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Filatova L.Y.,Moscow State University | Donovan D.M.,U.S. Department of Agriculture | Ishnazarova N.T.,Moscow State University | Foster-Frey J.A.,U.S. Department of Agriculture | And 5 more authors.
Applied Biochemistry and Biotechnology | Year: 2016

A staphylolytic fusion protein (chimeric enzyme K-L) was created, harboring three unique lytic activities composed of the LysK CHAP endopeptidase, and amidase domains, and the lysostaphin glycyl-glycine endopeptidase domain. To assess the potential of possible therapeutic applications, the kinetic behavior of chimeric enzyme K-L was investigated. As a protein antimicrobial, with potential antigenic properties, the biophysical effect of including chimeric enzyme K-L in anionic polymer matrices that might help reduce the immunogenicity of the enzyme was tested. Chimeric enzyme K-L reveals a high lytic activity under the following optimal (opt) conditions: pHopt 6.0–10.0, topt 20–30 °C, NaClopt 400–800 mM. At the working temperature of 37 °C, chimeric enzyme K-L is inactivated by a monomolecular mechanism and possesses a high half-inactivation time of 12.7 ± 3.0 h. At storage temperatures of 22 and 4 °C, a complex mechanism (combination of monomolecular and bimolecular mechanisms) is involved in the chimeric enzyme K-L inactivation. The optimal storage conditions under which the enzyme retains 100 % activity after 140 days of incubation (4 °C, the enzyme concentration of 0.8 mg/mL, pH 6.0 or 7.5) were established. Chimeric enzyme K-L is included in complexes with block-copolymers of poly-l-glutamic acid and polyethylene glycol, while the enzyme activity and stability are retained, thus suggesting methods to improve the application of this fusion as an effective antimicrobial agent. © 2016 Springer Science+Business Media New York

Ryzhov K.A.,Federal State Budgetary Institution | Nosik M.N.,Federal State Budgetary Institution | Kravtchenko A.V.,Federal Budget Institution of Science
Voprosy Virusologii | Year: 2015

In this work, a total of 200 samples from the HIV-infected individuals were analyzed: 50 samples from the Saha Republic (Yakutia), 50 samples from the Vologda Region (City of Cherepovets), and 100 samples from the Moscow Region (Moscow and Moscow Region). All samples were obtained from the patients who were not undergoing antiretroviral therapy. It was detected that the regulatory genes vif, vpr, vpu, rev, tat, and nef were amplified with moderate sensitivity after one-stage amplification. When those samples were analyzed by the nested PCR the detection ratio was much higher. While studying nef-gene the phenomena of the splicing in cells cores was detected at the advanced stages of the HIV-infection (3 and 4 stages). At the same time, the splicing was not detected at the earlier stages of the HIV-infection. This effect might be the cause of the transition from asymptomatic stage of the infection to the advanced stage. It was also shown for the first time that the variability of the regulatory genes correlated with the virus subtype.

Becker S.C.,U.S. Department of Agriculture | Swift S.,U.S. Department of Agriculture | Korobova O.,Federal Budget Institution of Science | Schischkova N.,Federal Budget Institution of Science | And 3 more authors.
FEMS Microbiology Letters | Year: 2015

Increases in the prevalence of antibiotic-resistant strains of Staphylococcus aureus have elicited efforts to develop novel antimicrobials to treat these drug-resistant pathogens. One potential treatment repurposes the lytic enzymes produced by bacteriophages as antimicrobials. The phage Twort endolysin (PlyTW) harbors three domains, a cysteine, histidine-dependent amidohydrolases/peptidase domain (CHAP), an amidase-2 domain and a SH3b-5 cell wall binding domain (CBD). Our results indicate that the CHAP domain alone is necessary and sufficient for lysis of live S. aureus, while the amidase-2 domain is insufficient for cell lysis when provided alone. Loss of the CBD results in ~10X reduction of enzymatic activity in both turbidity reduction and plate lysis assays compared to the full length protein. Deletion of the amidase-2 domain resulted in a protein (PlyTW Δ172-373) with lytic activity that exceeded the activity of the full length construct in both the turbidity reduction and plate lysis assays. Addition of Ca2+ enhanced the turbidity reduction activity of both the full length protein and truncation constructs harboring the CHAP domain. Chelation by addition of EDTA or the addition of zinc inhibited the activity of all PlyTW constructs.

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