Secretory expression of the human serum albumin gene in the yeast, Saccharomyces cerevisiae

We have fused a cDNA gene encoding mature human serum albumin (HSA) to several secretory leader-encoding sequences. The hybrid genes were cloned into an episomal vector under the control of several yeast promoters and then introduced into yeast cells. The GAL1 promoter in combination with either the native HSA pre-sequence or a modified HSA pre-sequence gave the highest production of immunoreactive HSA, 90 mg/liter being reached in a shake flask culture. The invertase pre-sequence, the mating factor alpha 1 prepro-sequence, and the modified HSA pre-sequence directed accurate processing. In contrast, the chicken lysozyme pre-sequence and the native HSA pre-sequence directed incorrect processing. Episomal vectors were unstable within the host cells under non-selective culture conditions. To improve the plasmid stability, the hybrid genes were incorporated into an integrative vector. Transformants carrying multicopies of the plasmid integrated at the LEU2 locus stably secreted HSA. The highest yield of 65 mg/liter in a shake flask culture was obtained with the combination of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter and the modified HSA pre-sequence. By constructing transformed strains containing multicopies of plasmids integrated at both the chromosome LEU2 and HIS4 loci, we have obtained a stable strain that continuously secretes as much as 85 mg HSA per liter of culture medium.     

Production of recombinant human serum albumin from Saccharomyces cerevisiae

Human serum albumin has been constitutively expressed in a Saccharomyces cerevisiae brewing yeast. After cell growth and disruption the product was associated with the insoluble fraction and represented approximately 1% of total cell protein. After the cell debris was extensively washed, the albumin was solubilized with 8 M urea and 28 mM 2-mercaptoethanol in 50 mM sodium carbonate buffer, pH 10. The denatured albumin was refolded by dialysis and further purified by anion exchange and gel filtration chromatography. Losses of renatured material could be reduced, or higher protein concentrations used during refolding, if the denatured product was purified by cation-exchange chromatography in urea prior to refolding. Apart from an additional N-terminal N-acetyl methionine, the refolded product proved identical to human serum albumin derived from plasma when compared by a variety of physical, chemical, and biological analytical methods.     

Construction of a human cytochrome c gene and its functional expression in Saccharomyces cerevisiae

The nucleotide sequences of a partial cDNA and three pseudogenes of human cytochrome c were determined. The complete nucleotide sequences which encode human cytochrome c were constructed on the basis of one of the pseudogenes by in vitro mutagenesis. The constructed human cytochrome c was functionally expressed in Saccharomyces cerevisiae. The recombinant human cytochrome c was purified and characterized.     

Proteolytic stability of recombinant human serum albumin secreted in the yeast Saccharomyces cerevisiae

 In order to direct the persistent expression of recombinant human serum albumin (HSA) from the GAL10 promoter in the yeast Saccharomyces cerevisiae, we carried out periodic feeding of galactose during shake-flask cultures. Unexpectedly, the recombinant protein secreted was observed to undergo rapid degradation, which was apparently accelerated by carbon-source feeding. The extracellular degradation of HSA occurred even in the strain deficient in the major vacuolar proteases PrA and PrB, and in the strain lacking the acidic protease Yap3p (involved in the generation of HSA-truncated fragments). Interestingly, the degradation correlated closely with the acidification of extracellular pH and thus was significantly overcome either by buffering the culture medium above pH 5.0 or by adding amino acid-rich supplements to the culture medium, which could prevent the acidification of medium pH during cultivation. Addition of arginine or ammonium salt also substantially minimized the degradation of HSA, even without buffering. The extracellular degradation activity was not detected in the cell-free culture supernatant but was found to be associated with intact cells. The results of the present study strongly suggest that the HSA secreted in S. cerevisiae is highly susceptible to the pH-dependent proteolysis mediated by cell-bound protease(s) whose activity and expression are greatly affected by the composition of the medium. Received: 23 August 1999 / Received revision: 8 November 1999 / Accepted: 12 December 1999     

Cloning and expression of a Saccharomyces diastaticus glucoamylase gene in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

