Purification of proteins with native terminal sequences using a Ni(II)-cleavable C-terminal hexahistidine affinity tag

Heba A.H. Abd Elhameed, Bálint Hajdu, Ria K. Balogh, Enikő Hermann, Éva Hunyadi-Gulyás, Béla Gyurcsik

Research output: Contribution to journalArticle

1 Citation (Scopus)


The role of the termini of protein sequences is often perturbed by remnant amino acids after the specific protease cleavage of the affinity tags and/or by the amino acids encoded by the plasmid at/around the restriction enzyme sites used to insert the genes. Here we describe a method for affinity purification of a metallonuclease with its precisely determined native termini. First, the gene encoding the target protein is inserted into a newly designed cloning site, which contains two self-eliminating BsmBI restriction enzyme sites. As a consequence, the engineered DNA code of Ni(II)-sensitive Ser-X-His-X motif is fused to the 3′-end of the inserted gene followed by the gene of an affinity tag for protein purification purpose. The C-terminal segment starting from Ser mentioned above is cleaved off from purified protein by a Ni(II)-induced protease-like action. The success of the purification and cleavage was confirmed by gel electrophoresis and mass spectrometry, while structural integrity of the purified protein was checked by circular dichroism spectroscopy. Our new protein expression DNA construct is an advantageous tool for protein purification, when the complete removal of affinity or other tags, without any remaining amino acid residue is essential. The described procedure can easily be generalized and combined with various affinity tags at the C-terminus for chromatographic applications.

Original languageEnglish
Pages (from-to)53-59
Number of pages7
JournalProtein Expression and Purification
Publication statusPublished - Jul 2019


  • Affinity chromatography
  • Circular dichroism
  • Colicin E7
  • Mass spectrometry
  • Ni(II)-Induced cleavage

ASJC Scopus subject areas

  • Biotechnology

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