[Purification of recombinant proteins with an example of tumor necrosis factor thymosin-alpha1].

Hybrid protein, cancer necrosis factor thymosin-alpha1 (CNF-T), when synthesizing in strain-producer of Escherichia coli SG200-50 with plasmid pThy315, was a part of "inclusion bodies" mostly in the form of a high-molecular complex with other proteins due to the S-S bonds formation. An approach of purification of CNF-T has been proposed, which is based on the destruction of the complex in the presence of sodium dodecylsulfate (DDS-NA) and dithiotreitol (DDT) followed by gel-filtration on Sephadex G-100 and renaturation by ultrafiltration on hollow fibers. The method allows the isolation of electrophoretically homogeneous CNF-T containing no DDS-Na and having high cytotoxic activity against cancer cells of mouse adenocarcinome L-929. The yield of CNF-T achieved 80% relative its content in biomass and 30% relative the total protein.

[Antiviral activity of human recombinant gamma-interferon and recombinant hybrid protein of tumor necrosis factor-alpha-thymosin-alpha1 on models of herpes-virus and cytomegalovirus infection in vitro]. / Protivovirusnaia aktivnost' rekombinantnogo interferona-gamma cheloveka i rekombinantnogo gibridnogo belka alpha-faktor nekroza opukholei-timozin-alpha1 na modeliakh gerpes-virusnoi i tsitomegalovirusnoi infektsii in vitro.

The conducted studies showed a certain efficiency of gene-engineering preparation of IFN-gamma and TNF-T in virus infections caused by herpes simplex virus 2 (HSV-2) and cytomegalovirus (CMV) in cell cultures of human embryo fibroblast (HEF). The drugs have no viricidal action. IFN-gamma, when used according to the treatment scheme in vitro, proved to be more effective versus TMF-T both in HSV-2 and in CMV. It inhibited significantly the HSV-2 reproduction within the dilution range of 1:50 to 1:500. It was also effective, when used in CMV, within the dilution range of 1:5000 and lower, whereas TNF-T was effective within the range of 1:500 and lower as well as in 0.1 multiple infection. A significantly higher effect was ensured when the drugs were used for prevention. In HSV-2, IFN-gamma inhibited the virus reproduction, like in the treatment scheme, within the dilution range of 1:50 to 1:500, whereas TNF-T was effective in the range of 1:50. In CMV, the drugs' effect, when used for prevention, was similar to that observed in the treatment scheme. The highest inhibition values were registered for HSV-2, when it was used 24 hours before infection (IFN-gamma--2.25 Ig, dilution range of 1:50; TNF-2--1.0 Ig, dilution range of 1:50). IFN-gamma and TNF-2 exert a synergic action on different stages of virus reproduction. A reliable additive effect was ensured in prevention made 4 hour before infection by IFN-gamma and TNF-T only in experimental CMV infection.

[Isolation of a group of hybrid proteins consisting of tumor necrosis factor alpha and thymosin alpha 1]. / Poluchenie gruppy gibridnykh belkov, sostoiashchikh iz faktora nekroza opukholei alpha i timozina alpha1.

New plasmid DNA coding for the synthesis of three hybrid proteins differing by the position of alpha 1-thymosin at N- and C-terminals of tumor necrosis factor were optimized and constructed. The instability of plasmids at culturing of E. coli strains stored in solid nutrient media was demonstrated. Newly obtained transformants and preserved cells were cultured, this providing a high level of synthesis of hybrid proteins. The effects of culturing temperature and protein structure on protein solubility were shown. Hybrid proteins were purified by chromatography on hydrophobic anion-exchange carriers and blue agarose in the presence of 7 M urea. After dialysis the proteins displayed different cytotoxicity in L-929 cells and were fit for immunobiological studies.

[Thymosin alpha-1 and hybrid proteins consisting of tumor necrosis factor-alpha and thymosin alpha-1 enhance the efficacy of vaccination against the causative agent of plague]. / Timozin al'fa-1 i gibridnye belki, sostoiashchie iz faktora nekroza opukholei al'fa i timozina al'fa-1, uvelichivaiut éffektivnost' vaktsinatsii protiv vozbuditelia chumy.

The influence of the preparation of chemical thymosin alpha 1 (T), recombinant thymosin alpha 1 (rT), tumor necrosis factor (TNF) and hybrid proteins on their basis (T-TNF, TNF-T and T-TNF-T) on the effectiveness of immunization against Y.pestis have been studied. The preparations of T and hybrid proteins exhibit immunostimulating action, enhancing specific immunity when injected at different periods of the vaccinal process against Y.pestis virulent strain 231 in experiments on mice and guinea pigs. The highest effectiveness and reproducibility of results is observed after the use of hybrid protein T-TNF-T. An increase in immunity after the use of the preparations of hybrid proteins is accompanied by the activation of its T-cell element. The influence of rT on the restoration of the immune system of white mice after their exposure to sublethal doses of gamma radiation has been shown.

