A rapid and sensitive assay for protein disulphide isomerase activity

Myllylä, Raili; Oikarinen, Jouko

doi:10.1016/0165-022x(83)90045-3

Cited by 11 publications

(8 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although the authors state that they did not observe similar effects during refolding of ribonuclease, purified PDI was used in their system. Our results suggest that the activity assay of Myllyla and Oikarinen (1983) is linear with purified PDI; however, it can be very nonlinear when using crude cell lysates due to concentration-dependent effects of other proteins on PDI foldase activity. These difficulties may explain why there are few reports in the literature which quantify PDI activity levels in cultured cells.…”

Section: Resultsmentioning

confidence: 87%

“…Protein Disulfide Isomerase Activity in the 9.2.27 Hybridoma during Batch Culture. The assay of Myllyla and Oikarinen (1983) was chosen for measuring PDI activity because its ease and sensitivity made it potentially suitable for analysis of multiple, crude cell samples. When purified PDI (Takara) was used, sRNase refolding was reproducible and proportional to the PDI concentration in the refolding mixture (Figure 2).…”

Section: Resultsmentioning

confidence: 99%

“…Quantitative measurement of PDI activity using the assay of Myllyla and Oikarinen (1983) on 9.2.27 hybridoma lysates varied using the same batch of randomly refolded ribonuclease. A recent in vitro study by Puig and Gilbert (1994) of lysozyme refolding by purified PDI showed that PDI may function as either an antichaperone or a chaperone depending on the relative concentrations of PDI and lysozyme and their order of addition to the reaction mixture.…”

Section: Resultsmentioning

confidence: 99%

“…Protein Disulfide Isomerase Activity Assay. The activity assay used was based on the procedure of Myllyla and Oikarinen (1983). Standard procedures were used for creating a ribonuclease-free environment in which to prepare reagents and run the assay (Sambrook et al, 1989).…”

Section: Methodsmentioning

confidence: 99%

“…Randomly reoxidized ribonuclease (sRNase) was prepared according to Myllyla and Oikarinen (19831, lyoph-ilized, gassed with nitrogen, and stored a t -80 or 4 "C. Before use, sRNase was reconstituted by adding ribonuclease-free water to a concentration of 1 mg/mL and stored in 20 pL aliquots at -20 "C. These aliquots were thawed immediately prior to use and never refrozen. Labeled RNA was prepared by feeding 9.2.27 cells with [5-3H]cytosine triphosphate (CTP), a modification of the method of Myllyla and Oikarinen (1983). A 100 pCi amount of [5-3H]CTP (Dupont-NEN, Wilmington, DE) was fed to a 10 mL culture containing 1.5 x lo7 exponentially growing 9.2.27 cells.…”

Section: Methodsmentioning

confidence: 99%

See 4 more Smart Citations

Alteration of Hybridoma Viability and Antibody Secretion in Transfectomas with Inducible Overexpression of Protein Disulfide Isomerase

Kitchin

Flickinger

1995

Biotechnology Progress

View full text Add to dashboard Cite

Monoclonal antibody (mAb)-secreting transfectomas with dexamethasone inducible expression of the mammalian endoplasmic reticulum foldase and chaperone protein disulfide isomerase (PDI, ERp59) were generated from the murine 9.2.27 hybridoma in order to obtain in vivo evidence of whether alteration of the level of PDI, believed to be involved in immunoglobulin (Ig) assembly, results in alteration of mAb secretion kinetics. Using an RNase refolding assay, the specific activity of endogenous PDI in the 9.2.27 hybridoma was found to be constant during batch growth. An expression vector for glucocorticoid-inducible overexpression of PDI, pMMTVPDI, was constructed from pMAMneo using a rat PDI cDNA. Cell lysates of stable transfectomas contained 2-4-fold higher levels of PDI mRNA and increased levels of PDI protein, detected by immunoblotting, following induction with 0.1 microM dexamethasone. Monoclonal antibody secretion kinetics were evaluated in 12.5 mL shake flasks, a 100 mL spinner, and a 1 L aerated batch reactor. A transfectoma was found with altered mAb secretion kinetics during cell growth following dexamethasone induction of PDI overexpression. Specific mAb secretion rate was not significantly increased following dexamethasone induction; however, hybridoma viability was sustained longer during the stationary phase of cell growth and hence total antibody yield was increased in comparison to the parent 9.2.27 hybridoma.

show abstract

Section: Resultsmentioning

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations