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Refolding of the human dihydrolipoamide dehydrogenase

Paper ID Volume ID Publish Year Pages File Format Full-Text
4206 214 2009 6 PDF Available
Title
Refolding of the human dihydrolipoamide dehydrogenase
Abstract

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 μM FAD and 10 mM DTT in 100 mM Tris, 150 mM 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.

Keywords
LADH, dihydrolipoamide dehydrogenase (protein); DLD, gene coding for LADH; MS, mass spectrometry; MW, molecular weight; MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; CD, circular dichroism; SDS-PAGE, sodium dodecyl sulfate polya
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Refolding of the human dihydrolipoamide dehydrogenase
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biochemical Engineering Journal - Volume 45, Issue 2, 15 July 2009, Pages 120–125
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us