[Immunobiologic characteristics of hybrid proteins consisting of tumor necrosis factor alpha and thymosin alpha 1]. / Izuchenie immunobiologicheskikh svoistv gibridnykh belkov sostoiashchikh iz faktora nekroza opukholei alpha i timozina alpha 1.

The preparations of alpha 1-thymosin (T), alpha-tumor necrotic factor (TNF) and their based hybrid proteins: T-TNF, TNF-T, and T-TNF-T were studied by using a wide spectrum of immunobiological tests. In the L-929 cells, T-TNF preserved cytotoxicity unique to TNF; TNF-T preserved it 10 times less, and T-NTF-T was completely inactive. TNF-T inhibited the growth of Molt-4, Jm-9, Raji cells by 63 = 84%, and TNF suppressed only Raji cells by 50%. Hybrid proteins caused cell proliferation of the thymus, spleen, and lymph nodes; H-2K, CD4, and CD8 differently increased the expression of thymocytic antigens. The authors found the different effects of the drugs on phagocytosis, the production of antibody-forming cells, delayed reaction, and activation of natural killer cells. The protein T-TNF-T produced the most pronounced action.

[Synthesis of an artificial gene coding for thymosin alpha1 and its expression in Escherichia coli as a hybrid with human tumor necrosis factor]. / Sintez iskusstvennogo gena, kodiruiushchego timozin alpha1, i ego ékspressiia v Escherichia coli v sostave gibridov s faktorom nekroza opukholei cheloveka.

Chemical-enzymatic synthesis and cloning in Escherichia coli of an artificial gene encoding the immunoactive peptide thymosin alpha 1 have been carried out. Recombinant plasmids were constructed which contain fusion genes coding for hybrids of human tumour necrosis factor (TNF) and thymosin alpha 1 as N- or C-terminal part of the hybrid protein. In the C-terminal hybrid protein, TNF and thymosin alpha 1 are linked through a methionine residue, thus allowing for thymosin alpha 1 to be cleaved off the rest of the hybrid protein with cyanogen bromide. In case of the N-terminal hybrid protein, the linker between the thymosin alpha 1 and TNF sequences is the acid-labile dipeptide Asp-Pro. Expression of the hybrid genes in E. coli and properties of the recombinant proteins were studied. The N-terminal hybrid protein was secreted into periplasmic space, in contrast with the C-terminal hybrid protein, which formed insoluble aggregates inside bacterial cells. Procedures for the isolation of both hybrid proteins were developed. The N-terminal hybrid protein displayed full biological activity in the cytotoxic assay on the mouse fibroblast L-929 whereas the C-terminal hybrid protein proved to be much less active. Treatment of the hybrid protein TNF-thymosin alpha 1 with cyanogen bromide lead to a mixture of two polypeptides, from which thymosin alpha 1 was purified to homogeneity by simple chromatographic procedures.

[Synthesis, secretion, and proteolytic degradation of diphtheria toxin in Escherichia coli]. / Sintez, sekretsiia i proteoliticheskaia degradatsiia difteriinogo toksina v Escherichia coli.

The recombinant plasmids have been constructed encoding the synthesis of a full-sized diphtheria toxin from its own or PR, PL-promoters of bacteriophage lambda in Escherichia coli cells. The high level constitutive synthesis of toxin results in slow cell growth and plasmid elimination. The toxin was mainly detected in the periplasm, partially in the membrane and to a less extent in the cytoplasm and culturing medium. The dimeric form of toxin was found in the cytoplasm. Participation of toxin B-subunit in secreting of the toxin into culturing medium is discussed. Proteolytic degradation of the synthesized toxin in different Escherichia coli strains was demonstrated. The process takes place in cytoplasm and periplasm mainly. The main enzyme participating in the process is a La-protease. The data on proteolysis obtained by immunoprecipitation immunoblotting, affinity chromatography and in mini-cells of Escherichia coli are summarized.

[The heterogeneity of collection strains of Yersinia pseudotuberculosis and their molecular biological properties]. / Geterogennost' muzeinykh shtammov Yersinia pseudotuberculosis i ikh molekuliarno-biologicheskie svoistva.

