Overexpression, purification, and characterization of the periplasmic space thiamin-binding protein of the thiamin traffic ATPase in Escherichia coli.
Thiamin (Vitamin B(1)) transport in Escherichia coli occurs by the superfamily of traffic ATPases in which the initial receptor is the periplasmic binding protein. We have cloned the periplasmic thiamin-binding protein (TBP) of the E. coli periplasmic thiamin transport system and purified the overexpressed protein to apparent homogeneity. A subsequent biochemical characterization demonstrates that TBP is a 34.205kDa monomer. TBP also contains one tightly bound thiamin species [thiamin, thiamin monophosphate (TMP), or thiamin diphosphate (TDP)] per monomer (K(D)=0.8 microM) when isolated under conditions that would remove any loosely bound ligands. We also demonstrate that thiamin is readily exchangeable in the presence of exogenous thiamin with a k(off)=0.12s(-1). The biochemical characteristics of the overexpressed, plasmid-derived TBP are indistinguishable from those determined for endogenous TBP purified from E. coli. The overexpression and purification of TBP that we present here allows the rapid isolation of large amounts of pure protein that are required for further mechanistic and structural studies and demonstrates a vast improvement over previously reported purifications.
Hollenbach
Andrew D
AD
Department of Biochemistry and Molecular Biology, The Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD 21205, USA. andrew.hollenbach@stjude.org
Dickson
Kimberly A
KA
Washabaugh
Michael W
MW
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019897:Periplasm / Q000737:chemistry*; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013831:Thiamine / Q000378:metabolism*
D002352:Carrier Proteins; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; C488915:tbpA protein, E coli; C026989:thiamine-binding protein; D000251:Adenosine Triphosphatases; D013831:Thiamine