PMID:8425112
| Citation |
Shenoy, BC, Magner, WJ, Kumar, GK, Phillips, NF, Haase, FC and Samols, D (1993) 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. Protein Expr. Purif. 4:85-94 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1006/prep.1993.1013 |
| Keywords |
Amino Acid Sequence; Amino Acids/analysis; Avidin/metabolism; Biotin; Carboxyl and Carbamoyl Transferases; Chromatography, Affinity; Chromatography, High Pressure Liquid; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Molecular Sequence Data; Propionibacterium/enzymology; Sepharose/metabolism; Transferases/chemistry; Transferases/isolation & purification; Transferases/metabolism |
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