Journal
BIOCHEMICAL ENGINEERING JOURNAL
Volume 45, Issue 2, Pages 120-125Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bej.2009.03.004
Keywords
alpha-Ketoglutarate dehydrogenase complex; Dihydrolipoamide dehydrogenase; Inclusion body; Strep-tag; Refolding
Funding
- Hungarian Scientific Research Fund [049851]
- Young Investigator Grant of Semmelweis University
- Young Investigator Grant of the Centennial Foundation of Gedeon Richter Pharmaceutical, Plc
- Bolyai Fellow of the Hungarian Academy of Sciences
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A refolding procedure was optimized for human dihydrolipoamide dehydrogenase (LADH) that can serve as a complementary method to isolation protocols for cytosolic or periplasmic expression of the enzyme. The reported procedure is especially useful if part of the protein is precipitated into inclusion bodies during expression and higher quantities of the protein would be required without the need to increase the expression volume, like during protein labeling. Optimized parameters included the preparation/purification strategy of inclusion bodies, unfolding conditions, the composition of the refolding cocktail, time-dependence of the regained activity and dialysis conditions; other parameters were applied according to optimized values from previous studies or recommendations of the applied kits. The most efficient refolding mixture included 10 mu M FAD and 10 mM DTT in 100 mM Tris, 150mM NaCl, 1 mM EDTA at pH 8.0; this solution condition is also compatible to the subsequent Strep-tag affinity chromatography step applied for purification. The protein became fully active after refolding overnight. The final protein preparation showed a correct folding and a high purity owing to our optimized Strep-tag affinity purification strategy. The reported refolding protocol is able to generate up to 2.3 mg/L (of bacterial culture) active protein after complete purification. (C) 2009 Elsevier B.V. All rights reserved.
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