Grant A. Bitter

Expression and Secretion Vectors for Yeast

Publisher Summary The chapter focuses on methods used to efficiently express and secrete biologically active proteins from Saccharomyces cereoisiae . The chapter describes yeast expression vectors utilizing episomal vectors. The expression cassettes from these vectors can also be integrated into the yeast chromosome, where they are presented at controlled copy number, exhibiting a high degree of mitotic stability. The chapter discusses extrachromosomal replication vectors, centromere plasmids, and the assembly of expression cassettes. One of the advantages of a secretion system is the production of heterologous proteins with authentic NH 2 termini. The methods utilized to analyze heterologous protein secretion are the same as those used for proteins expressed directly in the cytoplasm (direct expression vectors) The chapter describes several related procedures including the Mung Bean nuclease digestion, the S1 nuclease digestion of cohesive termini, and the analysis of carbohydrate additions to heterologous proteins.

A multi-component upstream activation sequence of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene promoter.

SummaryThe majority of the activation potential of the Saccharomyces cerevisiae TDH3 gene promoter is contained within nucleotides −676 to −381 (relative to the translation initiation codon). An upstream activation sequence (UAS) in this region has been characterized by in vitro and in vivo assays and demonstrated to be composed of two small, adjacent DNA sequence elements. The essential determinant of this upstream UAS is a general regulatory factor 1 (GRF1) binding site at nucleotides −513 to −501. A synthetic DNA element comprising this sequence, or an analogue in which two of the degenerate nucleotides of the GRF1 site consensus sequence were altered, activated 5′ deleted TDH3 and CYC1 promoters. The second DNA element of the UAS is a 7 by sequence which is conserved in the promoters of several yeast genes encoding glycolytic enzymes and occurs at positions −486 to −480 of the TDH3 promoter. This DNA sequence represents a novel promoter element: it contains no UAS activity itself, yet potentiates the activity of a GRF1 UAS. The potentiation of the GRFl UAS by this element occurs when placed upstream from the TATA box of either the TDH3 or CYC1 promoters. The characteristics of this element (termed GPE for GRF1 site potentiator element) indicate that it represents a binding site for a different yeast protein which increases the promoter activation mediated by the GRF1 protein. Site-specific deletion and promoter reconstruction experiments suggest that the entire activation potential of the −676 to −381 region of the TDH3 gene promoter may be accounted for by a combination of the GRF1 site and the GPE.

Functional genetic tests of DNA mismatch repair protein activity in Saccharomyces cerevisiae.

Hereditary non-polyposis colorectal cancer (HNPCC) is associated with mutations in four different genes encoding proteins involved in DNA mismatch repair (DMR). As many as 30% of the observed sequence variations in human DMR genes predict minor alterations in the encoded protein, such as amino acid (aa) replacements or small in-frame deletions/insertions. For such sequence variants, a functional genetic test will be required to discriminate mutations from polymorphisms. We have constructed a series of isogenic yeast strains in which individual genes involved in DMR are disrupted, and have standardized an assay which measures GT tract stability (Strand et al., 1993) to characterize these gene products. Disruptions of the yeast MSH2, MLH1, and PMS1 genes result in, respectively, a 290-, 450- and 390-fold increased tract instability over the wild type (wt) strain under optimized assay conditions. Expression of the wt MSH2 and PMS1 gene from plasmids results in complementation of the corresponding chromosomal gene disruption. Two different aa replacements which correspond to previously observed sequence variants of the human MSH2 gene, and implicated in HNPCC, were created in the conserved aa of the yeast MSH2 gene by site directed mutagenesis. Conversion of the Pro640 in the yeast protein to Leu resulted in a complete loss of protein function. In contrast, a yeast MSH2p protein in which the His658 is changed to Tyr retains full function in this in vivo assay. These results indicate that the Pro-->Leu and His-->Tyr variants observed in humans constitute, respectively, a mutation and a polymorphism. The system described here may be used for further structure/function analysis of yeast DMR proteins. Such studies may provide insight into the effects that specific sequence variations observed in human DMR proteins have on their function.

Characterization of purified hepatitis B surface antigen containing pre-S(2) epitopes expressed in Saccharomyces cerevisiae

The cloning and expression of the hepatitis B middle-protein surface antigen gene in the yeast Saccharomyces cerevisiae is described. A generalized expression vector carrying the yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter was used. Expressed material, in the form of supramolecular particles, was purified and characterized. Severe proteolysis within the pre-S(2) region was observed for material expressed in a wild-type yeast host. This proteolysis was substantially reduced by utilization of a protease-deficient host. Immunoblotting of sodium dodecyl sulfate-polyacrylamide gels with several antibodies of differing specificity was performed to characterize the various protein species present. All species were analyzed by N-terminal sequencing after electroelution from gels. Carbohydrate staining of gels and glycosidase treatments of the purified antigen material indicated that full-length antigen was present in both glycosylated and unglycosylated forms. Glycosylation appeared to be of both asparagine-linked and threonine/serine-linked types. Site-directed mutagenesis was used to convert two arginine residues in the pre-S(2) region of the antigen to glutamine residues. The changes abolished reactivity with one polyclonal and two monoclonal antibodies specific for epitopes within the pre-S(2) region.

