Green fluorescent protein (GFP) was used to study the regulation of the galactose-inducible GAL1 promoter in yeast Saccharomyces cerevisiae strains. GFP was cloned into the pGAL110 vector and transformed into the yeast strains. Time course studies comparing culture fluorescence intensity and GFP concentration were conducted along with on-line monitoring of GFP expression. Our results demonstrated that GFP fluorescence could be used as a quantifiable on-line reporter gene in yeast strains. The effect of an integrated GAL10pGAL4 transcription cassette was investigated. Induction time studies showed that there was no significant difference in GFP expression level by adding galactose at different culture times. A wide range of galactose concentrations was used to study the initial galactose concentration effect on GFP expression kinetics. A minimum of 0.05 g/L galactose doubled the GFP fluorescence signal as compared to the control, whereas 0.1 g/L gave the highest specific GFP yield. A simple analytical model was proposed to describe GFP expression kinetics based on the experimental results. In addition, this GFP-based approach was shown to have potential use for highthroughput studies. The use of GFP as a generic tool provided important insights to the GAL expression system and has great potential for further process optimization applications. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 70: 187–196, 2000.

The effect of host strain ploidy on the production of hepatitis B surface antigen (HBsAg) in Saccharomyces cerevisiae was evaluated at the pilot scale (75 L). We found that the accumulation of HBsAg normalized to cell protein was 2-fold higher for the diploid strain compared to its isogenic haploid. No detectable differences in many fermentation parameters were observed (e.g., rate of fermentation, growth rate, final cell yield). However, the enhancement of productivity in the diploid strain appeared to be associated with a slower rate of plasmid shedding (2 microns element) and, thus, a higher average copy number (2-fold at stationary phase) compared to those of the haploid strain.

Conference Article| February 01 1994 Expression and secretion of human protein disulphide isomerase in Saccharomyces cerevisiae José M. Luz; José M. Luz 1Biological Laboratory, University of Kent at Canterbury, Kent CT2 7NJ, U.K. Search for other works by this author on: This Site PubMed Google Scholar H. Markus; H. Markus ¶Merck Research Laboratories, West Point, PA 19496, USA Search for other works by this author on: This Site PubMed Google Scholar R. Farquhar; R. Farquhar 1Biological Laboratory, University of Kent at Canterbury, Kent CT2 7NJ, U.K. Search for other works by this author on: This Site PubMed Google Scholar L. D. Schultz; L. D. Schultz ¶Merck Research Laboratories, West Point, PA 19496, USA Search for other works by this author on: This Site PubMed Google Scholar R. W. Ellis; R. W. Ellis ¶Merck Research Laboratories, West Point, PA 19496, USA Search for other works by this author on: This Site PubMed Google Scholar R. B. Freedman; R. B. Freedman 1Biological Laboratory, University of Kent at Canterbury, Kent CT2 7NJ, U.K. Search for other works by this author on: This Site PubMed Google Scholar M. F. Tuite M. F. Tuite 1Biological Laboratory, University of Kent at Canterbury, Kent CT2 7NJ, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1994) 22 (1): 76S. https://doi.org/10.1042/bst022076s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation José M. Luz, H. Markus, R. Farquhar, L. D. Schultz, R. W. Ellis, R. B. Freedman, M. F. Tuite; Expression and secretion of human protein disulphide isomerase in Saccharomyces cerevisiae. Biochem Soc Trans 1 February 1994; 22 (1): 76S. doi: https://doi.org/10.1042/bst022076s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: PDI-protein disulphide isomerase, P4H-prolyl 4-hidroxylase, ER-endoplasmic reticulum, y-yeast, h-human, TCA-trichloroacetic acid © 1994 Biochemical Society1994 Article PDF first page preview Close Modal You do not currently have access to this content.

Moubatin, a new type of specific inhibitor of collagen-induced platelet aggregation, has been isolated from the soft tick Ornithodoros moubata (Waxman, L., and Connolly, T. M. (1993) J. Biol. Chem. 268, 5445-5449). A polymerase chain reaction-generated hybridization probe, produced using primers based on moubatin protein sequence, identified phage containing the entire cDNA sequence of moubatin. Analysis of the predicted amino acid sequence yielded a mature protein of 156 amino acids with a putative prepeptide of 15 amino acids. Comparison of the sequence of moubatin to that of other proteins in the Swiss PROT data base revealed no significant homology. The cDNA sequence was cloned into the yeast expression vector pKH4 alpha 2, producing a biologically active protein which inhibited collagen-stimulated aggregation of washed human platelets with an IC50 of about 100 nM, which is similar to the potency of native tick moubatin. A concentration of recombinant moubatin that fully inhibited collagen-stimulated aggregation did not inhibit aggregation induced by a variety of other platelet agonists, again demonstrating comparable properties of the recombinant and native proteins. Moubatin did not inhibit platelet adhesion to collagen even at a concentration up to 16 times its IC50 for the inhibition of aggregation. This specificity for inhibiting collagen-stimulated aggregation and not adhesion to collagen indicates that moubatin is unique among the natural product inhibitors of collagen stimulation of platelets. Further examination of the mechanism of moubatin-mediated inhibition of collagen-stimulated aggregation revealed that 1-6 microM moubatin diminished the second phase of aggregation induced by ADP, inhibited aggregation in response to submaximal concentrations of the thromboxane A2 mimetic U46619, and competed for the binding of a thromboxane A2 receptor antagonist to platelet membranes. Therefore, at higher concentrations, moubatin may affect more than one aspect of platelet signal transduction including the thromboxane A2 receptor. The availability of recombinant moubatin will allow further investigation of its unique activities in vitro and in vivo.

The rabbit plasminogen activator inhibitor-1 (PAI-1) cDNA has been isolated from a rabbit corneal cell cDNA library. The cDNA encodes a 402-amino acid (AA) protein that shares an overall 66% AA sequence identity with the rat, mouse, bovine and human forms of PAI-1 and exhibits the greatest AA sequence identity (85%) with human PAI-1. Three potential N-linked glycosylation sites and the P1, P1′ reactive centre of PAI-1 are conserved among all five species of animals. The cDNA encoding the proposed mature form of rabbit PAI-I was expressed in Saccharomyces cerevisiae as an intracellular, non-glycosylated protein. The purified, recombinant rabbit PAI-1 (R-rPAI-1) has an apparent Mr of 39 100 and exists primarily in a latent form which can be activated by guanidine HCI treatment. Activated R-rPAI-1 exhibits in vitro functional properties which are virtually indistinguishable from a recombinant, non-glycosylated form of human PAI-I and from fully glycosylated, native human PAI-1.

Salivary glands of the leech Haementeria officinalis contain a protein, leech antiplatelet protein (LAPP), that specifically blocks collagen-mediated platelet aggregation (Connolly, T. M., Jacobs, J. W., and Condra, C. (1992) J. Biol. Chem. 267, 6893-6898). Degenerate oligonucleotides whose sequences were derived from two short peptides from V8 digests of the native LAPP were used as primers to generate a polymerase chain reaction (PCR) product which contains the cDNA region coding for the sequence between these two peptides. Using this PCR product as a hybridization probe, phage containing cDNA clones were isolated containing the entire deduced amino acid sequence for LAPP. Computer analysis of the amino acid sequence predicts a peptidase cleavage site between a 21-residue pre-peptide and a mature protein of 126 amino acids. A DNA insert to express the predicted mature LAPP protein was generated by PCR amplification using phage-derived cDNA clones as a substrate. This insert encoded a fusion protein with the leader sequence of the yeast alpha mating factor and the mature LAPP cDNA. These PCR products were cloned into the yeast expression vector pKH4 alpha 2. A KEX 2 Lys-Arg endopeptidase cleavage site was placed NH2-terminal to the predicted mature protein. This vector transfected into the yeast Saccharomyces cerevisiae directs expression of a secreted mature protein at levels up to 200 mg of LAPP/liter of culture medium. The recombinant protein was comparable to native LAPP in its electrophoretic mobility, its reactivity with anti-LAPP antisera, and its biological activity including inhibition of collagen-stimulated platelet aggregation and the adhesion of platelets to collagen. Availability of significant quantities of recombinant LAPP opens the way to further biochemical structure/function studies and to studies on the effects of an inhibitor of collagen-stimulated platelet aggregation in vivo.

The rapidly acting inhibitor of plasminogen activators, PAI-1, was produced intracellularly in Saccharomyces cerevisiae by using the ADH2 promoter to drive the expression of the human PAI-1 cDNA. Approximately 8 mg of human PAI-1 was produced per liter of confluent yeast culture. A purification scheme which resulted in 20% recovery of isolated PAI-1 from the broken yeast cell homogenate was devised. Yeast-derived human PAI-1 differs from endothelial-type PAI-1 isolated from HT1080 fibrosarcoma cells in that the recombinant inhibitor does not contain carbohydrate side chains. Nevertheless, the activity and other functional attributes of yeast-derived PAI-1 are similar to those exhibited by HT1080 fibrosarcoma cell-derived PAI-1. Hence, this study demonstrates that expression of human PAI-1 in yeast is a viable strategy for the production of ample quantities of this key modulator of plasminogen activator-mediated proteolysis.

Yeast-expressed p55 precursor core protein of human immunodeficiency virus type 1 (HIV-1) was used to immunize chimpanzees. The animals developed high titers of antibodies to p55 as well as to the p24 and p17 mature cleavage products of the core precursor. Virus-neutralizing antibodies were not elicited. The induced immune responses did not prevent establishment of HIV-1 infection following challenge of one immunized chimpanzee with live virus.

In summary, we have shown that yeast is the preferred host for the expression of recombinant-derived hepatitis B vaccines, and that a yeast expression system which is productive, stable and scaleable can be developed for each of the three HBV envelope proteins. The versatility of regulated and integrated yeast expression systems in the production of foreign polypeptides with biomedical utility also has been highlighted. We also have shown that careful attention to the development of recombinant clones helps to optimize the entire production process leading to highly purified products which share many biochemical properties with the plasma-derived vaccine. Furthermore, immunization with PreS2 sequences is capable of protecting chimpanzees from HBV infection. The availability of PreS2 + S and PreS1 + PreS2 + S proteins expressed in yeast now provides the opportunity for establishing the relevance of such candidate vaccines in preventing human disease, thereby highlighting the utility of molecular biology in modern vaccine development.

Recombinant strains of Saccharomyces cerevisiae, expressing the PreS2 + S envelope protein of the hepatitis B virus (HBV), were constructed using either the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter or the regulated galactose-1-phosphate uridyltransferase (GAL10) promoter. These strains were compared by determining not only the yield of PreS2 + S antigen but also the retention of the plasmid during growth and expression. When the GAP system was examined, the expression of PreS2 + S was high, but plasmid levels decreased significantly during growth. For the GAL10 system, the expression of the PreS2 + S was significantly lower, but the plasmid copy number remained high. Much higher expression levels (comparable to those obtained for GAP promoter) were achieved with the GAL10 promoter when PreS2 + S was expressed in a host strain containing a chromosomally integrated expression cassette comprised of the GAL10 promoter fused to the GAL4 positive regulatory gene, and the plasmid level remained high.