Thermostable Lichenase from Clostridium thermocellum as a Host Protein in the Domain Insertion Approach.

Clostridium thermocellum lichenase (endo-ß-1,3;1,4-glucan-D-glycosyl hydrolase, EC 3.2.1.73 (P29716)) has been tested for the insertion of two model fluorescent proteins (EGFP and TagRFP) into two regions of this enzyme. Functional folding of the resulting proteins was confirmed by retention of lichenase activity and EGFP and TagRFP fluorescence. These results convincingly demonstrate that (i) the two experimentally selected lichenase loop regions may serve as the areas for domain insertion without disturbing enzyme folding in vivo; (ii) lichenase permits not only single but also tandem insertions of large protein domains. High specific activity, outstanding thermostability, and efficient in vitro refolding of thermostable lichenase make it an attractive new host protein for the insertional fusion of domains in the engineering of multifunctional proteins.

Expression of Soluble Active Interferon αA in Escherichia coli Periplasm by Fusion with Thermostable Lichenase Using the Domain Insertion Approach.

A recombinant DNA in which the interferon αA (IFN-αA) gene sequence is integrated into a loop region of the gene coding thermostable lichenase was constructed. This approach of insertion fusion with thermostable lichenase is advantageous in terms of increasing the solubility, stability, and production of the fusion partner in soluble form in general and in the periplasm of bacterial cells in particular. Thus, the insertion of IFN-αA into the loop (53 a.a.) of thermostable lichenase from Clostridium thermocellum resulted in effective expression of the soluble form of the recombinant protein in the periplasm of Escherichia coli without any compromise in biological activity of IFN-αA, while the thermostable lichenase retained its ability for functional folding without dramatic loss of its basic activity and thermostability.

Clostridium thermocellum thermostable lichenase with circular permutations and modifications in the N-terminal region retains its activity and thermostability.

The Clostridium thermocellum lichenase (endo-ß-1,3;1,4-glucan-D-glycosyl hydrolase) displays a high thermostability and specific activity and has a compact protein molecule, which makes it attractive, in particular, for protein engineering. We have utilized in silico analysis to construct circularly permuted (CP) variants and estimated the retained activity and thermostability. New open termini in the region of residues 53 or 99 in two lichenase CP variants (CN-53 and CN-99) had no effect on their activity and thermal tolerance versus another variant CP variant, CN-140 (cut in the region of residue 140), which displayed a dramatic decrease in the activity and thermostability. Construction and further activity and thermostability testing of the modified lichenase variants (M variants) and CP variants with peptides integrated via insertion fusion have demonstrated that the N-terminal regions in the lichenase catalytic domain (53 and 99 amino acid residues) that permit circular permutations with retention of activity and thermostability of the enzyme as well as the region between the C and N termini of the native lichenase in thermostable and active lichenase variants (CN-53 and CN-99) may be used for integrating small peptides without the loss of activity and thermostability. These findings not only suggest that CP predictions can be used in search for internal integration sites within protein molecule, but also form the background for further enzymatic engineering of the C. thermocellum thermostable lichenase aiming to create new fusion proteins.

[Range of Gene candidates involved in biosynthesis of laccase from basidiomycete Trametes hirsuta].

A comparative analysis of transcripts from the basidiomycota T. hirsuta grown with and without an inducer of the laccase biosynthesis was carried out. Methods of subtraction hybridization and massive parallel sequencing were used for this purpose. Unique transcripts encoded by genes that have a relatively high level of expression and belong to different gene ontology categories were identified. Also, a large number of transcripts were found to encode for predicted proteins, as well as noncoding transcripts. The latter may represent regulatory RNA molecules. Transcripts that increase their abundance when the laccase synthesis is induced are selected as gene-candidates involved in the laccase biosynthetic pathway.

[Set of module vectors for stable or transient expression of heterologous genes in plants].

A set of module vectors for stable or transient gene expression in plants was constructed with regard to the majority of factors ensuring efficient heterologous gene expression in plants. The vectors are convenient to clone new regulatory elements and genes of interest via simple molecular cloning procedures. The vectors can be used to obtain transgenic plants with stable heterologous gene expression as well as to achieve transient expression because one vector includes the gene for the tomato bushy stunt virus p19 protein, which acts as a suppressor of posttranscriptional gene silencing.

[Expression of heterologous genes in plant systems: new possibilities].

The basic methods used in current practice for stable and transient expression of heterologous genes in plants are presented and compared. The key areas of research in the heterologous expression of genes in plants have been identified by analyzing literature and experimental data: modeling of metabolic pathways; creation of marker-free transgenic plants; the search for new regulatory elements and plant genes influencing the efficiency of expression of heterologous genes in plants; development of new methods for analyzing of transgenic plants and new approaches to the expression of heterologous genes in plants.

[Efficiency of evaluating the carcinogenicity of chemical substances in short-term tests and the SAR model].

The efficiency of estimating the carcinogenic activity of chemical substances was compared for five short-term tests and structure-activity relationships (SAR) analysis. The sample included 84 substances with known biological testing results obtained by the Ames test, bacterial SOS chromotest (SOS), chromosome aberration (CA) cytogenetic test, sister chromatid exchange (SHE) test, and gene mutation (GM) test with mammalian cells in vitro and by carcinogenicity assays in rodents in vivo. Structural descriptors were selected using an original database, which included the structural formulas of substances with known carcinogenic activity in rodents. Original software was created to generate and select the descriptors that statistically coincided with carcinogenic activity. The descriptors that were associated exclusively with carcinogenic substances from the database and the tests that produced positive results exclusively with carcinogens were used to evaluate the carcinogenic activity of the substances. A combination of three short-term tests (Ames, SOS, and CA tests) and the SAR model proved to identify carcinogenicity for more than 60% of carcinogens.

[Dependence of the carcinogenicity of nitro compounds on their structural characteristics].

A new approach to the description of quantitative structure-activity relationships (QSAR analysis) based on compound descriptors has been used. The effect of the structural characteristics of nitric compounds on their carcinogenicity has been studied. It has been found that the carcinogenicity of nitric compounds is determined by the presence of furyl and/or azole heterocycles not condensed with benzene rings in their molecular structures. The carcinogenicity of the nitric compounds in which the benzene ring is the basic structure is determined by the presence of other substituents (halogens, amines, and methyl groups) and their positions relative to the nitro group.

[Use of ensemble structural descriptors for increasing the efficiency of QSAR study].

A new concept of describing the dependence of the mutagenic activity of a chemical substance on its structure (QSAR analysis) is presented. It involves ensemble descriptors, which are combinations of unrelated fragments of molecular structure. Software has been developed to generate various structural fragments of molecules and their combinations (ensembles) and select ensemble descriptors of statistical significance for the biological activity of a chemical. By examples of univocal ensemble descriptors consisting of two structural fragments and present only in active or only in inactive compounds, it has been shown that the efficiency of QSAR study can be increased fourfold or more. The approach has been applied to a set of 105 compounds whose mutagenic effect on rodent sex cells is known.