On the engineering of rDNA proteins for purification by immobilized metal affinity chromatography: applications to alternating histidine-containing chimeric proteins from recombinant Escherichia coli.
Recently we reported (D. B. Evans, W. G. Tarpley, and S. K. Sharma, 1991, Protein Expression Purif. 2, 205-213) the cloning, expression, and characterization of recombinant chimeric proteins with an N-terminal metal-binding peptide (mbp), His-Asp-His-Asp-His, and a renin cleavage site. Using these chimerics as examples, we describe here the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography (IMAC). In these chimerics, an alternate histidine-containing peptide was fused to the N-termini of HIV reverse transcriptase (HIV RT) and beta-galactosidase. These chimerics were retarded on immobilized nickel very strongly and could be completely eluted only by the use of 100 mM imidazole, whereas the wildtype HIV RT and Escherichia coli contaminating proteins were eluted between 10 and 35 mM imidazole. When the DNA coding for the mbp was removed, the resulting chimerics were recovered from the IMAC column at 35 mM imidazole. The strong and specific interaction between the chimeric protein and the immobilized metal ion was also abolished when the mbp was specifically cleaved by human renin. It is concluded from these studies that tailoring recombinant proteins with three or more alternate histidines should result in the isolation of such chimeric proteins from crude mixtures in a single step. Since IMAC is amendable to scale up, the tailored specificity engineered into the protein of interest via an mbp should allow one to achieve large-scale isolation of recombinant proteins from bacterial and nonbacterial hosts in a highly predictable manner.
Vosters
A F
AF
Biochemistry and Cancer and Infectious Diseases Research, Upjohn Company, Kalamazoo, Michigan 49001.
Evans
D B
DB
Tarpley
W G
WG
Sharma
S K
SK
D000595:Amino Acid Sequence; D002413:Cations, Divalent; D002614:Chelating Agents; D002846:Chromatography, Affinity / Q000379:methods*; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015684:Gene Products, pol / Q000235:genetics / Q000302:isolation & purification; D006678:HIV / Q000201:enzymology / Q000235:genetics; D006639:Histidine; D008969:Molecular Sequence Data; D015202:Protein Engineering; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification*
D002413:Cations, Divalent; D002614:Chelating Agents; D004274:DNA, Recombinant; D015684:Gene Products, pol; D011993:Recombinant Fusion Proteins; D006639:Histidine; D012194:RNA-Directed DNA Polymerase