Efficient secretion of human parathyroid hormone by Saccharomyces cerevisiae

A cDNA encoding mature human parathyroid hormone (hPTH) was expressed in Saccharomyces cerevisiae, after fusion to the prepro region of yeast mating factor alpha (MF alpha). Radioimmunoassay showed high levels of hPTH immunoreactive material in the growth medium (up to 10 micrograms/ml). More than 95% of the immunoreactive material was found extracellularly as multiple forms of hormone peptides. Three internal cleavage sites were identified in the hPTH molecule. The major cleavage site, after a pair of basic amino acids (aa) (Arg25Lys26 decreases Lys27), resembles that recognized by the KEX2 gene product on which the MF alpha expression-secretion system depends. The use of a protease-deficient yeast strain and the addition of high concentrations of aa to the growth medium, however, not only changed the peptide pattern, but also resulted in a significant increase in the yield of intact hPTH (1-84) (more than 20% of the total amount of immunoreactive material). The secreted hPTH (1-84) migrates like a hPTH standard in two different gel-electrophoretic systems, co-elutes with standard hPTH on reverse-phase high-performance liquid chromatography, reacts with two hPTH antibodies raised against different parts of the peptide, has a correct N-terminal aa sequence, and has full biological activity in a hormone-sensitive osteoblast adenylate cyclase assay.     

Isolation and characterization of biologically active murine interleukin-6 produced in Escherichia coli.

Interleukin-6 (IL-6) is a multi-functional cytokine produced and secreted by several different cell types, including those of the immune system. A cDNA coding for the mature murine IL-6 (mIL-6), which extends from amino acid (aa) 25 through 211, was cloned into a prokaryotic vector and then expressed in Escherichia coli. The recombinant mIL-6 (remIL-6) was isolated from bacterial inclusion bodies by solubilization in 4 M guanidine hydrochloride followed by gel-filtration chromatography. The protein was refolded to an active conformation by dialysis against 25 mM Na. acetate pH 5.5. A final step of purification and concentration on a cation exchange resin yielded pure and biologically active remIL-6. The purified preparation had the expected aa composition, as confirmed by aa analysis and pI of 7.0-7.1. The biological activity of the recombinant protein was measured in two systems; a proliferation assay employing 7TD1 cells, and a fibrinogen biosynthesis assay employing primary rat hepatocytes. Both assay systems demonstrated that the remIL-6 was active in the range of 10(8) units/mg, which is similar to that estimated for native cytokine. Antibodies raised in rabbits against remIL-6 neutralized the biological activity of both recombinant and native IL-6.     

Isolation and characterization of a glycosylated form of human insulin-like growth factor I produced in Saccharomyces cerevisiae

Expression and secretion of human insulin-like growth factor-I (IGF-I) in Saccharomyces cerevisiae was achieved by linking an actin (ACT) promoter to an MF alpha 1 prepro leader peptide/IGF-I gene fusion. Purified human IGF-I from yeast culture media was found to contain, in addition to the native form, also a glycosylated variant. Structural studies showed that both IGF-I forms were processed identically, resulting in 70-amino-acid long polypeptides, with intact N-terminal and C-terminal residues of glycine and alanine, respectively. The glycosylation site was determined to threonine-29 (Thr29), by 1H NMR spectroscopy and protein sequence analysis of an isolated tryptic peptide(22-36). No other glycosylation sites were found. Only mannose was detected in the sugar analysis, with an estimated content of 4.5% w/w corresponding to 2 mannose residues per molecule of IGF-I. The carbohydrate structure, determined by 1H and 13C NMR spectroscopy, was found to be alpha-D-Manp(1----2)alpha-D-Manp(1----3)Thr corresponding to an O-linked glycoprotein structure. No other post-translational modifications could be identified in the glycosylated IGF-I form. Furthermore, this form was highly active, comparable to native IGF-I, exhibiting a specific activity of 20,500 units/mg, as determined by a radio-receptor assay.     