Expression of interferon-gamma from hybrid yeast GPD promoters containing upstream regulatory sequences from the GAL1-GAL10 intergenic region

The expression of human immune interferon (IFN-gamma) is toxic to yeast, resulting in low plasmid stability and copy number. The Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene (GPD) promoter [Bitter and Egan, Gene 32 (1984) 263-274] has been modified by introduction of upstream regulatory sequences from the yeast GAL1-GAL10 intergenic region [UASG; Guarente et al., Proc. Natl. Acad. Sci. USA 79 (1982) 7410-7414] and utilized to express IFN-gamma. In contrast to the native GPD promoter, the GPD(G) hybrid promoters are regulated by the carbon source. With glucose as the carbon source, a level of expression is observed which is much lower than that obtained with the native GPD promoter. Expression of the hybrid promoters is induced approx. 150- to 200-fold in shaker flask cultures by growth in galactose and similar levels of expression are observed after growth in lactate plus galactose. However, full galactose induction is not observed in the presence of glucose.? Utilization of these regulated promoters has allowed maintenance of plasmids at high copy number with glucose as the carbon source and, after induction with galactose, production of IFN-gamma mRNA at levels more than ten times higher than the native yeast PGK gene transcript. In contrast, the native GPD promoter directs comparable levels of expression when grown in either glucose or galactose resulting in low plasmid copy number and a correspondingly lower IFN-gamma transcript abundance. It is demonstrated that nucleotide sequences more than 240 bp upstream from the TATA box are required for optimal activity of the native GPD promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Intergenic complementation truncation mutants of cyclin-dependent kinase

Abstract The Saccharomyces cerevisiae genes PHO80 and PHO85 encode, respectively, a cyclin and cyclin-dependent kinase, which negatively regulate PHO5 gene transcription by phosphorylating the transcription activator Pho4p. Cyclin-dependent kinases (CDKs) are highly conserved proteins, both within and between species. It was previously demonstrated, using reporter genes activated in yeast by Pho4p, that hybrid proteins in which over two-thirds of Pho85p were replaced with the homologous region from human Cdk2 retained the function of native Pho85p with respect to promoter repression. In the present study, various truncated forms of the hybrid human-yeast CDKs were tested for function. Surprisingly, truncations in which significant portions of the C-terminal region of the 291-residue hybrid CDK were deleted retained activity. Genes encoding human Cdk2 proteins which terminated after amino acids 151, 140, 130, 120 and 90 each complement a chromosomal pho85 gene disruption in which the HIS3 gene is inserted at codon 49. Truncated Cdk2 proteins containing less than 60 amino acids failed to complement the pho85::HIS3 gene disruption. Although the functional C-terminal truncations disrupt the ATP-binding and active sites of Cdk2, reporter gene repression mediated by these truncated proteins is apparently due to phosphorylation of Pho4p, since a gene in which the essential lysine codon at position 33 was converted to an arginine codon does not complement the chromosomal gene disruption. The human Cdk2 truncations were demonstrated to function through intergenic complementation. The intact Cdk2-Pho85 hybrid CDK complemented the pho85 mutation in yeast strains in which the entire PHO85 coding region was deleted from chromosome XVI. The C-terminal Cdk2 truncations, however, were non-functional in these strains and thus dependent for activity on the pho85 coding region which remained in the mutant pho85::HIS3 chromosomal locus. These genetic results are consistent with a model involving protein fragment complementation in which the active site of the CDK is bisected.

29 – Expression and Secretion Vectors for Yeast

Publisher Summary This chapter focuses on the methods used to efficiently express and secrete biologically active proteins from Saccharomyces cerevisiae. The first yeast genes to be molecularly cloned were those encoding biosynthetic enzymes and these genes are currently routinely utilized as selectable markers on recombinant yeast plasmids. A number of DNA sequence elements have been isolated that confer the capability of autonomous replication of colinear DNA in yeast. High-level expression of heterologous genes is generally facilitated by increasing the copy number of the expression vectors. Plasmid stability of a culture is determined by plating appropriate culture dilutions on nonselective medium, then replica plating the resulting colonies onto medium that is selective for cells containing the plasmid. High copy number plasmids may be visualized by ethidium bromide staining of restriction enzyme-digested whole-cell DNA that has been size fractionated by agarose gel electrophoresis.