Purification and characterization of the L-Ara4N transferase protein ArnT from Salmonella typhimurium. Protein Expr Purif 2006 Mar;46(1):33-9
Date
10/18/2005Pubmed ID
16226890DOI
10.1016/j.pep.2005.08.028Scopus ID
2-s2.0-32644459308 (requires institutional sign-in at Scopus site) 16 CitationsAbstract
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 CSAuthor
Candice S. Klug PhD Professor in the Biophysics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Amino Acid SequenceBacterial 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