Production of Rat Protein Disulfide Isomerase in Saccharomyces cerevisiae

Laboissiere, Martha C. A.; Chivers, Péter T.; Raines, Ronald T.

doi:10.1006/prep.1995.1092

Cited by 16 publications

(11 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Western blot analysis of intracellular soluble proteins showed two distinct molecular weight Na-ASP1 products. The nature of these bands could be a result of unprocessed and incomplete processing of the a-factor leader (Laboissiere et al, 1995), 52 and 54 kDa, respectively. The 52 kDa accounts for cleavage of the pre-region from the a-mating factor signal sequence with the pro-region still attached to Na-ASP1 protein.…”

Section: Discussionmentioning

confidence: 99%

Enhancement of protein secretion inPichia pastoris by overexpression of protein disulfide isomerase

İnan

Aryasomayajula

Sinha

et al. 2006

Biotechnol. Bioeng.

158

View full text Add to dashboard Cite

A potential vaccine candidate, Necator americanus secretory protein (Na-ASP1), against hookworm infections, has been expressed in Pichia pastoris. Na-ASP1, a 45 kDa protein containing 20 cysteines, was directed outside the cell by fusing the protein to the preprosequence of the alpha-mating factor of Saccharomyces cerevisiae. Most of the protein produced by single copy clones was secreted outside the cell. However, increasing gene copy number of Na-ASP1 protein in P. pastoris saturated secretory capacity and therefore, decreased the amount of secreted protein in clones harboring multiple copies of Na-ASP1 gene. Overexpression of the endoplasmic reticulum (ER) resident, homologous chaperone protein, protein disulfide isomerase (PDI) was able to increase the secretion of (Na-ASP1) protein in high copy clones. The effect of PDI levels on secretion of Na-ASP1 protein was examined in clones with varying copy number of PDI gene. Increase in secreted Na-ASP1 secretion is correlated well with the PDI copy number. Increasing levels of PDI also increased overall Na-ASP1 protein production in all the clones. Nevertheless, there was still accumulation of intracellular Na-ASP1 protein in P. pastoris clones over-expressing Na-ASP1 and PDI proteins.

show abstract

Section: Discussionmentioning

confidence: 99%

Enhancement of protein secretion inPichia pastoris by overexpression of protein disulfide isomerase

İnan

Aryasomayajula

Sinha

et al. 2006

Biotechnol. Bioeng.

158

View full text Add to dashboard Cite

show abstract

“…[For a detailed discussion of this expression system, see Laboissiere et al (1995a).] First, experiments were designed so that the same plasmid could be used for both complementation analysis (in pdilA strain YPH274) and protein production (in vacuolar protease-deficient strain BJ2168).…”

Section: Function Of Trx In Vivomentioning

confidence: 99%

“…Mutant and wild-type Trxs were produced in vacuolar protease-deficient S.cerevisiae BJ2168 using a protocol similar to that of Laboissiere et al (1995a). Briefly, to select for plasmid YEpWL.TRX, starter cultures (10 ml) were grown at 30°C in SDtrp medium.…”

Section: Protein Purificationmentioning

confidence: 99%

The CXXC motif: imperatives for the formation of native disulfide bonds in the cell.

1996

View full text Add to dashboard Cite

The rapid formation of native disulfide bonds in cellular proteins is necessary for the efficient use of cellular resources. This process is catalyzed in vitro by protein disulfide isomerase (PDI), with the PDI1 gene being essential for the viability of Saccharomyces cerevisiae. PDI is a member of the thioredoxin (Trx) family of proteins, which have the active‐site motif CXXC. PDI contains two Trx domains as well as two domains unrelated to the Trx family. We find that the gene encoding Escherichia coli Trx is unable to complement PDI1 null mutants of S.cerevisiae. Yet, Trx can replace PDI if it is mutated to have a CXXC motif with a disulfide bond of high reduction potential and a thiol group of low pKa. Thus, an enzymic thiolate is both necessary and sufficient for the formation of native disulfide bonds in the cell.

show abstract

“…The rPDI gene (taken as a Tth III- Sal I fragment from pMAL5.1, kindly donated by Dr. R.T. Raines, Department of Chemistry, University of Wisconsin-Madison) [50] was subcloned into the Nde I- Sal I sites of pET28a (Novagen) (after blunting the Nde I site) and expressed in Escherichia coli BL21(DE3). The PDI-treated TLPs were incubated at 37° for 1 h, diluted to 0.05 final MOI and inoculated on cells which had been previously treated (or not) with 2 m M DTNB or 20 m M bacitracin for 30 min at 37°.…”

Section: Methodsmentioning

confidence: 99%

Inhibiting Rotavirus Infection by Membrane-Impermeant Thiol/Disulfide Exchange Blockers and Antibodies against Protein Disulfide Isomerase

Calderón¹,

Guerrero

Acosta

et al. 2012

Intervirology

View full text Add to dashboard Cite

Objectives: Determining the effect of membrane-impermeant thiol/disulfide exchange inhibitors on rhesus rotavirus infectivity in MA104 cells and investigating protein disulfide isomerase (PDI) as a potential target for these inhibitors. Methods: Cells were treated with DTNB [5,5-dithio-bis-(2-nitrobenzoic acid)], bacitracin or anti-PDI antibodies and then infected with virus. Triple-layered particles (TLPs) were also pretreated with inhibitors before inoculation. The effects of these inhibitors on α-sarcin co-entry, virus binding to cells and PDI-TLP interaction were also examined. FACS analysis, cell-surface protein biotin-labeling, lipid-raft isolation and ELISA were performed to determine cell-surface PDI expression. Results: Infectivity became reduced by 50% when cells or TLPs were treated with 1 or 6 mM DTNB, respectively; infectivity became reduced by 50% by 20 mM bacitracin treatment of cells whereas TLPs were insensitive to bacitracin treatment; anti-PDI antibodies decreased viral infectivity by about 45%. The presence of DTNB (2.5 mM) or bacitracin (20 mM) was unable to prevent virus binding to cells and rotavirus-induced α-sarcin co-entry. Conclusions: It was concluded that thiol/disulfide exchange was involved in rotavirus entry process and that cell-surface PDI was at least a potential target for DTNB and bacitracin-induced infectivity inhibition.

show abstract

Production of Rat Protein Disulfide Isomerase in Saccharomyces cerevisiae

Cited by 16 publications

References 35 publications

Enhancement of protein secretion inPichia pastoris by overexpression of protein disulfide isomerase

Enhancement of protein secretion inPichia pastoris by overexpression of protein disulfide isomerase

The CXXC motif: imperatives for the formation of native disulfide bonds in the cell.

Inhibiting Rotavirus Infection by Membrane-Impermeant Thiol/Disulfide Exchange Blockers and Antibodies against Protein Disulfide Isomerase

Contact Info

Product

Resources

About