Immobilized metal affinity chromatography of bacterially expressed proteins engineered to contain an alternating-histidine domain

Academic Article

Abstract

  • A genetic approach to purifying chimeric recombinant proteins by immobilized metal affinity chromatography (IMAC) is described. Plasmid expression vectors have ben constructed to direct the synthesis in Escherichia coli of human immunodeficiency virus (HIV-1) reverse transcriptase (RT) designed to possess alternating histidine residues at either the N- or the C-terminus. As shown below, HIV-1 RT containing six adjacent histidines (S. K. Sharma et al., 1991, Biotechnol. Appl. Biochem.14, 69-81) was also included for comparison purposes: (a) Met-Glu-(His-Glu)4-lle-Pro- (-2)-HIV-1 RT; (b) HIV-1 RT-Asn-Gly-(His-Asp)4-His-Leu; and (c) Pro-lle-(His)6-Pro-Phe-His-Leu-Val-Lle-His-HIV-1 RT. Protocols in which the histidine-containing domain allows the single-step purification of the chimeric proteins, under nondenaturing conditions, by IMAC on Ni2+ immobilized to iminodiacetic acid-Sepharose are described. Most of the contaminating E. coli proteins were eliminated by washing the IMAC column with 30-35 mm imidazole. Linear gradients of imidazole utilized to recover purified chimerics resulted in elution of the alternating histidine-containing recombinant proteins between 50 and 80 mm imidazole. In contrast, the chimeric with six adjacent histidines remained bound to immobilized Ni2+ even in the presence of 100 mm imidazole. An imidazole concentration of 300 mm was required to elute this chimeric protein from the IMAC column. It is concluded that elution by a competitive counterligand during IMAC of engineered proteins is determined by the sequence of the IMAC-specific tag. Characterization of the IMAC-purified chimerics shows that these relatively small N- or C-terminal fusions have an insignificant effect on the RNA-dependent DNA polymerase activity of HIV-1 RT. A strategy for utilizing the other alternating amino acid for stepwise trimming of the His-X dipeptides from chimeric proteins by dipeptidylpeptidases is discussed. © 1992.
  • Published In

  • Methods  Journal
  • Digital Object Identifier (doi)

    Author List

  • Sharma SK; Evans DB; Vosters AF; Chattopadhyay D; Hoogerheide JG; Campbell CM
  • Start Page

  • 57
  • End Page

  • 67
  • Volume

  • 4
  • Issue

  • 1