Expression of Highly Active Bacterial Phospholipase A2 in Yeast Using Intein-Mediated Delayed Protein Autoactivation.

Phospholipase A2 (PLA2) has found extensive use in industry. However, recombinant PLA2 production in different expression systems is a difficult task because of its toxicity to cell membranes. We report here the development of an effective method for production of highly active PLA2 from Streptomyces violaceoruber strain A-2688 in the yeast Saccharomyces cerevisiae. The method is based on the use of the PRP8 mini-intein (from Penicillium chrysogenum) inserted into the phospholipase sequence with the purpose of temporal inactivation of the enzyme and its subsequent delayed autoactivation. We demonstrate that the most effective site for intein insertion is Ser76 of the mature phospholipase. As a result of intein-containing precursor secretion from yeast cells and its subsequent autocatalytic splicing, highly active enzyme accumulated in the yeast culture fluid. The properties of the obtained recombinant phospholipase A2 protein were similar to those of the native Streptomyces violaceoruber PLA2 protein. A possible evolutionary role of delayed autoactivation of intein-containing proteins is also discussed.

Intranasal Human Recombinant Modified Glucagon-Like Peptide-1: High Antihyperglycemic Activity and Duration of Action in Mice.

The study compared effectiveness of intranasal administration of glypin (human recombinant modified glucagon-like peptide-1) and reference drug Victoza in BALB/c mice. The minimum effective dose of intranasal glypin was 0.5 mg/kg, and a 2-fold elevation of this dose increased the parameters of glypin activity up to the maximal levels. During the first 2 h after intranasal administration, the effectiveness of glypin greatly surpassed that of Victoza. Duration of action and the time course of antihyperglycemic activity of intranasal glypin (1 mg/kg) matched to the best parameters attained during its subcutaneous application. A high effectiveness of intranasal glypin opens the vistas to its further examination and employment.

The Modified Heparin-Binding L-Asparaginase of Wolinella succinogenes.

The modified asparaginase Was79 was derived from the recombinant wild-type L-asparaginase of Wolinella succinogenes. The Was79 contains the amino acid substitutions V23Q and K24T responsible for the resistance to trypsinolysis and the N-terminal heparin-binding peptide KRKKKGKGLGKKR responsible for the binding to heparin and tumor K562 cells in vitro. When tested on a mouse model of Fischer lymphadenosis L5178Y, therapeutic efficacy of Was79 was significantly higher than that of reference enzymes at all single therapeutic doses used (125-8000 IU/kg). At Was79 single doses of 500-8000 IU/kg, the complete remission rate of 100 % was observed. The Was79 variant can be expressed intracellularly in E. coli as a less immunogenic formyl-methionine-free form at high per cell production levels.

[New antibiotics produced by Bacillus subtilis strains].

Two Bacillus subtilis strains isolated from the fruiting body of a basidiomycete fungus Pholiota squarrosa exhibited a broad range of antibacterial activity, including those against methicillin-resistant Staphylococcus aureus INA 00761 (MRSA) and Leuconostoc mes6nteroides VKPM B-4177 resistant to glycopep-> tide antibiotics, as well as antifungal activity. The strains were identified as belonging to the "B. subtilis" com- plex based on their morphological and physiological characteristics, as well as by sequencing of the 16S rRNA gene fragments. Both strains (INA 01085 and INA 01086) produced insignificant amounts of polyene antibiotics (hexaen and pentaen, respectively). Strain INA 01086 produced also a cyclic polypeptide antibiotic containing Asp, Gly, Leu, Pro, Tyr, Thr, Trp, and Phe, while the antibiotic of strain INA 01085 contained, apart from these, two unidentified nonproteinaceous amino acids. Both polypeptide antibiotics were new compounds efficient against gram-positive bacteria and able to override the natural bacterial antibiotic resistance.

[Anti-peptide antibodies specifically recognize the L1 protein of human papilloma virus type 31].

Antibodies that specifically recognize the capsid protein (L1) of human papillomavirus (HPV) are an important tool necessary for designing vaccines against HPV infection. In this work, we have predicted and synthesized peptide fragments mimicking B cell epitopes of L1 HPV type 31 (sequences 49-65, 131-145, 172-189, 349-362 and 402-414), and conjugated their to KLH and BSA to generate the L1-31-specific anti-peptide antibodies in mice. Variants of recombinant L1-31, including full-size and mutants with C-terminal single amino acid changes and deletions and full-size L1-16 were produced in the yeast using monitoring with L1 HPV16-specific monoclonal antibody. Testing of anti-peptide antisera in ELISA showed that antibodies to peptides 49-65 and 172-189 were capable to recognize specifically L1-31 protein, but not L1-16 one. Such antibodies may be used for assay of L1-31 production in various expression systems.