Protein O-glycosylation in Saccharomyces cerevisiae. Purification and characterization of the dolichyl-phosphate-D-mannose-protein O-D-mannosyltransferase

The enzyme dolichyl-phosphate-D-mannose:protein O-D-mannosyltransferase has been solubilized from Saccharomyces cerevisiae membranes and its mannosyltransferase activity demonstrated using short peptides. The specific activity of the protein was enriched 130-fold before it was further purified by native and SDS gel chromatography. A 92-kDa band correlated well with the enzyme activity; an antibody raised against this protein precipitated the mannosyltransferase. The 92-kDa band was hydrolysed to 84 kDa after treatment with endoglycosidase F, indicating that the protein is a glycoprotein which may contain four carbohydrate chains. The purified mannosyltransferase is distinctly influenced in transfer specificity by amino acids next to serine and threonine within the acceptor peptides. Thus acidic amino acids strongly inhibit acceptor activity as do glycine and proline residues as amino-terminal and carboxy-terminal neighbours, respectively.     

Expression and secretion of biologically active human atrial natriuretic peptide in Saccharomyces cerevisiae

A hybrid gene was constructed containing a fusion between the DNA sequences encoding the secretory precursor of the yeast mating pheromone alpha-factor and a synthetic sequence encoding a biologically active 24-amino acid carboxyl-terminal portion of the human atrial natriuretic peptide (hANP) precursor. Transformation of Saccharomyces cerevisiae with the hybrid gene resulted in the yeast cells secreting biologically active hANP into the extracellular medium. The secreted hANP was purified and found to be accurately processed at the junction in the chimeric alpha-factor/hANP protein, producing the desired mature hANP amino terminus. The secreted product was also folded correctly with respect to the single disulfide bond. However, the carboxyl terminus of the secreted hANP material was heterogeneous such that the major form lacked the last two amino acids of the peptide while the minor form was the full length material. The observed processing at the carboxyl terminus of the secreted hANP may reflect a normal processing event involved in alpha-factor peptide maturation.     

Purification and characterization of human caseinomacropeptide produced by a recombinant Saccharomyces cerevisiae

Caseinomacropeptide (CMP) is a biologically active polypeptide derived from the C-terminal of milk kappa-casein. CMP is heterogeneous since it is modified differently by glycosylation and phosphorylation after translation. Recently, recombinant human CMP (hCMP) has been produced as a secretory product in yeast. The present study aimed at the purification and characterization of recombinant hCMP. By sequential molecular cut-off ultrafiltration and anion-exchange chromatography, the recombinant hCMP in the culture broth could be purified to an HPLC purity over 94%. The authenticity of the purified hCMP was confirmed by sequence analysis of N-terminal amino acids. The recombinant hCMP was estimated to be 7.0kDa by SDS-PAGE, and showed a lower glycosylation than the natural bovine CMP.     

Expression and secretion of biologically active echistatin in Saccharomyces cerevisiae

A synthetic gene coding for a platelet aggregation inhibitor, echistatin (ECS), was inserted into a Saccharomyces cerevisiae expression vector utilizing the alpha-mating factor pre-pro leader sequence and galactose-inducible promoter, GAL10. Cleavage of the pre-pro leader sequence in vivo results in the secretion of a properly processed recombinant ECS with the native N-terminal glutamic acid residue. Recombinant ECS was recovered from yeast supernatants and purified by reverse phase high performance liquid chromatography. Recombinant ECS expressed and purified from yeast was identical to native ECS in its ability to inhibit platelet aggregation.     

Preparation and properties of biologically active radioiodinated parathyroid hormone

A constant-current microelectrolytic radioiodination method was used to label bovine parathyroid hormone (BPTH) with ¹²⁵I to an overall iodination ratio of 1:1 iodide atoms per PTH molecule. Such iodinated preparations were shown to be fully active in several bioassay systems: in vitro adenylate cyclase activation in rat renal and skeletal membranes, in vitro calcium release from rat calvaria, and the in vivo hypercalcemic response in chickens. Analysis by Sephadex G-15 chromatography after enzymatic digestion showed the radioiodine to be incorporated predominantly as monoiodotyrosine. Bioassay of iodinated preparations from which uniodinated hormone had been removed by isoelectric focusing showed essentially full hormonal activity. Such methods can be used to consistently produce radioiodinated biologically active preparations of BPTH 1–84 with high specific activity (2000 Ci/mmol).     

Isolation and characterization of a dinucleoside triphosphatase from Saccharomyces cerevisiae.

An enzyme able to cleave dinucleoside triphosphates has been purified 3,750-fold from Saccharomyces cerevisiae. Contrary to the enzymes previously shown to catabolize Ap4A in yeast, this enzyme is a hydrolase rather than a phosphorylase. The dinucleoside triphosphatase molecular ratio estimated by gel filtration is 55,000. Dinucleoside triphosphatase activity is strongly stimulated by the presence of divalent cations. Mn2+ displays the strongest stimulating effect, followed by Mg2+, Co2+, Cd2+, and Ca2+. The Km value for Ap3A is 5.4 microM (50 mM Tris-HCl [pH 7.8], 5 mM MgCl2, and 0.1 mM EDTA; 37 degrees C). Dinucleoside polyphosphates are substrates of this enzyme, provided that they contain more than two phosphates and that at least one of the two bases is a purine (Ap3A, Ap3G, Ap3C, Gp3G, Gp3C, m7Gp3A, m7Gp3G, Ap4A, Ap4G, Ap4C, Ap4U, Gp4G, and Ap5A are substrates; AMP, ADP, ATP, Ap2A, and Cp4U are not). Among the products, a nucleoside monophosphate is always formed. The specificity of cleavage of methylated dinucleoside triphosphates and the molecular weight of dinucleoside triphosphatase indicate that this enzyme is different from the mRNA decapping enzyme previously characterized (A. Stevens, Mol. Cell. Biol. 8:2005-2010, 1988).     

Characterization of two forms of asparaginase in Saccharomyces cerevisiae.

Saccharomyces cerevisiae X2180-1A synthesizes two forms of asparaginase: L-asparaginase I, an internal constitutive enzyme, and asparaginase II, an external enzyme which is secreted in response to nitrogen starvation. The two enzymes are biochemically and genetically distinct. The structural gene for asparaginase I (asp 1) is closely linked to the trp 4 gene on chromosome IV. The gene controlling the synthesis of asparaginase II is not linked to either the trp 4 or asp 1 genes. The rate of biosynthesis of asparaginase II is unaltered in yeast strains carrying the structural gene mutation for asparaginase I. Asparaginase II has been purified approximately 300-fold from crude extracts of Saccharomyces by heat and pH treatment, ethanol fractionation, ammonium sulfate fractionation followed by Sephadex G-25 chromatography, and DEAE-cellulose chromatography. Multiple activity peaks were obtained which, upon gas chromatographic analysis, exhibit varying mannose to protein ratios. Asparaginase I has been purified approximately 100-fold from crude extracts of Saccharomyces by protamine sulfate treatment, ammonium sulfate fractionation, gel permeation chromatography, and DEAE-cellulose chromatography. No carbohydrate component was observed upon gas chromatographic analysis. Comparative kinetic and analytic studies show the two enzymes have little in common except their ability to hydrolyze L-asparagine to L-aspartic acid and ammonia.     

Purification and partial characterization of a factor, a mating hormone produced by mating-type-a cells from Saccharomyces cerevisiae.

