Purification and characterization of the L-Ara4N transferase protein ArnT from Salmonella typhimurium.

Abstract:

:The covalent addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) groups to lipid A, which resides in the outer membranes of bacteria such as Salmonella typhimurium and Escherichia coli, is the final step in the polymyxin-resistance pathway in these organisms. This modification is catalyzed by the inner membrane protein 4-amino-4-deoxy-L-arabinose transferase (ArnT). Little is known about the ArnT protein structure because it has not previously been purified. We report here the first expression and purification of 6 x His-tagged S. typhimurium ArnT in NovaBlue cells. The enzyme was purified using sequential Q-Sepharose anion exchange and HisLink nickel affinity column chromatography. The purified protein has an apparent molecular weight of 62 kDa on SDS-PAGE and the identity of the purified ArnT was confirmed by Western blot using a monoclonal antibody against the His-tag and by MALDI-TOF mass spectrometry. Purified ArnT protein was shown to be highly alpha-helical as determined by circular dichroism analysis. A chromosomal ArnT knockout strain of E. coli BL21(DE3) was developed to allow in vivo functional analysis of plasmid-encoded ArnT constructs, and a polymyxin assay was used to confirm that the cloned ArnT proteins retained full activity. These studies provide an essential foundation for further analysis of ArnT structure and function using mutagenesis and biophysical techniques.

journal_name

Protein Expr Purif

authors

Bretscher LE,Morrell MT,Funk AL,Klug CS

doi

10.1016/j.pep.2005.08.028

keywords:

subject

Has Abstract

pub_date

2006-03-01 00:00:00

pages

33-9

issue

1

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(05)00286-X

journal_volume

46

pub_type

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