Metal affinity chromatography of recombinant HIV-1 reverse transcriptase containing a human renin cleavable metal binding domain.
A metal binding peptide, hexahistidine, preceding a renin cleavage sequence (Pro-Phe-His-Leu-Val-Ile-His-) was engineered on to the N-terminus of HIV-1 reverse transcriptase (RT). The chimeric protein was expressed in Escherichia coli and characterized after purification by DEAE chromatography and HPLC. Amino-terminal sequencing confirmed the presence of the first 15 amino acids of the chimeric protein. The chimeric exhibited RT activity like that of HIV-1 RT and was cleaved by human renin at the expected site. The potential of a hexa-histidine fusion in the purification of recombinant HIV-1 RT by immobilized metal affinity chromatography (IMAC) on the commonly used resin (IDA-Ni2+) was investigated. The chimeric gene product from a crude E. coli extract was strongly retarded on a immobilized nickel column, while most of the contaminating E. coli proteins were eliminated after elution with 20-35 mM imidazole. The bound chimeric protein was eluted with 300 mM imidazole and appeared predominantly as a single band on an SDS-polyacrylamide gel. The remarkable specificity of this affinity tail was further demonstrated by separating the chimeric protein from HIV-1 RT in a crude extract prepared by mixing extracts from cells expressing HIV-1 RT and the hexahistidine recombinant chimeric protein. The usefulness of a enzymatically cleavable metal binding peptide in the rapid purification and production of HIV-1 RT without proteolysis to a heterodimer is discussed.
Sharma
S K
SK
Upjohn Company, Kalamazoo, Michigan 49001.
Evans
D B
DB
Vosters
A F
AF
McQuade
T J
TJ
Tarpley
W G
WG
D000595:Amino Acid Sequence; D001665:Binding Sites; D002614:Chelating Agents / Q000737:chemistry; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004800:Enzymes, Immobilized / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015497:HIV-1 / Q000201:enzymology*; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D008670:Metals / Q000737:chemistry*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012083:Renin / Q000235:genetics
D002614:Chelating Agents; D004800:Enzymes, Immobilized; D008670:Metals; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D012194:RNA-Directed DNA Polymerase; D012083:Renin