Cells of Saccharomyces cerevisiae exhibiting the a mating type secrete into the culture medium a mating-type-specific hormone activity (a factor), which specifically causes a transient arrest of DNA replication and cell division in cells of the opposite mating type, alpha. Three compounds exhibiting a factor activity have been found in culture filtrates from a cells. The most active compound has been purified more than 10(5)-fold and appears to be homogeneous on the basis of thin-layer chromatography and thin-layer electrophoresis in different systems. We propose that this compound, which exhibits in alpha cells the biological activities that have been attributed to a factor, represents pure a factor. a factor has been characterized as a very hydrophobic undecapeptide with the following amino acid composition: H2N-Tyr (Asx1, Gly1, Ala1, Val1, Ile2, Phe1, Lys1, Trp1, Pro1). Although in their respective target cells the biological effects of a factor and of alpha factor, the corresponding mating hormone of mating-type-alpha cells, are remarkably similar, the primary structures of both hormones appear to be quite different.     

Expression and characterization of biologically active human Fas ligand produced in CHO cells

We describe an expression system for high-yield production of recombinant soluble human FasL (rsh-FasL) in CHO cells. After one round of selection for gene amplification, cell lines producing rsh-FasL up to 60 μg/L × 10⁶ cells in 24 h were obtained. Cell lines were grown in protein-free medium as suspension cultures. The protein secreted into growth medium was purified by immunoaffinity. The rsh-FasL thus obtained was further fractionated by gel filtration and a form of approx 140 kDa was isolated and characterized. Mass spectral analysis yielded a main peak of 28,321.15 Da, while, although to a lesser extent, dimeric and trimeric forms were also detected according to the described oligomerized state of native FasL. Our procedure permits consistent production of biologically active rsh-FasL as shown in tests on FasL-sensitive cells and in in vitro binding assays. S. Zappitelli and L. D’Alatri contributed equally to this work.     

Isolation and characterization of two distinct myo-inositol transporter genes of Saccharomyces cerevisiae

By the complementation of a yeast mutant defective in myo-inositol transport (Nikawa, J., Nagumo, T., and Yamashita, S. (1982) J. Bacteriol. 150, 441-446), we isolated two myo-inositol transporter genes, ITR1 and ITR2, from a yeast gene library. The ITR1 and ITR2 genes contained long open reading frames capable of encoding 584 and 612 amino acids with calculated relative molecular masses of 63,605 and 67,041, respectively. The sequence similarity between the ITR1 and ITR2 products was extremely high, suggesting that the two genes arose from a common ancestor. Both gene products show significant sequence homology with a superfamily of sugar transporters, including human HepG2 hepatoma/erythrocyte glucose transporter and Escherichia coli xylose transporter. Hydropathy analysis indicated that the ITR1 and ITR2 products are both hydrophobic and contain 12 putative membrane-spanning regions. Thus, yeast myo-inositol transporters could be classified into the sugar transporter superfamily. Gene disruption and tetrad analysis showed that yeast cells contain two separate myoinositol transporters. The ITR1 product was the major transporter and the ITR2 product the minor one in cells grown in minimum medium containing glucose. Northern blot analysis showed that ITR1 mRNA was much more abundant than ITR2 mRNA. The previously isolated myo-inositol transport mutant was determined to be defective in ITR1.     

Isolation and characterization of biologically active and inactive cholecystokinin-octapeptides from human brain

Cholecystokinin-like immunoreactivity (CCK-LI) in 0.9 kg human brain was extracted by 2% trifluoroacetic acid at 4 degrees C. Sephadex G50 gel filtration of crude extract revealed one main molecular form of CCK, detected by a carboxy-terminal antibody (5135), that eluted in the position of CCK8. When the CCK-LI in the extract was purified by affinity chromatography using another carboxyl-terminal CCK antibody followed by several steps of reverse phase high pressure liquid chromatography (HPLC), a component was isolated that was found by sequence analysis to be identical to the carboxyl-terminal CCK-octapeptide of porcine CCK33, isolated from intestinal mucosa, and to CCK-octapeptide, isolated from sheep brain. This component possessed comparable biological potencies to synthetic sulfated CCK8 in eliciting amylase release and in competitively displacing radioiodinated CCK33 from isolated mouse pancreatic acini. Furthermore, it exhibited a similar binding characteristic to CCK8 in binding to specific receptors on mouse brain cortical particulate preparations. On high pressure liquid chromatography another minor, earlier eluting immunoreactive peak was observed, which had the same amino acid composition and sequence as CCK8. These findings suggested that this material was oxidized CCK8. This earlier eluting component, exhibiting CCK8-like immunoreactivity, did not induce amylase release from acini and had no or minimal effect in inhibiting tracer CCK33 binding to receptors on isolated acini or on mouse brain cortical particulate preparations at the concentrations tested.     

