Secretion of glycosylated human erythropoietin from yeast directed by the α-factor leader region

Elliott, Steve; Giffin, James C.; Suggs, Sid; Lau, Edward; Banks, Allen R.

doi:10.1016/0378-1119(89)90102-9

Cited by 54 publications

(29 citation statements)

References 27 publications

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“…Intracellular retention of insulin required endoproteolytic cleavage of the fusion protein by Kex2 in the Golgi. Expression of an ␣MF leader/glycosylated erythropoietin fusion protein in yeast identified several rate-limiting steps, including cleavage of the ␣MF-erythropoietin precursor protein by Kex2 and transport of the protein through the secretory pathway (65).…”

Section: Discussionmentioning

confidence: 99%

Supplying Copper to the Cuproenzyme Peptidylglycine α-Amidating Monooxygenase

Meskini

Culotta

Mains

et al. 2003

Journal of Biological Chemistry

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We explored the role of known copper transporters and chaperones in delivering copper to peptidylglycine-␣-hydroxylating monooxygenase (PHM), a copper-dependent enzyme that functions in the secretory pathway lumen. We examined the roles of yeast Ccc2, a P-type ATPase related to human ATP7A (Menkes disease protein) and ATP7B (Wilson disease protein), as well as yeast Atx1, a cytosolic copper chaperone. We expressed soluble PHMcc (catalytic core) in yeast using the yeast pre-pro-␣-mating factor leader region to target the enzyme to the secretory pathway. Although the yeast genome encodes no PHM-like enzyme, PHMcc expressed in yeast is at least as active as PHMcc produced by mammalian cells. PHMcc partially co-migrated with a Golgi marker during subcellular fractionation and partially co-localized with Ccc2 based on immunofluorescence. To determine whether production of active PHM was dependent on copper trafficking pathways involving the CCC2 or ATX1 genes, we expressed PHMcc in wild-type, ccc2, and atx1 mutant yeast. Although ccc2 and atx1 mutant yeast produce normal levels of PHMcc protein, it lacks catalytic activity. Addition of exogenous copper yields fully active PHMcc. Similarly, production of active PHM in mouse fibroblasts is impaired in the presence of a mutant ATP7A gene. Although delivery of copper to lumenal cuproproteins like PAM involves ATP7A, lumenal chaperones may not be required.

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Section: Discussionmentioning

confidence: 99%

Supplying Copper to the Cuproenzyme Peptidylglycine α-Amidating Monooxygenase

Meskini

Culotta

Mains

et al. 2003

Journal of Biological Chemistry

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“…It has also been introduced into insect cells (Quelle et al, 1989), bacteria (LeeHuang, 1984), and yeast (Elliott et al, 1989). In case of non-mammalian systems, the carbohydrate moieties were lacking in the recombinant EPO compared to the human EPO, making these recombinant EPOs biologically inactive in vivo.…”

Section: Introductionmentioning

confidence: 97%

Overexpression of human erythropoietin (EPO) affects plant morphologies: retarded vegetative growth in tobacco and male sterility in tobacco and Arabidopsis

Cheon

Kim

et al. 2004

Transgenic Res

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Erythropoietin (EPO) is a glycoprotein used for curing human anemia by regulating the differentiation of erythroid progenitors and the production of red blood cells. To examine the expression of recombinant EPO in plants, pPEV-EP21, in which human epo cDNA under the control of the CaMV 35S promoter, was introduced into tobacco and Arabidopsis via Agrobacterium tumefaciens-mediated transformation. The RNA expression level of epo in the transgenic lines was initially estimated by Northern blot analysis. Two transgenic lines, which exhibited a high expression level of epo mRNA determined by Northern analysis, were chosen for Western blot analysis to examine the production of EPO proteins. Those two lines, EP21-12 and EP21-14, revealed detectable bands on the immunoblot. Interestingly, constitutive expression of the human epo gene affected the morphologies in transgenic plants such that vegetative growth of transgenic tobacco was retarded, and male sterility was induced in transgenic tobacco and Arabidopsis.

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“…Unfortunately, despite numerous reports on a-factor-mediated secretion of heterologous proteins (for review see Brake, 1989), pulse/chase kinetic experiments to study formation, conversion and translocation of precursors have not been carried out in other cases. From Western blot analysis of extra-and intra-cellular material, evidence can be obtained that at least for a consensus interferon species (Zsebo et al, 1986), transforming growth factor CI (Barr et al, 1987) or erythropoietin (Elliott et al, 1989), all expressed as a-factor-leader fusions, secretion seems to be limiting, leading to accumulation of larger amounts of presumed precursors inside yeast cells. In many cases this inefficient secretion was accompanied by the occurrence of unprocessed, hyperglycosylated fusion protein in the medium (Zsebo et al, 1986), Stetler et al, 1989).…”

Section: Discussionmentioning

confidence: 99%

α‐Factor‐leader‐directed secretion of recombinant human‐insulin‐like growth factor I from Saccharomyces cerevisiae

Steube

Chaudhuri

Märki

et al. 1991

European Journal of Biochemistry

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A synthetic gene coding for human-insulin-like growth factor I (IGFI) was fused to the leader sequence of yeast prepro-a-factor and expressed in Saccharomyces cerevisiae under the control of a glyceraldehyde-3-phosphate dehydrogenase promoter fragment. Recombinant IGFI was found inside yeast cells and secreted into the medium. The secreted IGFI migrated on SDS gels with the same electrophoretic mobility as authentic IGFI, i.e. at about 7.5 kDa. HPLC analysis of secreted IGFI revealed the presence of the correctly folded, genuine molecule as well as an isomeric byproduct of equal molecular mass but with two of the three disulfide bonds interchanged.Inside exponentially growing cells the 7.5-kDa IGFI was also found, along with up to four additional IGFIrelated polypeptides of higher molecular mass. By endoglycosidase F treatment the three polypeptides between 19-26 kDa were converted to a single peptide of 17 kDa. Since this peptide also reacted with an anti-a-factor antibody, it represents most likely the unglycosylated a-factor -IGFI fusion precursor. Pulse-chase experiments established the precursor nature of the intracellular higher-molecular-mass IGFI species. Conversion of the primary translation product to the differently glycosylated IGFI precursor proteins and into the mature form occurred very rapidly, within 2 min. Rapid maturation was, however, not followed by an equally rapid secretion of the mature form into the medium: only after 30-40 min did IGFI appear outside the cells.We therefore postulate the presence of an as yet undefined Golgi or post-Golgi bottleneck representing a major obstacle in secretion of recombinant IGFI from S. cerevisiae cells.

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Secretion of glycosylated human erythropoietin from yeast directed by the α-factor leader region

Cited by 54 publications

References 27 publications

Supplying Copper to the Cuproenzyme Peptidylglycine α-Amidating Monooxygenase

Supplying Copper to the Cuproenzyme Peptidylglycine α-Amidating Monooxygenase

Overexpression of human erythropoietin (EPO) affects plant morphologies: retarded vegetative growth in tobacco and male sterility in tobacco and Arabidopsis

α‐Factor‐leader‐directed secretion of recombinant human‐insulin‐like growth factor I from Saccharomyces cerevisiae

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