Isolation and functional analysis of histidine-tagged elongation factor Tu.
The study of the structure/function relationships of the Escherichia coli elongation factor Tu (EF-Tu) via mutagenesis has been hampered by difficulties encountered in separating the mutated factor from other proteins, in particular native EF-Tu. Here we describe a novel system for the purification of EF-Tu mutant species, based on metal-ion affinity chromatography. To facilitate rapid and efficient purification we designed a recombinant EF-Tu with an additional C-terminal sequence of one serine and six histidine residues. A cell extract containing the His-tagged EF-Tu (EF-TuHis) is applied to a Ni(2+)-nitrilotriacetic acid column. EF-TuHis can be selectively eluted with an imidazole containing buffer, yielding a preparation of more than 95% purity, free of wild-type EF-Tu. In-vitro and in-vivo functional analyses show that EF-TuHis resembles the wild-type EF-Tu, which makes this one-step isolation procedure a promising tool for the study of the interactions of mutant EF-Tu with the various components of the elongation cycle. The new isolation procedure was successfully applied for the purification of a mutant EF-TuHis with a Glu substitution for Lys237, a residue possibly involved in the binding of aminoacyl-tRNA.
Boon
K
K
Department of Biochemistry, Gorlaeus Laboratories, Leiden University, The Netherlands.
Vijgenboom
E
E
Madsen
L V
LV
Talens
A
A
Kraal
B
B
Bosch
L
L
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism; D006639:Histidine / Q000737:chemistry*; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides; D010444:Peptide Elongation Factor Tu / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D009841:Oligonucleotides; D006639:Histidine; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu