The nonbiotinylated form of the 1.3 s subunit of transcarboxylase binds to avidin (monomeric)-agarose: purification and separation from the biotinylated 1.3 S subunit.
Avidin-biotin technology is used routinely to purify biotin-containing carboxylases and also proteins that have been chemically coupled to biotin. The 1.3 S subunit of transcarboxylase (TC) studied here is the biotin-containing subunit of TC which not only acts as a carboxyl carrier between the CoA ester sites on the central 12 S subunit of TC and keto acid sites on the outer 5 S subunit of TC but also links the 12 S and 5 S subunits together to form a 26 S multisubunit TC complex. The 1.3 S subunit has been cloned, sequenced, and expressed in Escherichia coli. A method for purifying recombinant 1.3 S subunits from E. coli using avidin (monomeric)-agarose column chromatography has been developed. This affinity-purified 1.3 S was found to be homogeneous by SDS-PAGE, amino acid composition, and N-terminal sequence analysis but had a biotin content of only 28% based on moles of biotin per mole of 1.3 S. This lack of stoichiometry was found to be due to copurification of apo-1.3 S as evidenced by the holocarboxylase synthetase reaction. A procedure for separating the apo- and biotinylated 1.3 S forms using hydrophobic interaction chromatography on an Ether 5 PW column is described. The method is based on the difference in hydrophobicity between apo and biotinylated 1.3 S forms. The copurification of apo and biotinylated forms of 1.3 S on the avidin (monomeric)-agarose column was found to be due to specific interaction with avidin rather than to interaction between apo- and biotinylated 1.3 S forms as demonstrated by the fluorescence quenching studies. The results suggest that the avidin-biotin system by itself may not be sufficient to obtain homogeneous biotinyl proteins as nonbiotinyl protein can also bind avidly to such columns.
Shenoy
B C
BC
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106-4935.
Magner
W J
WJ
Kumar
G K
GK
Phillips
N F
NF
Haase
F C
FC
Samols
D
D
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001360:Avidin / Q000378:metabolism*; D001710:Biotin; D019878:Carboxyl and Carbamoyl Transferases; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008969:Molecular Sequence Data; D011424:Propionibacterium / Q000201:enzymology; D012685:Sepharose / Q000378:metabolism; D014166:Transferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D001360:Avidin; D001710:Biotin; D012685:Sepharose; D014166:Transferases; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase