Automation of yeast two-hybrid screening.

We have developed an automated format for screening yeast two-hybrid libraries for protein-protein interactions. The format consists of a liquid array in which pooled library subsets of yeast, expressing up to 1000 different cDNAs, are mated to a yeast strain of the opposite mating type, expressing a protein of interest. Interactors are detected by a liquid assay for beta-galacsidase following prototrophic selection. The method is demonstrated by the detection of interactions between two encoded yeast RNA polymerase subunits in simulated libraries of varied complexity. To demonstrate its utility for large scale screening of complex cDNA libraries, two nuclear receptor ligand-binding domains were screened through two cDNA libraries arrayed in pooled subsets. Screening these libraries yielded clones which had previously been identified in traditional yeast two hybrid screens, as well as several new putative interacting proteins. The formatting of the cDNA library into pooled subsets lends itself to functional subtraction of the promiscuous positive class of interactor from the library. Also, the liquid arrayed format enables electronic handling of the data derived from interaction screening, which, together with the automated handling of samples, should promote large-scale proteome analysis.

Development of infrared imaging to measure thermogenesis in cell culture: thermogenic effects of uncoupling protein-2, troglitazone, and beta-adrenoceptor agonists.

PURPOSE: Although the effects of thermogenic agents in cell culture can be measured by direct microcalorimetry, only a few samples can be analyzed over several hours. In this report, we describe a robust non-invasive technique to measure real-time thermogenesis of cells cultured in microtiter plates using infrared thermography. METHODS: Yeast were transformed with uncoupling protein-2 (UCP2) or exposed to carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) or rotenone. Adipocytes were exposed to rotenone, FCCP, cycloheximide. troglitazone, or CL316243. Thermogenesis was measured using infrared thermography. RESULTS: Thermogenesis increased after exposing yeast to the mitochondrial uncoupler, FCCP, or transforming the cells with UCP2. Further, thermogenesis in adipocytes was stimulated by CL316243, a beta3-adrenoceptor agonist being developed to treat obesity. The protein synthesis inhibitor, cycloheximide, did not inhibit CL316243-mediated thermogenesis. In contrast, the mitochondrial proton transport inhibitor, rotenone, inhibited thermogenesis in yeast and adipocytes. Similarly, the antidiabetic agent, troglitazone, suppressed thermogenesis in adipocytes. Although increased UCP synthesis resulted in increased thermogenesis in yeast, UCP expression did not correlate with thermogenesis in adipocytes. CONCLUSIONS: The results, taken together with the high resolution (0.002 degrees C) and robustness (384-well format) of the approach, indicate infrared-imaging is a rapid and effective method for measuring thermogenesis in vitro.

The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein, gp120 (ENV), is required in large quantities for immunological studies and as a potential vaccine component. We have expressed the DNA encoding gp120 in a highly efficient expression system based on the methylotrophic yeast, Pichia pastoris. The native gene was found to contain a sequence which resembled a Saccharomyces cerevisiae polyadenylation consensus and acted as a premature polyadenylation site in P. pastoris, resulting in the production of truncated mRNA. As full-length mRNA was produced in S. cerevisiae, this indicates differences in mRNA 3'-end formation between the two yeasts. Inactivation of this site by site-directed mutagenesis revealed several additional fortuitous polyadenylation sites within the gene. We have designed and constructed a 69%-synthetic gene with increased G + C content which overcomes this transcriptional problem, giving rise to full-length mRNA. High levels of intracellular, insoluble, unglycosylated ENV were produced [1.25 mg/ml in high-density (2 x 10(10) cells per ml) fermentations]. ENV also was secreted from P. pastoris using the S. cerevisiae alpha-factor prepro secretion leader and the S. cerevisiae invertase signal sequence. However, a high proportion of the secreted product was found to be hyperglycosylated, in contrast to other foreign proteins secreted from P. pastoris. There also was substantial proteolytic degradation, but this was minimized by maintaining a low pH on induction. Insoluble, yeast-derived ENV proteins are being considered as vaccine antigens and the P. pastoris system offers an efficient method of production.

Yeast systems for the expression of heterologous gene products.

Significant advances have been made over the past year in our understanding of some of the critical parameters affecting high-level production of heterologous proteins in yeast. Recent studies of plasmid stability, promoter strength and secretion efficiency are yielding potential improvements in expression.

The allantoinase (DAL1) gene of Saccharomyces cerevisiae.

