Application of Ni(II)-Assisted Peptide Bond Hydrolysis to Non-Enzymatic Affinity Tag Removal

By Edyta Kopera, Agnieszka Belczyk-Ciesielska, W. Bal. 2012

24 citations recorded by the source corpus.

In this study, we demonstrate a non-enzymatic method for hydrolytic peptide bond cleavage, applied to the removal of an affinity tag from a recombinant fusion protein, SPI2-SRHWAP-His6. This method is based on a highly specific Ni(II) reaction with (S/T)XHZ peptide sequences. It can be applied for the protein attached to an affinity column or to the unbound protein in solution. We studied the effect of pH, temperature and Ni(II) concentration on the efficacy of cleavage and developed an analytical protocol, which provides active protein with a 90% yield and ∼100% purity. The method works well in the presence of non-ionic detergents, DTT and GuHCl, therefore providing a viable alternative for currently used techniques.

Subfields: Molecular Biology, Radiology, Nuclear Medicine and Imaging

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In Molecular Biology

1. W. Bal
2. Edyta Kopera
3. Mariusz Jaskolski

Citations in the corpus

• Atomic Resolution Structure of a Protein Prepared by Non-Enzymatic His-Tag Removal. Crystallographic and NMR Study of GmSPI-2 Inhibitor (2014, methodology): “However, despite prolonged incubations in the presence of Ni(II) ions at elevated temperatures no cleavage was observed for the natively folded protein, while Ubi denaturated by GuHCl was hydrolyzed [14].”