Medical College of Wisconsin
CTSICores SearchResearch InformaticsREDCap

Purification and characterization of the L-Ara4N transferase protein ArnT from Salmonella typhimurium. Protein Expr Purif 2006 Mar;46(1):33-9

Date

10/18/2005

Pubmed ID

16226890

DOI

10.1016/j.pep.2005.08.028

Scopus ID

2-s2.0-32644459308 (requires institutional sign-in at Scopus site)   16 Citations

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.

Author List

Bretscher LE, Morrell MT, Funk AL, Klug CS

Author

Candice S. Klug PhD Professor in the Biophysics department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Amino Acid Sequence
Bacterial Proteins
Circular Dichroism
Cloning, Molecular
Escherichia coli
Escherichia coli Proteins
Gene Deletion
Hexosyltransferases
Molecular Sequence Data
Protein Structure, Secondary
Recombinant Proteins
Salmonella typhimurium
Sequence Alignment
Sequence Homology, Amino Acid