Expression of proteins encoded by foreign genes in Saccharomyces cerevisiae

The yeast, Saccharomyces cerevisiae is currently used for the production of recombinant DNA-generated proteins derived from a variety of eukaryotic organisms. The applications of a yeast-based technology in the production of proteins for pharmaceutical and industrial purposes is discussed including current methods for introducing recombinant genes into yeast and strategies for maximizing their expression.     

Expression of foreign antigenic determinants on bacterial envelope proteins

Conclusions and perspectives We have demonstrated that the LamB153 vector was expressed in attenuated Salmonella typhi and typhimurium (Charbit et al., unpublished) so that the development of live oral vaccines based on this vector can be envisaged.Using the same procedure for detecting permissive sites, we have constructed a number of other expression vectors based on other sites in LamB (Charbit et al., in preparation), but also based on other envelope proteins of the system for maltose and maltodextrins transport (Duplay et al., 1987; Martineau, unpublished results; Dassa, unpublished results). This provides a useful material to study the influence on the immune response of the site of insertion of the foreign epitope into a protein and of the location of the protein within the bacterial cell.     

Phospholipid transfer proteins in microorganisms

Phospholipid transfer activity has been demonstrated in cell lysates of Saccharomyces cerevisiae, Rhodopseudomonas sphaeroides and Bacillus subtilis, and proteins facilitating phospholipid transfer from the first two organisms have recently been purified. The phospholipid transfer protein from S. cerevisiae has mol. wt. 35 000 with a specificity of transfer for phosphatidylinositol and phosphatidylcholine. The purified phospholipid transfer protein from R. sphaeroides has mol. wt. 27 000 and, although it has the ability to transfer all phospholipid species tested it displays a preference for phosphatidylglycerol. The cellular levels of phospholipid transfer activity in both S. cerevisiae and R. sphaeroides are not strictly related to the level of subcellular membranes. However, in photosynthetically grown R. sphaeroides, the distribution of the activities between soluble and membrane-associated forms is correlated with the level of intracytoplasmic membrane (a postulated membrane substrate).     

Heat-shock proteins and stress tolerance in microorganisms.

Heat-shock proteins help microorganisms cope with the toxic effects of a wide variety of stresses. Some help the organism grow under moderately stressful conditions, others help it to survive more extreme conditions. Surprisingly, the relative importance of individual proteins differs between organisms.     

Novel proteins for homocysteine biosynthesis in anaerobic microorganisms

The metabolic network for sulfide assimilation and trafficking in methanogens is largely unknown. To discover novel proteins required for these processes, we used bioinformatics to identify genes co‐occurring with the protein biosynthesis enzyme SepCysS, which converts phosphoseryl‐tRNA^Cys to cysteinyl‐tRNA^Cys in nearly all methanogens. Exhaustive analysis revealed three conserved protein families, each containing molecular signatures predicting function in sulfur metabolism. One of these families, classified within clusters of orthologous groups (COG) 1900, possesses two conserved cysteine residues and is often found in genomic contexts together with known sulfur metabolic genes. A second protein family is predicted to bind two 4Fe‐4S clusters. All three genes were also identified in more than 50 strictly anaerobic bacterial genera from nine distinct phyla. Gene‐deletion and growth experiments in Methanosarcina acetivorans, using sulfide as the sole sulfur source, demonstrate that two of the proteins (MA1821 and MA1822) are essential to homocysteine biosynthesis in a background lacking an additional gene for sulfur insertion into homocysteine. Mutational analysis confirms the importance of several structural elements, including a conserved cysteine residue and the predicted 4Fe‐4S cluster‐binding domain.     

Characteristics of proteins synthesized by hydrogen-oxidizing microorganisms

The study was conducted to determine the biological value of proteins synthesized by hydrogen-oxidizing microorganisms—the hydrogen bacteria Alcaligenes eutrophus Z1 and Ralstonia eutropha B5786 and the CO-resistant strain of carboxydobacterium Seliberia carboxydohydrogena Z1062. Based on a number of significant parameters characterizing the biological value of a product, the proteins of hydrogen-oxidizing microorganisms have been found to occupy an intermediate position between traditional animal and plant proteins. The high total protein in biomass of these microorganisms, their complete amino acid content, and availability to proteolytic enzymes allow for us to consider these microorganisms as potential protein producers.     

Integrin-like proteins in Candida spp. and other microorganisms

The vertebrate integrins provide a paradigm for cell surface proteins involved in adhesion and morphogenesis. However, homologs of integrins have been found in more primitive organisms. This review will discuss the evidence for surface proteins in Candida albicans and Candida tropicalis that contain motifs reminiscent of integrins and will analyze the contributions of one of these proteins, Int1p, to adhesion, morphogenesis, and virulence. Other microorganisms thought to express integrin-like proteins will also be addressed.     

