Abstract: Developing simple and reliable methods to purify recombinant proteins is among the most important problems of modern biotechnology. It is of particular interest to take advantage of protein splicing for this purpose. Affinity tagging of inteins allows the use of regular protocols for protein purification. Autocatalytic excision of the tagged intein yields the pure protein lacking N-terminal formylmethionine. A new purification technique was developed on the basis of protein splicing for the human growth hormone. The Mxe GyrA intein with the histidine tag or cellulose-binding domain was used as a self-removable affinity unit. The resulting twostep purification protocol makes it possible to obtain the human growth hormone having the native N terminus with minimal losses.

Abstract: Protein splicing is a post-translational autocatalytic excision of internal protein sequence (intein) with the subsequent ligation of the flanking polypeptides (exteins). This process doesn't require any cofactors or enzymes, which distinguish it from other variants of protein processing. Protein splicing is catalyzed by Hint-domain - internal intein's domain. In review the main molecular mechanisms of this process are described. Function of analogous Hint-domains of other proteins families (Hh-proteins, BIL-domains etc) are considered. The analysis of inteins characteristic to different branches of life illustrates the role of horizontal transfer in intein's distribution and evolution. A possible role of inteins in regulation of different cell processes is discussed.

Abstract: Protein splicing is a post-translational autocatalytic process that results in excision of internal peptide (intein) from a precursor protein and the ligation of the flanking protein sequences (exteins). High specificity of the intein-mediated excision of protein precursors allows the use of protein splicing in biotechnology. This work was aimed at the obtaining of human growth hormone with a native N-terminus in E. coli. Chimerical protein consisting of a short N-terminal peptide, Mxe GyrA intein and human growth hormone was created. During the translation formyl-methionine modified N-terminal peptide should have been removed by splicing. Intein was shown to mediate the cleavage of exteins, but their subsequent ligation was not observed. That allowed the preparation of human growth hormone with a native N-terminus. The effect of various factors on cleavage efficiency was studied. The most efficient cleavage of chimeric protein (60-80%) was achieved in the presence of inductor (100 mM beta-mercaptoethanol) upon the incubation for 4-6 days.

Abstract: The elaboration of methods of gene delivery into somatic cells is the foundation of gene therapy development. There is necessity to develop new and more effective vectors as well as corresponding methods of gene delivery into the target tissues. The analysis of distribution of vector molecules in tissues and transgene expression are the constituent parts of evaluation of effectiveness of gene therapy methods for a target gene delivery. The study on the transfer of the plasmid DNA, containing human àpîÀ-1 gene, in complex with polyetilenimine (PEI) into rat and rabbit liver parenchyma has been performed. The expression of human àpîÀ-1 gene in vivo has been examined.

Abstract: 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 N-terminus, without formyl-metionine. Key words: intein, human growth hormone, protein splicing

Abstract: Summary of theoretical and experimental data of insulin buccal permeability is presented in the article. The hypothetical mechanism of insulin buccal permeability which complies with published experimental data is proposed. The basic barriers for insulin before getting into blood through the mucous membrane of mouth are indicated and described. On the basis of the proposed hypothetical mechanism the application of surfactants for facilitating the buccal permeability of insulin is substantiated. The proposed mechanism will be useful for the development of new protein buccal preparations.

Abstract: The technique of permeability assessment for high-molecular compounds is presented in present work. Isolated hamster cheek pouches as a simple and convenient model for the transport studies were used. It was shown that a mucous membrane is permeable for alpha-interferon, insulin but not for nucleic acids and peroxidase. Buccal permeability for insulin is provided by its transport through the intercellular ways. Data obtained in experiments with use of rodent keratinized mucous membrane could not be directly extrapolated to human buccal mucous permeability due to significant differences in the nature and organization of intercellular lipids in these species. However, we supposed that freshly isolated hamster cheek pouch could be employed as initial step of assessment for the fast screening of buccal permeability for the high-molecular compounds.

Abstract: Drug delivery across the buccal mucosa is convenient and safe transport method. The efficiency of the buccal system of peptide delivery is, however, not yet satisfactory. To improve the buccal transport new absorption promoters should be developed to be sufficiently active and at the same time causing no side effects like irritation or unpleasant taste. We have found that lysalbinic acid, a product of the alkaline hydrolysis of egg albumin and a mild detergent, meets those requirements. The preparation and some physicochemical properties of lysalbinic acid are described. Hamster cheek pouch was used as a model for the penetration process studies lysalbinic acid was shown to increase significantly an oral mucosa permeability for alpha-interferon and insulin. So this substance of the natural origin can be applied as an absorption enhancer for the buccal delivery of peptide drugs.

Abstract: Inteins are internal polypeptide sequences that are posttranslationally excised from a protein precursor by a self-catalyzed protein-splicing reaction. Most of inteins consist of N- and C-terminal protein splicing domain and central endonuclease domain. The endonuclease domain can initiate mobility of the intein gene, this process being named intein homing. This review is focused on the recent data about the structure and function of inteins. Main intein-mediated protein-engineering applications, such as protein purification, ligation and cyclization, new forms of biosensors, are presented.