[Cloning and expression of bacteriophage FMV lysocyme gene in cells of yeasts Saccharomyces cerevisiae and Pichia pastoris].

Cloning, sequencing, and expression of the gene for soluble lysozyme of bacteriophage FMV from Gram-negative Pseudomonas aeruginosa bacteria were conducted in yeast cells. Comparable efficiency of two lysozyme expression variants (as intracellular or secreted proteins) was estimated in cells of Saccharomyces cerevisiae and Pichia pastoris. Under laboratory conditions, yeast S. cerevisiae proved to be more effective producer of phage lysozyme than P. pastoris, the yield of the enzyme in the secreted form being significantly higher than that produced in the intracellular form.

Antibody fragments may be incorrectly processed in the yeast Pichia pastoris.

We have studied the efficiency of N-terminal processing of the antibody light chain depending on the structure of the leader sequence when expressed in the yeast Pichia pastoris. The humanized light kappa-chain of the murine antibody H3-1 and the Saccharomyces cerevisiae alpha-factor pre-pro-leader sequence (pre-pro-alpha-F) were used as models. The use of pre-region of the pre-pro-alpha-F alone or together with the Glu-Ala-linker leads to the slightly increased yield of the secreted L-chain but was accompanied by the incomplete N-terminal processing of the secreted product.

The p1 protein of the yeast transposon Ty1 can be used for the construction of bi-functional virus-like particles.

Virus-like particles (VLPs) containing heterologous proteins are often used as vaccines. Two approaches for the construction of bi-functional VLPs using hybrid protein pl-380 of the TY1 transposon of Saccharomyces yeast are described. We have shown that both C- and N-termini of p1-380 can be used for the expression of heterologous peptides. Peptides from A. Fumigatus Asp f 2, expressed at the C- and/or N-termini of p1-380, did not interfere with VLP self-assembling, were accessible for antibodies and hence were exposed at the VLP surface. Another way to obtain bivalent VLPs is the formation of mixed particles, which co-express two hybrid pl proteins with different heterologous protein fragments at the C-terminus. To do it the yeast cells were transfected with a mixture of two recombinant DNA coding Asp f 2 peptide and green fluorescent protein (Gfp). We have shown that both Asp f 2 peptide and Gfp are expressed within the same particle. To evaluate biological activity of bi-functional VLP a construction containing peptides representing dominant T- and B-cell epitopes of Asp f 2 was produced. Bi-functional particles were more potent in stimulating memory immune responses. These results demonstrate new possibilities of pl-380 based expression system to produce multifunctional VLPs.

[Coregulation of alternative transcripts of the STA2 gene in Saccharomyces cerevisiae]. / Koreguliatsiia al'ternativnykh transkriptov gena STA2 drozhzhei Saccharomyces cerevisiae.

It is known that upon STA2 gene expression in Saccharomyces cerevisiae cells, two transcripts of different lengths are formed. These fragments contain different start codons of translation (AUG1 and AUG2) located in the same reading frame. The ratio between expression levels of proteins translated from AUG1 and AUG2 was invariably about 2:7 and did not depend on the choice of the reporter product (secreted glucoamylase (GA) or beta-galactosidase accumulated in cells). Neither this ratio depended on glucose repression/derepression. Based on the assumed proportional relationship between levels of transcription and translation of the STA2 gene in yeast cells, all these results cumulatively indicate that the two STA2 transcripts are coregulated. The production of secreted GA was also shown to be markedly stimulated at the posttranscriptional level under conditions of glucose repression.

Destabilase from the medicinal leech is a representative of a novel family of lysozymes.

Intrinsic lysozyme-like activity was demonstrated for destabilase from the medicinal leech supported by (1) high specific lysozyme activity of the highly purified destabilase, (2) specific inhibition of the lysozyme-like activity by anti-destabilase antibodies, and (3) appreciable lysozyme-like activity in insect cells infected with recombinant baculoviruses carrying cDNAs encoding different isoforms of destabilase. Several isoforms of destabilase constitute a protein family at least two members of which are characterized by lysozyme activity. The corresponding gene family implies an ancient evolutionary history of the genes although the function(s) of various lysozymes in the leech remains unclear. Differences in primary structures of the destabilase family members and members of known lysozyme families allow one to assign the former to a new family of lysozymes. New proteins homologous to destabilase were recently described for Caenorhabditis elegans and bivalve mollusks suggesting that the new lysozyme family can be widely distributed among invertebrates. It remains to be investigated whether the two enzymatic activities (isopeptidase and lysozyme-like) are attributes of one and the same protein.

Inulase-secreting strain of Saccharomyces cerevisiae produces fructose.

