Purification and characterization of the L-Ara4N transferase protein ArnT from Salmonella typhimurium.
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.
Bretscher
Lynn E
LE
Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Morrell
Megan T
MT
Funk
Andrea L
AL
Klug
Candice S
CS
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017353:Gene Deletion; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D006602:Hexosyltransferases; C442226:L-Ara4N transferase