The allantoinase (DAL1) gene from Saccharomyces cerevisiae has been cloned, sequenced, and found to encode a 472 amino acid protein with a Mr of 52,028. DAL1 is expressed in an inducer-independent manner in strain M970 (sigma 1278b genetic background) and modestly responds to mutation of the dal80 locus. Expression was also sensitive to nitrogen catabolite repression (NCR). Correlated with these expression characteristics, the upstream region of DAL1 contained five copies of a sequence that is homologous to the DAL UASNTR element previously shown to be required for transcriptional activation and NCR sensitivity of the DAL5 and DAL7 genes. Missing from the DAL1 5' flanking region were any sequences with significant homology to the DAL7 UIS element required for response to inducer. These observations further support the roles of UASNTR and DAL7 UIS in the regulation of allantoin pathway gene expression.

Yeast systems for the commercial production of heterologous proteins.

Yeasts are attractive hosts for the production of heterologous proteins. Unlike prokaryotic systems, their eukaryotic subcellular organization enables them to carry out many of the post-translational folding, processing and modification events required to produce "authentic" and bioactive mammalian proteins. In addition, they retain the advantages of a unicellular microorganism, with respect to rapid growth and ease of genetic manipulation. The vast majority of yeast expression work has focused on the well-characterized baker's yeast Saccharomyces cerevisiae. However, with the development of DNA transformation technologies, a growing number of non-Saccharomyces yeasts are becoming available as hosts for recombinant polypeptide production. These include Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Schizosaccharomyces pombe, Schwanniomyces occidentalis and Yarrowia lipolytica. The performance of these alternative yeast expression systems is reviewed here relative to S. cerevisiae, and the advantages and limitations of these systems are discussed.

High-level secretion of biologically active aprotinin from the yeast Pichia pastoris.

A synthetic gene encoding aprotinin (bovine pancreatic trypsin inhibitor) was fused to the Saccharomyces cerevisiae prepro alpha mating factor leader sequence at the dibasic amino acid processing site. Pichia pastoris strains were developed to express one or multiple copies of a methanol-inducible expression cassette containing the gene fusion. P. pastoris containing a single copy of the vector secreted approximately 150 mg/l of immunoreactive protein. A construct bearing five copies of the expression cassette secreted 930 mg/l of aprotinin. The purified aprotinin molecule was equipotent with the native molecule in a trypsin inhibition assay. Protein sequence analysis showed that the alpha factor-aprotinin fusion was not processed at the basic amino acid residues Lys-Arg. Instead, recombinant aprotinin had additional N-terminal amino acids derived from prepro alpha factor. The N-terminal extension was variably 11 or 4 amino acids. Inclusion of the spacer DNA sequence encoding Glu and Ala between aprotinin and the Lys-Arg processing site led to the secretion of a biologically active aprotinin containing only a Glu-Ala N-terminal extension.

Oxalurate induction of multiple URA3 transcripts in Saccharomyces cerevisiae.

The URA3 gene from Saccharomyces cerevisiae is localized on a 1.1-kilobase (kb) DNA fragment. By using this fragment as a hybridization probe, we found that oxalurate, a gratuitous inducer of the allantoin degradative system, also serves to induce URA3 specific RNA. This response is restricted to oxalurate; other conditions which bring about high-level synthesis of the allantoin degradative enzymes did not produce the effect. Two classes of RNA (1.0 and 1.5 kb) were found to be oxalurate induced. Both classes are encoded by the URA3 gene, overlap, and probably do not significantly differ at their 5' termini. Northern blot mapping of the transcripts indicated that the 1.5-kb transcript was likely encoded by sequences extending up to 0.5 kb downstream from the 3' terminus of the 1.0-kb transcript. Analysis of the endpoints of the major 1.0-kb URA3 transcript by S1 nuclease mapping revealed the existence of two 5' termini, separated by 5 to 10 nucleotides, and seven 3' termini, separated by 5 to 20 nucleotides each, over a range of about 70 bases.

Induction and genetics of two alpha-galactosidase activities in Saccharomyces cerevisiae.

The induction of alpha-galactosidase of Saccharomyces cerevisiae was investigated. We have demonstrated the existence of inducible internal and external alpha-galactosidase activities and have studied the relationship between the two alpha-galactosidases by examining a mutant strain which lacks both the internal and external activities. The mutant possesses a mutation in a single locus (mel1-1) which does not affect the synthesis of the other galactose pathway enzymes or the ability of the yeast to grow on media containing only galactose as the carbon source. Genetic studies of the mutant indicate that mel 1-1 is recessive and allelic to the wild tye allele for melibiose fermentation Mel 1.