The capacity of Y. pseudotuberculosis strains for disassociation with the appearance of S- and P-forms has been studied. Strains 852 and 9547 show high stability in S-forms, their conversion into R-forms occurring at 40-42 degrees C. Strain 6953 shows pronounced polymorphism and instability of its associations at different growth temperatures. Strain 9532 exists in S- and R-forms which retain their stability during numerous subculturings at different growth temperatures and prolonged storage. This strain has plasmids of 130, 72.2, 5.7 kb. All plasmids are retained in S- and R-forms, i. e. the dissociation of the strain is not accompanied by the loss of plasmids. The conversion of the strain from the S-form into the R-form leads to changes in the structure of lipopolysaccharide and the composition of low-molecular (less than 23 kD) proteins in the outer and inner membranes. In tests on guinea pigs the LD50 of the R-form of the strain is tenfold greater than that of its S-form. The dissociants of strain 9532 are transformed by plasmid DNA with equal efficiency and equally inherit them without selective pressure.

[Specific proteolysis by fibrinolysin-coagulase from Yersinia pestis of Yersinia pseudotuberculosis outer membrane proteins coded by the Ca(2+)-dependence plasmid]. / Spetsificheskii proteoliz fibrinolizinokoagulazoi Yersinia pestis belkov vneshnei membrany, kodiruemykh plazmidoi Ca(2+)-zavisimosti v Yersinia pseudotuberculosis.

The pesticinogenicity 9.5 kb plasmid from Yersinia pestis strain EV76 has been marked by the kanamycin phosphotransferase gene inserted into PstI site and designated pP3. The obtained plasmid pP3 determines the synthesis of 45 kd pesticin, alpha and beta-forms of fibrinolysin coagulase (37 and 35 kd) and the 29, 19 and 13 kd proteins in Escherichia coli mini cells. When transferred into Yersinia pseudotuberculosis strain 6933 the plasmid causes the proteolysis of outer membrane proteins. The 150 kd protein is reduced to 138 kd, the 48.5 kd protein is reduced to 45 kd. The proteins secreted into the cultural medium (51 and 38 kd) are also cleaved. The proteolysis of the 150 kd protein was found to occur at the stage of secretion via the inner membrane. The purified fibrinolysin coagulase from Escherichia coli strain JM83 harbouring the plasmid pP3 induces the proteolysis in vitro of the isolated membrane proteins from Yersinia pseudotuberculosis strain 6953 similar to the proteolysis registered in vivo.

[Cloning of Yersinia pseudotuberculosis calcium-dependence plasmid pYV6953 BamHI fragments and their analysis in Escherichia coli mini-cells]. / Klonirovanie BamHI-fragmentov plazmidy kal'tsiizavisimosti pYV 6953 Yersinia pseudotuberculosis i ikh analiz v mini-kletkakh Escherichia coli.

Calcium dependence plasmid pYV6953 (70.4 kb) in Yersinia pseudotuberculosis cells codes for the major quantities synthesis of 150; 48.5; 19.4 Kd outer membrane proteins and the 51, 38, 27 Kd proteins secreted into the culturing medium. These outer membrane and secreted proteins are synthesized in considerable amounts in Yersinia pseudotuberculosis strains 6953 and 9547 at 37 degrees C and in the absence of calcium ions in the culturing medium. BamHI fragments of the plasmid pYV6953 as components of the recombinant plasmids code for the synthesis of 150; 66.6; 51; 48.5; 47; 38 and 21.5 Kd proteins in Escherichia coli mini cells. The synthesis of 150 and 48.5 Kd proteins is determined by the BamHI fragment 9 of the plasmid pYV6953 (3.3 kb). Addition of up to 8% of ethanol inhibiting the protein synthesis eliminates the 150 Kd protein but not the 48.5 Kd synthesis. The 48.5 Kd protein is concluded to be a subunit of the 150 Kd protein. The plasmid pYV6953 is different from the known plasmids pIB1 and pCD1 plasmids as far as the outer membrane and secreted proteins coded by the plasmids are concerned.

[Plasmids of Yersinia pseudotuberculosis]. / Plazmidy Yersinia pseudotuberculosis.

Plasmids with the sizes of 5.7; 51; 70-77; and 120-130 kb were found in six strains among the ten strains collection of Yersinia pseudotuberculosis. The restriction endonucleases analysis. Southern-blot hybridization and physical maps construction were performed for the plasmids. The 70-77 kb plasmids were found to be analogous to the Ca2(+)-dependence plasmid pYVO19 from Yersinia pestis EV76. The difference between the plasmids of this type is in the insertions or deletions located on the similar fragments of the restriction maps. The 51 kb plasmid has no common fragments with the Ca2(+)-dependence plasmids and does not code for virulence properties of the strain harbouring it. No homology is shared by the 5.7 kb plasmid and the 10 kb plasmid from Yersinia pestis EV76. Replicon of the 5.7 kb plasmid has been used to construct the pVS11 vector plasmid.