A. B. Shevelev

Heterologous expression, purification, and properties of a potato protein inhibitor of serine proteinases

The gene PKPI-B10 [AF536175] encoding in potato (Solanum tuberosum L., cv. Istrinskii) a Kunitz-type protein inhibitor of proteinases (PKPI) has been cloned into the pET23a vector and then expressed in Escherichia coli. The recombinant protein PKPI-B10 obtained as inclusion bodies was denatured, separated from admixtures by ion-exchange fast protein liquid chromatography (FPLC) on MonoQ under denaturing conditions, and renatured. The native protein was additionally purified by ion-exchange FPLC on DEAE-Toyopearl. The PKPI-B10 protein effectively inhibits the activity of trypsin, significantly weaker suppresses the activity of chymotrypsin, and has no effect on other serine proteinases: human leukocyte elastase, subtilisin Carlsberg, and proteinase K, and also the plant cysteine proteinase papain.

IgA-specific proteins of pathogenic bacteria

Data on structure and specificity of bacterial IgA receptors (IgA-binding M-like proteins Arp4 and Sir22 from hemolytic streptococci of serogroup A, β-antigen from hemolytic streptococci of serogroup B, and SSL family proteins from Staphylococcus aureus) are surveyed in this review. The principal conclusion derived from comparison is the fact that all bacterial receptors bind the same site in the IgA molecule overlapping with the binding site of endogenous human IgA receptor CD89. We assume that this site, consisting of spatially close amino acid strands Leu257-Gly259 in domain Cα2 and Pro440-Phe443 in domain Cα3, is subject to conformational rearrangement induced by the binding of antigen in the IgA active site.

Subtilisin protein inhibitor from potato tubers.

A protein with molecular weight of 21 kD denoted as PKSI has been isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii). The isolation procedure includes precipitation with (NH4)2SO4, gel chromatography on Sephadex G-75, and ion-exchange chromatography on CM-Sepharose CL-6B. The protein effectively inhibits the activity of subtilisin Carlsberg (Ki = 1.67 ± 0.2 nM) by stoichiometric complexing with the enzyme at the molar ratio of 1 : 1. The inhibitor has no effect on trypsin, chymotrypsin, and the cysteine proteinase papain. The N-terminal sequence of the protein consists of 19 amino acid residues and is highly homologous to sequences of the known inhibitors from group C of the subfamily of potato Kunitz-type proteinase inhibitors (PKPIs-C). By cloning PCR products from the genomic DNA of potato, a gene denoted as PKPI-C2 was isolated and sequenced. The N-terminal sequence (residues from 15 to 33) of the protein encoded by the PKPI-C2 gene is identical to the N-terminal sequence (residues from 1 to 19) of the isolated protein PKSI. Thus, the inhibitor PKSI is very likely encoded by this gene.

Unknown functions of immunoglobulins A

Traditionally, the function of immunoglobulins A (IgA), the major type of secreted antibodies, has been thought to be restricted to binding antigens outside the epithelium basal membrane. Therefore, effector mechanisms eliminating IgA-opsonized targets have not been investigated so far. However, some indirect observations of infectious agents penetrating into tissues and blood from the environment suggest such mechanisms (analogous to IgG/IgM-dependent activation of complement and natural killers). In the present review, we examine details of IgA structure that might contribute to elucidation of IgA-dependent effector functions in human and animal immunity. Special attention is given to a putative transduction of signal about antigen binding in the active center of IgA from the Fab-to the Fc-superdomain via intramolecular conformational rearrangements. Different structure of the IgA subclasses (IgA1 and IgA2) is examined taking into account probable divergence of their functions in immune response.

Extended plant protection by an epiphytic Pseudomonas sp. derivative carrying the cry9Aa gene from Bacillus thuringiensis galleriae against the pine processionary moth Thaumetopoea pityocampa

Abstract A strain of Pseudomonas sp. isolated from the phyllosphere of Pinus nigra in northern Italy was used for the introduction and high expression level of the gene encoding the Cry9Aa entomocidal toxin from Bacillus thuringiensis spp. galleriae. Laboratory tests showed that the resulting bacterial construct was more efficacious in terms of LC50 when compared to the purified toxin alone, against the first instar larvae of the pine processionary moth (Thaumetopoea pityocampa), suggesting that the encapsulation of the toxin within the bacterial cell may prevent the degradation of the protein. When the efficacy of the strain was compared in a long-term greenhouse experiment (102 days) with that of a commercial preparation of Btk (Foray 48B), the latter was superior in terms of total mortality, but its effectiveness decreased with time at a faster rate than that of the cry9Aa-Pseudomonas. Overall data indicate that the expression of Bt toxins in heterologous epiphytic bacteria offers potential for more efficient and persistent delivery of toxins to the target insect pests.

Molecular cloning of kunitz-type proteinase inhibitor group B genes from potato

Eighteen clones representing copies of four Kunitz-type proteinase inhibitor group B genes (PKPI-B) obtained by polymerase chain reaction cloning of potato (Solanum tuberosum L. cv. Istrinskii) genomic DNA were sequenced and analyzed. Three new genes were found and named PKPI-B1, PKPI-B2, and PKPI-B10: these were represented by five, two, and seven clones, respectively. The remaining four clones corresponded to the formerly characterized PKPI-B9 gene. These data show that at least four PKPI-B encoding genes are harbored in the genome of potato cv. Istrinskii. Their analysis suggests that variability of PKPI-B encoding genes in potato is limited and could be explained by cross-hybridization events in the ancestor forms rather than by random mutagenesis.

The study of adaptation mechanisms of Yarrowia lipolytica yeast to alkaline conditions by means of proteomics

Using proteomic technologies—two-dimensional electrophoresis in denaturing conditions in combination with mass spectroscopy of MALDI-TOF proteins—we demonstrated, for the first time, that the most noticeable alteration of protein composition of a Yarrowia lipolytica cell during adaptation to alkaline conditions was an increase of mitochondrial proteins relatively to proteins of cytoplasm.

Sup35p yeast prion-like protein as an adapter for production of the Gag-p55 antigen of HIV-1 and the L-chain of botulinum neurotoxin in Saccharomyces cerevisiae.

Effective expression of the HIV-1 core protein Gag-p55 was obtained in Saccharomyces cerevisiae under control of the inducible UASgal/CYC1 promoter as a translational fusion with the prion-forming NM domain of the translation terminator Sup35p (eRF3) of S. cerevisiae. where only poor expression of the original-type Gag-p55 was observed. A deletion within the Sup35NM prion-forming domain altering Sup35-associated [PSI] inheritance did not compromise expression of the Sup35NM Gag-p55 fusion protein. Therefore, either the mechanism of this phenomenon is not directly related to the effect of Sup35p prion-formation or the modified protein maintains residual prion-forming abilities. The recombinant Sup35p-Gag-p55 protein was quite stable under boiling in an alkali/sodium dodecyl sulfate (SDS) solution and completely retained its antigenic properties. Moreover, 10-min boiling of the native yeast cells in this solution allowed immediate inhibition of lysosomal and other yeast proteases, responsible for autolysis of many natural and recombinant proteins. The use of this method of preliminary enrichment for the recombinant fusion protein Sup35p-Gag-p55 with the SDS-alkaline extraction could be useful for yeast heterologous expression and purification of other of insoluble and unstable proteins. A translational fusion with the NM domain of Sup35p was also used to produce another poorly soluble protein, the L-chain of botulinum exotoxin A, in S. cerevisiae. When the Sup35p fragment was removed from the recombinant construct encoding a fused Sup35/BoNT protein, a dramatic drop in both transformation efficiency and growth rate of transformants was shown.

Identification of Proteins Involved in pH Adaptation in Extremophile Yeast Yarrowia lipolytica

Extremophile yeast Yarrowia is now commonly acknowledged as a prospective industrial microorganism and a highly promising cell model. This is due to several unique properties of this organism. First, it is able to grow rapidly on a broad range of organic substrates including waste water, oil paraffin and non-natural substances. This property, due to the presence of peroxisomes, allows the application of Y. lipolytica for waste management, water and soil bioremediation and for conversion of fossil organic compounds and pollutants to feed ingredients. Second, due to a high metabolic activity and resistance to chemical stresses, Y. lipolytica is able to produce aggressive organic compounds (e.g. succinate) at high yields. Third, Y. lipolytica provides a well-established model of a dimorphiс transition between a yeast-like state and a mycelium forming fungi. This property enables the application of Y. lipolytica as a model for drug discovery for therapeutic control of Candida albicans (the most common fungal pathogen in humans) and other pathogenic fungi. In contrast to C.albicans, Y.lipolytica is easily cultivated and has a complete sexual cycle. Thus, genetic mating analyses are readily applicable. Dimorphic transition in Y. lipolytica is also considered as the simplest model of cell differentiation in eukaryotes. Taken together, these factors provided the incentive for complete sequencing of the Y. lipolytica genome. This has been carried out by GenoLevures Consortium in France (Dujon et al, 2004). Availability of the complete genomic sequence opened access to proteomic assays to be combined with functional studies of Y. lipolytica. The proteomic approach has been successfully used by Morin et al (2007) for the identification of major proteins involved in the dimorphic transition of Y. lipolytica.

Molecular cloning and expression of genes of Kunitz-type C protease inhibitors from potato

We cloned the products of polymerase chain reaction of the genome DNA of potato (Solanum tuberosum L., Istrinskii cultivar) and isolated 35 clones, which represent copies of eight genes encoding Kunitz type C proteases. Their nucleotide sequences were established. All the genes were found for the first time and designated as PKPI-C1-PKPI-C8. The gene PKPI-C2, which we had earlier presumed to encode the subtilisin PKSI inhibitor, was cloned into pQE30 vector and then expressed in Escherichia coli cells. The recombinant protein PKPI-C2 underwent spontaneous folding and transformation into a soluble state. We purified it to homogeneity by affinity chromatography. The PKPI-C2 protein efficiently inhibited subtilisin Carlsberg activity and did not act on trypsin, chymotrypsin, or papain.