Addition of a poly-(6X) His tag to Milk Bundle-1 and purification using immobilized metal-affinity chromatography.
Milk Bundle 1 (MB-1) is a de novo designed protein enriched in M, T, K, and L. Its future application is as a high-quality dietary protein source for ruminants. The protein is currently expressed in Escherichia coli and is being characterized to solve its folded conformation. MB-1 has marginal stability at room temperature, which has hindered our attempts at characterization. To increase the stability of the protein at room temperature, the purification procedure was examined and changed to hopefully increase its effectiveness. We describe here the production and purification of a new MB-1 with six His residues at the C-terminal end. This allows the new mutant (MB-1-His) to bind metal ions and to be purified with immobilized metal-affinity chromatography (IMAC). MB-1-His obtained using IMAC was purer on SDS-PAGE than both MB-1 or MB-1-His isolated using the current protocol. The IMAC protocol is more economical and more efficient; preliminary results show that the protein purified by this method is also quite stable at room temperature.
Grundy
J E
JE
Department of Chemistry, University of Prince Edward Island, Charlottetown, Canada.
Wirtanen
L Y
LY
Beauregard
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular; D004044:Dietary Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010455:Peptides / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D004044:Dietary Proteins; C107457:MB-1synthetic dietary protein; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine