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Refolding and activation of recombinant N-carbamoyl-d-amino acid amidohydrolase from Escherichia coli inclusion bodies

Paper ID Volume ID Publish Year Pages File Format Full-Text
36694 45142 2005 7 PDF Available
Title
Refolding and activation of recombinant N-carbamoyl-d-amino acid amidohydrolase from Escherichia coli inclusion bodies
Abstract

To recover active DCaseH, a recombinant N-carbamoyl-d-amino acid amidohydrolase C-terminally fused with a six-His peptide, from Escherichia coli inclusion bodies, a dilution-additive-type refolding strategy was employed for both urea-solubilized inclusion solutions and purified denatured DCaseH. All refoldings of the inclusion solutions led to an increase (blue shift) in the average emission energy of the intrinsic tryptophan fluorescence, while only the ones with the addition of 30–50% glycerol effectively restored DCaseH activity, with a best activity achieved at 40 μg/mL total protein concentration, 50% glycerol, and pH 8 for 22 days. A further native purification using metal affinity resins yielded pure DCaseH of 2.54 U/mg, which is 37% of the native activity. On the other hand, the refolding of the purified denatured DCaseH was best yielded at 50% glycerol for 3 days, with a 20% renaturation yield. Circular dichroism analyses of the refolded pure DCaseH revealed that approximately 38% of the native secondary structure was reformed. Therefore, the refolding with the addition of glycerol partially recovered both the activity and the native-like secondary structure of denatured DCaseH. As for better renaturation, a refolding of solubilized inclusion solutions followed by a native purification is suggested.

Keywords
Activation; N-carbamoyl-d-amino acid amidohydrolase; Dilution; Glycerol; Inclusion body; Refolding
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Refolding and activation of recombinant N-carbamoyl-d-amino acid amidohydrolase from Escherichia coli inclusion bodies
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 40, Issue 6, May 2005, Pages 2135–2141
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