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Production of 2-keto-3-deoxy-d-glycero-d-galacto-nonopyranulosonic acid (KDN) using fusion protein of N-acetyl-d-neuraminic acid aldolase

Paper ID Volume ID Publish Year Pages File Format Full-Text
5061 279 2006 6 PDF Available
Title
Production of 2-keto-3-deoxy-d-glycero-d-galacto-nonopyranulosonic acid (KDN) using fusion protein of N-acetyl-d-neuraminic acid aldolase
Abstract

A recombinant vector was constructed to overproduce a glutathione S-transferase (GST) tagged N-acetyl-d-neuraminic acid aldolase (Neu5Ac aldolase) fusion protein in Escherichia coli. A solid-phase cloning method was proposed for the recombination of a GST-containing plasmid and Neu5Ac aldolase gene amplified from E. coli K12. Although the over-expressed fusion protein was largely present as aggregate or inclusion body, soluble GST-Neu5Ac aldolase fusion protein possessing the enzymatic activity could be purified to homogeneity by the affinity of GST tag. Results from sequencing the reconstruction of Neu5Ac aldolase gene fusion and gel electrophoresis of its expressing product suggested that the insertion of DNA was in the proper reading frame. Using N-acetyl-d-neuraminic acid (Neu5Ac) as the substrate, the purified fusion protein showed a Km value of 2.8 mM that is close to those reported in the literature for Neu5Ac aldolase, indicating that the addition of GST tag in the N terminus did not change the affinity of Neu5Ac aldolase for substrate. The GST-Neu5Ac aldolase fusion protein, which was applied even in a very small dosage, could effectively catalyze the production of 2-keto-3-deoxy-d-glycero-d-galacto-nonopyranulosonic acid (KDN) from d-mannose and pyruvic acid.

Keywords
Fusion protein; N-acetyl-d-neuraminic acid aldolase; KDN; GST tag
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Production of 2-keto-3-deoxy-d-glycero-d-galacto-nonopyranulosonic acid (KDN) using fusion protein of N-acetyl-d-neuraminic acid aldolase
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biochemical Engineering Journal - Volume 29, Issues 1–2, 1 April 2006, Pages 75–80
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