Quantitative evaluation of refolding conditions for a disulfide-bond-containing protein using a concise 18O-labeling technique
✍ Scribed by Hiromasa Uchimura; Yusam Kim; Takaaki Mizuguchi; Yoshiaki Kiso; Kazuki Saito
- Book ID
- 105356643
- Publisher
- Cold Spring Harbor Laboratory Press
- Year
- 2011
- Tongue
- English
- Weight
- 374 KB
- Volume
- 20
- Category
- Article
- ISSN
- 0961-8368
- DOI
- 10.1002/pro.637
No coin nor oath required. For personal study only.
✦ Synopsis
Abstract
A concise method was developed for quantifying native disulfide‐bond formation in proteins using isotopically labeled internal standards, which were easily prepared with proteolytic ^18^O‐labeling. As the method has much higher throughput to estimate the amounts of fragments possessing native disulfide arrangements by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) than the conventional high performance liquid chromatography (HPLC) analyses, it allows many different experimental conditions to be assessed in a short time. The method was applied to refolding experiments of a recombinant neuregulin 1‐β1 EGF‐like motif (NRG1‐β1), and the optimum conditions for preparing native NRG1‐β1 were obtained by quantitative comparisons. Protein disulfide isomerase (PDI) was most effective at the reduced/oxidized glutathione ratio of 2:1 for refolding the denatured sample NRG1‐β1 with the native disulfide bonds.