Cloning, sequencing, expression, and single-step purification of the adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase (CobU) from Salmonella typhimurium ATCC 19585.
The bi-functional enzyme, adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase (CobU), is involved in the biosynthesis of cobalamin in Salmonella typhimurium, and, therefore, can be used for the in vitro synthesis of analogs of B(12). Previously, five different steps were required to purify the recombinant enzyme from Escherichia coli. Here, we describe the cloning, sequencing, and expression of the cobU gene from S. typhimurium ATCC 19585 and, without introducing a purification tag sequence to the N- or C-terminus of the recombinant enzyme, a new single-step purification method based on hydrophobic interaction chromatography.
Hsu
Fang-Ciao
FC
Institute of Biotechnology and Department of Chemical Engineering, National Taipei University of Technology, Taiwan.
Ho
Tsung-Jung
TJ
Lai
Chien-Chen
CC
Lin
Chin-Fen
CF
Chen
Hao-Ping
HP
D002847:Chromatography, Agarose / Q000379:methods; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D057927:Hydrophobic and Hydrophilic Interactions; D009097:Multienzyme Complexes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010430:Pentosyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry; D017422:Sequence Analysis, DNA
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C020927:phenyl-sepharose; D012685:Sepharose; D010430:Pentosyltransferases; C028015:nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase; D009713:Nucleotidyltransferases