Jan 09, 2023
Deglycosylation of N-glycosylated proteins using PNGase F
- Kaia Kukk1
- 1Tallinn University of Technology
Protocol Citation: Kaia Kukk 2023. Deglycosylation of N-glycosylated proteins using PNGase F . protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9dr84g3e/v1
Manuscript citation:
Kukk, K. (2023) Identification, characterisation and recombinant expression of flavonoid 3’,5’-hydroxylases and cytochrome P450 reductases fromVaccinium species. bioRxiv 2023.01.09.523147; doi: https://doi.org/10.1101/2023.01.09.523147
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 09, 2022
Last Modified: January 09, 2023
Protocol Integer ID: 73770
Keywords: cytochrome p450 reductase from helianthus annuus, glycosylated protein, cytochrome p450 reductase, deglycosylation, pichia pastori, helianthus annuus
Funders Acknowledgements:
European Regional Development Fund
Grant ID: Grant ID: 1.1.1.2/VIAA/2/18/286
Abstract
The described protocol was used to confirm that NADPH-cytochrome P450 reductase from Helianthus annuus was N-glycosylated when recombinantly expressed in Pichia pastoris (Komagataella phaffii).
Materials
PNGase F from Elizabethkingia meningosepticaMerck MilliporeSigma (Sigma-Aldrich)Catalog #P7367-50UN
Troubleshooting
12,5 µl of yeast lysate containing sufficient amount of target protein for detecting by Western analysis was pipetted into a tube. Two samples were prepared in parallel, one for negative control without N-glycosidase and the other with PNGase F.
0,5 µl of 10% sodium dodecyl sulfate (SDS) and 1 µl of 1M dithiothreitol (DTT) were added.
The mixtures were incubated at 95 °C for 5 min.
The mixtures were cooled to room temperature after which 2 µl of 0,5 M Tris-HCl, pH 8 and 2 µl of 10% Triton X-100 were added.
2 µl of PNGase F (500 U/ml) was added to one of the samples. Instead of N-glycosidase, water was added to the negative control sample.
The samples were incubated overnight at 37 °C.
SDS-PAGE sample buffer was added and the samples were heated for 5 min at 99 °C. SDS-PAGE and Western analysis were carried out to see whether the molecular weight of the target protein had decreased.