Immobilization and affinity purification of recombinant proteins using histidine peptide fusions.
A gene fusion approach to simplify protein immobilization and purification is described. A gene encoding the protein of interest is fused to a gene fragment encoding the affinity peptide Ala-His-Gly-His-Arg-Pro. The expressed fusion proteins can be purified using immobilized metal affinity chromatography. A vector, designed to ensure obligate head-to-tail polymerization of oligonucleotide linkers was constructed by in vitro mutagenesis. A linker encoding the affinity peptide, was synthesized and polymerized to two, four and eight copies. These linkers were fused to the 3' end of a structural gene encoding a two-domain protein A molecule, ZZ, and to the 5' end of a gene encoding beta-galactosidase. Fusion proteins, of both types, with zero or two copies of the linker showed little or no binding to immobilized Zn2+, while a relatively strong interaction could be observed for the fusions based on four or eight copies of the linker. Using a pH gradient, the ZZ fusions were found to be eluted from the resin at different pHs depending on the number of the affinity peptide. These results demonstrate that genetic engineering can be used to facilitate purification and immobilization of proteins to immobilized Zn2+ and that the multiplicity of the affinity peptide is an important factor determining the binding characteristics.
Ljungquist
C
C
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Breitholtz
A
A
Brink-Nilsson
H
H
Moks
T
T
Uhlén
M
M
Nilsson
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005696:Galactosidases / Q000302:isolation & purification*; D005822:Genetic Vectors; D006639:Histidine; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D009842:Oligopeptides; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification*; D001616:beta-Galactosidase / Q000235:genetics / Q000302:isolation & purification*
D015345:Oligonucleotide Probes; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D006639:Histidine; D005696:Galactosidases; D001616:beta-Galactosidase