[35] Use of heterologous and homologous signal sequences for secretion of heterologous proteins from yeast

Hitzeman, Ronald A.; Chen, Christina Y.; Dowbenko, Donald; Renz, Mark; Liu, Chung; Pai, Roger; Simpson, Nancy; Kohr, William J.; Singh, Arjun; Chisholm, Vanessa; Hamilton, Robert W.; Chang, Chung Nan

doi:10.1016/0076-6879(90)85037-o

Cited by 40 publications

(16 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The full-length cDNA for human ␣2(I) preprocollagen has been described (25). The PDI gene used was from either chicken (26) or human (27) utilizing the yeast invertase secretion signal (23), replacing the first 22 amino acids of the chicken PDI gene. The ␣PH gene cDNA was from chicken (28) or human (29).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Production of Recombinant Human Type I Procollagen Trimers Using a Four-gene Expression System in the Yeast Saccharomyces cerevisiae

Toman¹,

Chisholm²,

McMullin³

et al. 2000

Journal of Biological Chemistry

Self Cite

112

View full text Add to dashboard Cite

The expression of stable recombinant human collagen requires an expression system capable of post-translational modifications and assembly of the procollagen polypeptides. Two genes were expressed in the yeast Saccharomyces cerevisiae to produce both propeptide chains that constitute human type I procollagen. Two additional genes were expressed coding for the subunits of prolyl hydroxylase, an enzyme that post-translationally modifies procollagen and that confers heat (thermal) stability to the triple helical conformation of the collagen molecule. Type I procollagen was produced as a stable heterotrimeric helix similar to type I procollagen produced in tissue culture. A key requirement for glutamate was identified as a medium supplement to obtain high expression levels of type I procollagen as heatstable heterotrimers in Saccharomyces. Expression of these four genes was sufficient for correct assembly and processing of type I procollagen in a eucaryotic system that does not produce collagen.Collagen is the single most abundant protein found in animals. It is found in all animals, including sponges. It is not expressed in yeast. In mammals, it is expressed in most tissues and plays both a structural as well as a signaling role in the development, maintenance, and repair of tissues and organs. More than 30 gene products compose the collagen family of molecules (1). Procollagens have several features and require numerous steps for production of functional molecules, including post-translational modifications (2). Key features in the collagen family are the formation of a triple helix composed of three polypeptide chains and the post-translational modification of proline residues to hydroxyproline, which provides stability of the triple helix against thermal denaturation and unfolding (T m ) 1 at the animal's body temperature (3). The content of proline and hydroxyproline is correlated with the temperature of an animal's environment (4). The triple helical domain of procollagen consists of -(GXY) n -repeats, where X and/or Y is frequently proline or hydroxyproline in the mature molecule. Prolyl 4-hydroxylase, an ␣ 2 ␤ 2 tetrameric enzyme composed of the prolyl hydroxylase ␣-subunit (␣PH) and the protein-disulfide isomerase (PDI) subunit in higher eucaryotes, is the enzyme that modifies proline residues to hydroxyproline. Additional steps for procollagen production include carbohydrate attachment, folding into a triple helix, secretion into the extracellular matrix, and cleavage by specific proteases to remove the propeptide domains to form mature collagen helices. A C-terminal non-helical propeptide facilitates the assembly of trimeric collagen molecules, leading to helix formation (5); the N-terminal propeptide may limit fiber diameter (6). The association and folding steps of three polypeptide chains that compose the triple helix potentially require chaperone functions in the endoplasmic reticulum, with PDI (7) and Hsp47 (8) as two proteins that have been implicated in the assembly of a procollagen trimer.A fundam...

show abstract

Section: Methodsmentioning

confidence: 99%

“…The prepro-␣-factor signal was isolated using PCR, whereas the prepro-HSA signal was constructed from synthetic oligonucleotides. Both sequences were isolated as EcoRI/SalI fragments with the SalI site containing the Arg-Arg KEX2 protease cleavage site (23) at the end of these prosequences to give authentic procollagen protein.…”

Section: Methodsmentioning

confidence: 99%

Production of Recombinant Human Type I Procollagen Trimers Using a Four-gene Expression System in the Yeast Saccharomyces cerevisiae

Toman¹,

Chisholm²,

McMullin³

et al. 2000

Journal of Biological Chemistry

Self Cite

112

View full text Add to dashboard Cite

show abstract

“…It has been recommended for the efficient expression of heterologous genes in yeast to remove most of the 3'-flanking sequence on the gene (< 30 bp remain; Hitzeman et al, 1990). The 5'-flanking region of the start codon could also influence the initiation of translation and thereby affect gene expression (Schneider and Guarente, 1991 ;Cullin and Pompon, 1988).…”

Section: B E B E H 4 12mentioning

confidence: 99%

Cloning of Arabidopsis thaliana Glutathione Synthetase (GSH2) by Functional Complementation of a Yeast Gsh2 Mutant

Ullmann

Gondet

Potier

et al. 1996

European Journal of Biochemistry

View full text Add to dashboard Cite

Glutathione (L-y-glutamyl-L-cysteinylglycine, GSH) plays an important role in the protection of plants against various types of stress caused by reactive oxygen species, gazeous pollutants, heavy metals and xenobiotics. A cDNA fragment containing the entire coding unit for glutathione synthetase (GSH2) of Ambidopsis thaliunu was cloned by complementation of the methylglyoxal sensitivity of a gsh2 mutant of the yeast Succharomyces cerevisiae. The cDNA encodes a protein of 478 amino acids (deduced M,: 53 783), bearing clear sequence similarities to GSH2 products from frog embryos (Xenopus laevis), rat kidney (Rattus norvegicus) and from the fission yeast (Schizosaccharonzyces pombe). A highly conserved glycine-rich domain close to the carboxy-terminus was found in the GSH2 product and appears to be typical for eukaryotic glutathione synthetases. The M , is similar to those of soluble animal enzymes, suggesting that the Amhidopsis gene also codes for a cytosolic protein. Genomic DNA-blot analysis indicates the presence of a single GSH2 gene. The yeast gsh2 mutant becomes resistant to methylglyoxal and cadmium after transformation with the plasmid bearing the Arabidopsis GSH2 cDNA. Moreover, this increased resistance is correlated to the restoration of GSH content from below detectibility in mutants to about 50% of the wild-type levels in transformed cells.Keywords: glutathione synthetase; expression cloning; Arabidopsis thaliana; evolutionary relationships; cadmium.Glutathione (L-y-glutamyl-L-cysteinylglycine, GSH) is an ubiquitous tripeptide (Rennenberg, 1982;Fahey and Sundquist, 1991: Penninckx andElskens, 1993) that plays an important role in the protection of plants against various types of stress caused by reactive oxygen species and gazeous pollutants (Alscher, 19891, low temperature (De Kok and Oosterhuis, 1983) and heavy metals (Grill et al., 1985). GSH is also involved in the detoxification of xenobiotics through formation of conjugates via the action of' various GSH S-transferases (Timmerman, 1989). For some safeners, compounds which increase the selectivity of some herbicides, it was reported that the protection of the crop plant is partially due to a stimulation of GSH accumulation (Farago et al., 1994), followed by increased conjugation and sequestration into the vacuole (Gaillard et al., 1994). Furthermore, GSH, which is chemically stable, represents the most abundant form of reduced sulphur within the cell and, in plants, it appears to be involved in the control of uptake and xylem loading of sulfate (Herschbach and Rennenberg, 1994 Ahhreviations. GSH, glutathione; GSH-I, y-glutamylcysteine synthetase; GSHI, y-glutamylcysteine synthetase gene; GSH-11, glutathione synthetase; GSH2, glutathione synthetase gene.Enzymes. Glutathione synthetase, y-L-glutamyl-L-cysteine: glycine ligase (ADP forming) (EC 6.3.2.3); pglutamylcysteine synthetase (EC 6.3.2.2).Note. The nucleotide sequence mentioned here has been submitted to the EMBL sequence data bank and is available under accession number XX3411.GSH is syn...

show abstract

“…This sequence promotes secretion of the mating factor ␣ in yeast cells. Processing by the KEX2-protease has been reported to be a rate-limiting step in secretion (30). In order to best mimic the authentic KEX2-processing site (Lys-Arg2Glu-Ala), the Glu-Ala dipeptide which belongs to the signal sequence of preprocathepsin B was not deleted during construction of the fusion protein.…”

Section: Resultsmentioning

confidence: 99%

Cathepsin B of Schistosoma mansoni

Lipps

Füllkrug

Beck

1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

Procathepsin B from the parasitic trematode Schistosoma mansoni was expressed as a glycosylation-minus mutant in yeast cells and purified by means of a histidine affinity tag which was added to the carboxyl terminus of the recombinant protein. The purified zymogen underwent autoprocessing but required an assisting protease for activation. Pepsin-activated schistosomal cathepsin B was further characterized with the cathepsin B-specific substrates N-benzyloxycarbonyl (Z)-Arg-Arg-p-nitroanilide, Z-Arg-Arg-7-amido-4-methylcoumarin, and Z-Phe-Arg-7-amido-4-methylcoumarin. A proteolytic activity comparable to mammalian cathepsin B was observed. In addition, we analyzed the degradation of human hemoglobin by schistosomal cathepsin B, which has been suggested to be the physiological target of the protease.

show abstract

[35] Use of heterologous and homologous signal sequences for secretion of heterologous proteins from yeast

Cited by 40 publications

References 43 publications

Production of Recombinant Human Type I Procollagen Trimers Using a Four-gene Expression System in the Yeast Saccharomyces cerevisiae

Production of Recombinant Human Type I Procollagen Trimers Using a Four-gene Expression System in the Yeast Saccharomyces cerevisiae

Cloning of Arabidopsis thaliana Glutathione Synthetase (GSH2) by Functional Complementation of a Yeast Gsh2 Mutant

Cathepsin B of Schistosoma mansoni

Contact Info

Product

Resources

About