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Using native hydantoinase promoter to induce d-carbamoylase soluble expression in Escherichia coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
4409 225 2008 5 PDF Available
Title
Using native hydantoinase promoter to induce d-carbamoylase soluble expression in Escherichia coli
Abstract

By use of PCR, the genes encoding d-carbamoylase from A. radiobacter TH572 were cloned in plasmid pET30a and transformed into Escherichia coli BL21 (DE3) to overexpress d-carbamoylase. However, almost all of the protein remained trapped in inclusion bodies. To improve the expression of the properly folded active enzyme, a constitutive plasmid of pGEMT-DCB was constructed using the native hydantoinase promoter (PHase) whose optimal length was confirmed to 209 bp. Furthermore, the RBS region in the downstream of PHase was optimized to increase the expression level, so the plasmid pGEMT-R-DCB was constructed and transformed into E. coli strain Top10F′. The enzyme activity of Top10F′/pGEMT-R-DCB grown at 37 °C was found to be 0.603 U/mg (dry cell weight, DCW) and increase 58-fold over cells of BL21 (DE3) harboring the plasmid pET-DCB grown at 28 °C.

Keywords
Hydantoinase promoter; d-Carbomoylase; Constitutive expression; d-p-Hydroxyphenylglycine (d-HPG)
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Using native hydantoinase promoter to induce d-carbamoylase soluble expression in Escherichia coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biochemical Engineering Journal - Volume 41, Issue 1, 1 August 2008, Pages 12–16
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