Production and secretion of a biologically active Closterium sex pheromone by Saccharomyces cerevisiae

The cDNA fragment coding for the Closterium sex pheromone (protoplast-release-inducing protein inducer, PR-IP inducer) was inserted into a Saccharomyces cerevisiae–Escherichia coli shuttle vector, under the control of the glucose-repressible alcohol dehydrogenase (ADH2) promoter of yeast. The yeast cells transformed by this construct produced and secreted recombinant PR-IP inducer into the surrounding culture medium. The release of PR-IP from mt⁺ cells was induced by the recombinant pheromone, whereas amino- and carboxy-terminal truncated forms did not show any biological activity. High levels of asparagine-linked glycosylation in the recombinant pheromone were also confirmed after the treatment by deglycosylation enzymes.     

Isolation and characterization of Saccharomyces cerevisiae glycolytic pathway mutants.

Yeast strains carrying recessive mutations representing four different loci that cause defects in pyruvate kinase, pyruvate decarboxylase, 3-phosphoglycerate kinase, and 3-phosphoglycerate mutase were isolated and partially characterized. Cells carrying these mutations were unable to use glucose as a carbon source as measured in turbidimetric growth experiments. Tetrad analysis indicated that these mutations were not linked to each other; one of the mutations, that affecting phosphoglycerate kinase, was located on chromosome III.     

a-Factor from Saccharomyces cerevisiae: partial characterization of a mating hormone produced by cells of mating type a.

Conjugation between haploid cells of Saccharomyces cerevisiae is mediated through the action of diffusible mating hormones, two of which have been designated as a-factor and alpha-factor. Partially purified fractions exhibiting a-factor activity have been obtained from culture filtrates of a cells by ultrafiltration, ion-exchange chromatography, and gel filtration. The a-factor preparations specifically caused both G1 arrest and morphological alterations in cells of alpha-mating type, whereas a cells, a/alpha diploids, and nonmating alpha mutants were not affected. The a-factor activity was found in the culture filtrates of all a strains tested, but not in filtrates of alpha or a/alpha cell cultures. The hormone is sensitive to various proteases, showing that it is associated with a peptide or protein. Gel filtration studies suggest an apparent molecular weight greater than 600,000; however, this result may be due to aggregation with carbohydrate present in the preparations. Although the biological activities of a-factor are analogous to those described previously for alpha-factor, the chemical properties of these two hormones appear to be quite different.     

Isolation and preliminary characterization of Saccharomyces cerevisiae proline auxotrophs.

Proline-requiring mutants of Saccharomyces cerevisiae were isolated. Each mutation is recessive and is inherited as expected for a single nuclear gene. Three complementation groups cold be defined which are believed to correspond to mutations in the three genes (pro1, pro2, and pro3) coding for the three enzymes of the pathway. Mutants defective in the pro1 and pro2 genes can be satisfied by arginine or ornithine as well as proline. This suggests that the blocks are in steps leading to glutamate semialdehyde, either in glutamyl kinase or glutamyl phosphate reductase. A pro3 mutant has been shown by enzyme assay to be deficient in delta 1-pyrroline-5-carboxylate reductase which converts pyrroline-5-carboxylate to proline. A unique feature of yeast proline auxotrophs is their failure to grown on the rich medium, yeast extract-peptone-glucose. This failure is not understood at present, although it accounts for the absence of proline auxotrophs in previous screening for amino acid auxotrophy.