Designing new metal affinity peptides by random mutagenesis of a natural metal-binding site.
The metal-binding site of a Helicobacter pylori ATPase 439 (heli(WT)-tag) was successfully used as a new fusion peptide for immobilized metal ion affinity chromatography (IMAC). It produced higher yields than the frequently used his6-tag. Due to stronger binding of the peptide to metal ions, harsher elution conditions were, however, necessary. This undesired side-effect was overcome by modifying the heli(WT)-tag by polymerase chain reaction-directed mutagenesis. The modified tags were screened by an automated high-throughput IMAC system, leading to a heliM14-tag peptide that could be eluted under conditions similar to those of the his6-tag but at the same time produced 20% higher yields of the desired protein.
Enzelberger
M M
MM
Institut für Technische Biochemie, Stuttgart, Germany.
Minning
S
S
Schmid
R D
RD
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D017931:DNA Primers; D004274:DNA, Recombinant; D049452:Green Fluorescent Proteins; D016480:Helicobacter pylori / Q000201:enzymology; D008164:Luminescent Proteins / Q000737:chemistry; D008567:Membranes, Artificial; D008670:Metals / Q000378:metabolism*; D016296:Mutagenesis; D010455:Peptides / Q000737:chemistry*; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D004274:DNA, Recombinant; D008164:Luminescent Proteins; D008567:Membranes, Artificial; D008670:Metals; D010455:Peptides; D049452:Green Fluorescent Proteins; D000251:Adenosine Triphosphatases