A recombinant plasmid pool of the Saccharomyces diastaticus genome was constructed in plasmid YEp13 and used to transform a strain of Saccharomyces cerevisiae. Six transformants were obtained which expressed amylolytic activity. The plasmids each contained a 3.9-kilobase (kb) BamHI fragment, and all of these fragments were cloned in the same orientations and had identical restriction maps, which differed from the map of the STA1 gene (I. Yamashita and S. Fukui, Agric. Biol. Chem. 47:2689-2692, 1983). The glucoamylase activity exhibited by all S. cerevisiae transformants was approximately 100 times less than that of the donor strain. An even lower level of activity was obtained when the recombinant plasmid was introduced into Schizosaccharomyces pombe. No expression was observed in Escherichia coli. The 3.9-kb BamHI fragment hybridized to two sequences (4.4 and 3.9 kb) in BamHI-digested S. diastaticus DNA, regardless of which DEX (STA) gene S. diastaticus contained, and one sequence (3.9 kb) in BamHI-digested S. cerevisiae DNA. Tetrad analysis of crosses involving untransformed S. cerevisiae and S. diastaticus indicated that the 4.4-kb homologous sequence cosegregated with the glucoamylase activity, whereas the 3.9-kb fragment was present in each of the meiotic products. Poly(A)+ RNA fractions from vegetative and sporulating diploid cultures of S. cerevisiae and S. diastaticus were probed with the 3.9-kb BamHI fragment. Two RNA species, measuring 2.1 and 1.5 kb, were found in both the vegetative and sporulating cultures of S. diastaticus, whereas one 1.5-kb species was present only in the RNA from sporulating cultures of S. cerevisiae.     

Cloning of human lysozyme gene and expression in the yeast Saccharomyces cerevisiae

cDNA clones encoding human lysozyme were isolated from a human histiocytic cell line (U-937) and a human placenta cDNA library. The clones, ranging in size from 0.5 to 0.75 kb, were identified by direct hybridization with synthetic oligodeoxynucleotides. The nucleotide sequence coding for the entire protein was determined. The derived amino acid sequence has 100% homology with the published amino acid (aa) sequence; the leader sequence codes for 18 aa. Expression and secretion of human lysozyme in Saccharomyces cerevisiae was achieved by placing the cloned cDNA under the control of a yeast gene promoter (ADH1) and the alpha-factor peptide leader sequence.     

Cloning of Candida pelliculosa beta-glucosidase gene and its expression in Saccharomyces cerevisiae

Summary Candida pelliculosa var. acetaetherius is a strain of yeast which can utilize cellobiose as the carbon source. From a gene library prepared from this yeast, the -glucosidase gene has been cloned in a S. cerevisiae host using a chromogenic substrate, 5-bromo-4-chloro-3-indolyl--glucoside as an indicator. It was proved by Southern analysis that the DNA fragment carrying the -glucosidase gene originated from C. pelliculosa. -Glucosidase produced by S. cerevisiae transformants was secreted into the periplasmic space. In Candida, -glucosidase was not induced by cellobiose but was derepressed by lowering the concentration of glucose. The regulation of -glucosidase synthesis in S. cerevisiae carrying the cloned -glucosidase was not clear compared with that in Candida, however, the enzyme activity in low glucose medium (0.05%) was reproducibly higher than in high glucose medium (2%). We have found the sequence that controls the expression of the -glucosidase gene negatively in S. cerevisiae.     

Construction of an alpha-amylase/glucoamylase fusion gene and its expression in Saccharomyces cerevisiae.

A fusion gene which encoded a polypeptide comprised of 1116 amino acids was constructed using the alpha-amylase and glucoamylase cDNAs of Aspergillus shirousamii. When the fusion gene was expressed in Saccharomyces cerevisiae using a yeast expression plasmid under the control of the yeast ADH1 promoter, a bifunctional fusion protein (145 kDa) having both alpha-amylase and glucoamylase activities was secreted into the culture medium. The fusion protein had higher raw-starch-digesting activity than those of the original alpha-amylase and glucoamylase, and adsorbed onto raw starch like the glucoamylase. It was suggested that the characteristics are a result of the raw-starch-affinity site in the glucoamylase domain of the fusion protein.     

The sequence of human serum albumin cDNA and its expression in E. coli

A recombinant plasmid has been constructed which contains the mature protein coding region of the human serum albumin (HSA) gene. Bacteria containing this plasmid synthesize HSA protein under control of the E. coli trp promoter-operator. The DNA sequence and predicted protein sequence of HSA were determined from the cDNA plasmid and are compared to existing data obtained from direct protein sequencing. The DNA sequence predicts a mature protein of 585 amino acids preceded by a 24 amino acid "prepro" peptide.     

酿酒酵母组蛋白乙酰化修饰及其对基因表达的调控

真核生物核小体组蛋白修饰引起染色质重塑(Chromatin remodeling)是表观遗传的重要调控机制.乙酰化修饰(Acetylation modification)是其中一种重要的方式.组蛋白乙酰化修饰位点集中在各种组蛋白N末端赖氨酸残基上.细胞内存在功能拮抗的多种乙酰基转移酶和去乙酰化酶,二者相互竞争,共同调节组蛋白的乙酰化状态,通过影响核小体结构的致密性,并在多种效应分子的参与下,实现对基因的表达调控.以真核模式生物酿酒酵母(Saccharomyces cerevisiae)为对象,综述乙酰基转移酶和去乙酰化酶的种类、作用特点以及其基因调控的分子机制等方面的最新研究进展.     

Synthesis of human initiation factor-2 alpha in Saccharomyces cerevisiae.

A human eIF-2 alpha cDNA (encoding alpha-subunit of the eukaryotic initiation factor-2) was expressed under the control of the galactose-regulated GAL1, 10 promoter, in Saccharomyces cerevisiae, in order to study the possible interactions of human eIF-2 alpha with the yeast protein synthesis apparatus. Isoelectric focusing coupled with Western-blot analysis demonstrated that the human eIF-2 alpha subunit synthesized in yeast under a variety of growth conditions was detected as two bands which co-migrated with the phosphorylated and unphosphorylated forms of rabbit eIF-2 alpha, suggesting covalent modification in vivo. Cell fractionation studies further demonstrated that the synthesised human eIF-2 alpha protein, though present in the cytoplasm, was largely associated with the yeast ribosomes, but could be removed from these by washing with 0.3 M KCl. This possible association of the synthesised human subunit into a three-subunit (alpha, beta and gamma) eIF-2 complex was further examined by partial purification of the yeast eIF-2 complex and estimation of the molecular mass of this complex. Immunoreactive eIF-2 alpha was found in fractions with eIF-2 activity and the estimated molecular mass (130 kDa) corresponded to that predicted for the eIF-2 trimer. These analyses suggest that human eIF-2 alpha subunit synthesised in yeast can become involved with the yeast protein synthetic apparatus, though whether this is a functional incorporation requires further genetic studies.     

The gene for a major exopolyphosphatase of Saccharomyces cerevisiae.

The gene encoding a major exopolyphosphatase (scPPX1) in Saccharomyces cerevisiae (H. Wurst and A. Kornberg, J. Biol. Chem. 269:10996-11001, 1994) has been isolated from a genomic library. The gene, located at 57 kbp from the end of the right arm of chromosome VIII, encodes a protein of 396 amino acids. Overexpression in Escherichia coli allowed the ready purification of a recombinant form of the enzyme. Disruption of the gene did not affect the growth rate of S. cerevisiae. Lysates from the mutants displayed considerably lower exopolyphosphatase activity than the wild type. The enzyme is located in the cytosol, whereas the vast accumulation of polyphosphate (polyP) of the yeast is in the vacuole. Disruption of PPX1 in strains with and without deficiencies in vacuolar proteases allowed the identification of exopolyphosphatase activity in the vacuole. This residual activity was strongly reduced in the absence of vacuolar proteases, indicating a dependence on proteolytic activation. A 50-fold-lower protease-independent activity could be distinguished from this protease-dependent activity by different patterns of expression during growth and activation by arginine. With regard to the levels of polyP in various mutants, those deficient in vacuolar ATPase retain less than 1% of the cellular polyP, a loss that is not offset by additional mutations that eliminate the cytosolic exopolyphosphatase and the vacuolar polyphosphatases dependent on vacuolar protease processing.     

Regulated high efficiency expression of human interferon-alpha in Saccharomyces cerevisiae.

The 5' control region of the yeast phosphoglycerate kinase gene (PGK) was fused to the coding sequence of a human interferon-alpha. This PGK-interferon fusion was then introduced into yeast on a high copy number 2mu-based plasmid vector. Strains containing this plasmid produced a PGK-interferon-alpha fusion protein as 1-2% of cell protein and the expression of interferon activity was regulated by the availability of a fermentable carbon source. The system is capable of making as much as 15 mg of human interferon-alpha per litre of batch culture.