The gene encoding inulase of the yeast Kluyveromyces marxianus (INU1Km) was cloned and expressed in the inulin-negative yeast Saccharomyces cerevisiae. Cells of S. cerevisiae transformed with the INU1Km gene have acquired extracellular inulase activity and were able to grow in the medium with inulin as a sole carbon source. The S. cerevisiae strain was constructed that is capable of heterologous expression of secreted K. marxianus inulase and is defective in fructose uptake due to null-mutations of the hexokinase structural genes HXK1 and HXK2. When grown in inulin-containing media, this strain is capable of accumulating at least 10% glucose-free fructose in the culture liquid.

[Cloning the Saccharomycopsis fibuligera alpha-amylase gene and its expression in Saccharomyces cerevisiae]. / Klonirovanie gena alpha-amilazy drozhzhei Saccharomycopsis fibuligera i ego ékspressiia v Saccharomyces cerevisiae.

The alpha-amylase gene of yeast Saccharomycopsis fibuligera was cloned, partially sequenced, and expressed in Saccharomyces cerevisiae. Four amino acid substitutions were identified in the enzyme structure as compared to the earlier cloned gene. The Saccharomycopsis fibuligera alpha-amylase gene was expressed in Saccharomyces cerevisiae under the control of its own promoter. The native alpha-amylase signal peptide provided an efficient secretion of the enzyme by Saccharomyces cerevisiae. The efficiency of the alpha-amylase secretion was 98%. The pH optimum of the enzyme secreted by Saccharomyces cerevisiae was 4.0.

Host cell properties and external pH affect proinsulin production by Saccharomyces yeast.

The expression of a hybrid gene encoding an alpha-factor prepro leader peptide-miniproinsulin (MPI) fusion [MPI is the same as the LysArg human insulin precursor described by Thim et al. (1986)] was tested in a series of isogenic yeast strains to investigate the influence of some genetic and physiological factors on heterologous production in yeast. We found that: (i) an MF alpha 1 gene disruption in haploid cells, as well as MF alpha 1 gene product expression in diploid cells, do not affect the MPI secretion level; (ii) under conditions of exogenous leucine availability, MPI production is hindered by leucine auxotrophy (a leu2 mutation); (iii) rho- mutations increase the per-cell MPI yield approximately three-fold; (iv) the MPI yield is apparently dependent on the pH of the culture medium: the higher the external pH, the larger the per-cell MPI yield.

A strategy for construction of industrial strains of distiller's yeast.

A procedure was developed for construction of industrial strains of distiller's yeast (Saccharomyces cerevisiae). It includes several steps: construction of congenic genetically marked haploid strains of opposite mating types starting from an industrial strain of hybrid nature, integrative transformation of the above haploid strains with a DNA fragment containing an expression cassette responsible for new technological facilities, and hybridization of transformants and isolation of final industrial homozygous strains under experimental conditions simulating commercial fermentation processes. This strategy permits the generation of strains that have desirable characteristics of traditional races of distiller's yeast along with new technological facilities determined by the particular expression cassette. Using this procedure, we have constructed an industrial strain with improved amylolytic activity. (c) 1995 John Wiley & Sons, Inc.

[Cloning and expression of the gene for thermostable pullulanase from Clostridium thermohydrosulfuricum in Escherichia coli]. / Klonirovanie i ékspressiia gena termostabil'noi pullulanazy Clostridium thermohydrosulfuricum v kletkakh Escherichia coli.

Using a pUC19-based genomic library of the anaerobic thermophilic bacterium C. thermohydrosulfuricum a DNA fragment that confers pullulanase activity to E. coli cells has been identified. Subcloning and restriction mapping procedures was carried out and the primary structure of the 5'-region of the pullulanase gene (pul) was determined. The pul enzyme was shown to be a protein with molecular weight of approximately 60,000. It was found that both pullulanase and glucoamylase activities resides in pullulanase. The intracellular distribution of pullulanase was studied. An E. coli strain that produces large amounts of thermostable pullulanase has been constructed.

[Isolation and characteristics of thermostable pullulanase from Clostridium thermohydrosulfuricum produced by Escherichia coli cells]. / Vydelenie i kharakteristika termostabil'noi pullulanazy Clostridium thermohydrosulfuricum, produtsiruemoi kletkami Escherichia coli.

The expressed gene (pul) for a thermostable pullulanase from Clostridium thermohydrosulfuricum was cloned into Escherichia coli. The enzyme was purified from cell extracts of E. coli by thermoinactivation, ammonium sulphate precipitation and gel exclusion. The purified enzyme was characterized as monomer with both pullulanase and glucoamylase activities. The general physico-chemical and catalytic properties of this enzyme were obtained. In particular, pullulanase and glucoamylase activities were stable and optimally active at 65 degrees C. The pH optimum for activity was 5.8. The amino acid composition and amino acid sequence of N-terminal end were estimated.