was affinity-immobilized on the microcrystalline cellulose CC31 and was used for purification of antibodies. The purity of antibodies in eluted fractions was more than 95 %. The designed bioaffinity sorbent provides obtaining pure poly and monoclonal antibodies in functionally active form and can be useful for the fractionation of mouse immunoglobulin G. Introduced C-terminal cysteine provided oriented covalent immobilization of SPA on maleimide-activated matrix. In addition to affinity chromatography, the use of SPA, fused with a tag (enzymes, fluorescent proteins) is promising for diagnostics. As a fusion partner for the SPA was selected BAPmut. SPA-BAPmut was expressed in a soluble form in E. coli system. The target protein was obtained with purity more than 95 % using metal affinity chromatography. SPA-BAPmut is thermostable, and both parts of fusion protein (SPA and BAPmut) retain their IgG binding and alkaline phosphatase activity for a long time. As little as 0,2 ng of the antigen could be detected in Western blotting. The possibility of using SPA-BAPmut as universal secondary immunoreagent for different types of immunoassays was shown.