Uncoupling proteins in mitochondria of plants and some microorganisms.

Uncoupling proteins, members of the mitochondrial carrier family, are present in mitochondrial inner membrane and mediate free fatty acid-activated, purine-nucleotide-inhibited H+ re-uptake. Since 1995, it has been shown that the uncoupling protein is present in many higher plants and some microorganisms like non-photosynthetic amoeboid protozoon, Acanthamoeba castellanii and non-fermentative yeast Candida parapsilosis. In mitochondria of these organisms, uncoupling protein activity is revealed not only by stimulation of state 4 respiration by free fatty acids accompanied by decrease in membrane potential (these effects being partially released by ATP and GTP) but mainly by lowering ADP/O ratio during state 3 respiration. Plant and microorganism uncoupling proteins are able to divert very efficiently energy from oxidative phosphorylation, competing for deltamicroH+ with ATP synthase. Functional connection and physiological role of uncoupling protein and alternative oxidase, two main energy-dissipating systems in plant-type mitochondria, are discussed.     

Various aspects of topogenesis of foreign proteins

The paper considers some aspects of the processing of precursors of foreign proteins, their interaction with the secretory system, directed changes of protein compartmentalization, and protection of foreign proteins from proteolysis.     

Use of synthetic DNA in expression of foreign proteins

Synthetic DNAs and oligonucleotides, which can be prepared conveniently by combining chemical synthesis and enzymatic methods, have been used extensively in recombinant DNA research. Examples include total gene synthesis, probes for the isolation of specific genes from cDNA or genomic libraries, linkers containing specific restriction sites for cloning, primers for DNA and RNA sequencing, and primers for the construction of specific mutations (either deletion, insertion or point mutations) by oligonucleotide-directed site-specific mutagenesis.This article reviews recent advances in the chemical and enzymatic synthesis of oligo- and polynucleotides and the application of synthetic DNA to the expression of foreign proteins. The synthesis of genes, including structural genes and regulatory genes are reviewed. Oligonucleotide-directed site-specific mutagenesis and use of synthetic DNA to optimize foreign protein expression are also discussed.     

Production of foreign proteins using plastid transformation

In the past decades, the progress made in plant biotechnology has made possible the use of plants as a novel production platform for a wide range of molecules. In this context, the transformation of the plastid genome has given a huge boost to prove that plants are a promising system to produce recombinant proteins. In this review, we provide a background on plastid genetics and on the principles of this technology in higher plants. Further, we discuss the most recent biotechnological applications of plastid transformation for the production of enzymes, therapeutic proteins, antibiotics, and proteins with immunological properties. We also discuss the potential of plastid biotechnology and the novel tools developed to overcome some limitations of chloroplast transformation.     

Merits of secretion of heterologous proteins from industrial microorganisms

The heterologous expression of proteins is without doubt one of the most fascinating applications of the recombinant DNA technique. Despite clear successes many attempts to produce a certain protein in a heterologous host cell have met with technical difficulties. Secretion from cells has been used as a solution to overcome the intracellular formation of inactive protein. Microorganisms with a history of use in the fermentation industry exhibit clear advantages over the frequently usedEscherichia coli as host cells for secreted products. Interleukin-3, chymosin and phytase are examples of commercial products that are produced efficiently with the aid of industrial microorganisms. Presented at the FEMS Symposium "Novel Methods and Standardization in Microbiology", Košice (Slovakia) 1996. Part of this study was conducted with sponsoring from theEU Commission (project CT9302540).     

Expression of foreign genes in transgenic yellow-poplar plants

Cells of yellow-poplar (Liriodendron tulipifera L.) were transformed by direct gene transfer and regenerated into plants by somatic embryogenesis. Plasmid DNA bearing marker genes encoding β-glucuronidase (GUS) and neomycin phosphotransferase (NPT II) were introduced by microprojectile bombardment into single cells and small cell clusters isolated from embryogenic suspension cultures. The number of full-length copies of the GUS gene in independently transformed callus lines ranged from approximately 3 to 30. An enzyme-linked immunosorbent assay for NPT II and a fluorometric assay for GUS showed that the expression of both enzymes varied by less than fourfold among callus lines. A histochemical assay for GUS activity revealed a heterogeneous pattern of staining with the substrate 5-bromo-4-chloro-3-indoyl-β-d-glucuronic acid in some transformed cell cultures. However, cell clusters reacting positively (blue) or negatively (white) with 5-bromo-4-chloro-3-indoyl-β-d-glucuronic acid demonstrated both GUS activity and NPT II expression in quantitative assays. Somatic embryos induced from transformed cell cultures were found to be uniformly GUS positive by histochemical analysis. All transgenic plants sampled expressed the two marker genes in both root and shoot tissues. GUS activity was found to be higher in leaves than roots by fluorometric and histochemical assays. Conversely, roots expressed higher levels of NPT II than leaves.