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

Abstract

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 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.

Zinc binding of a Cys2His2-type zinc finger protein is enhanced by the interaction with DNA

“…Intact protein analysis was performed on an LTQ-Orbitrap Elite (Thermo Scientific) mass spectrometer coupled with a TriVersa NanoMate (Advion) chip-based electrospray ion source as described previously [40]. During top-down analysis R = 30,000 resolution was used at 400 m/z.…”

Interactions of an Artificial Zinc Finger Protein with Cd(II) and Hg(II): Competition and Metal and DNA Binding

“…Intact protein analysis was performed on an LTQ-Orbitrap Elite (Thermo Scientific, Thousand Oaks, CA, USA) mass spectrometer coupled with a TriVersa NanoMate (Advion, Ithaca, NY, USA) chip-based electrospray ion source. Measurements were carried out in positive mode at 120,000 resolution in 8.2 mM ammonium hydrogen carbonate buffer (pH ~7.8), as described previously [72]. Fitting of the ESI-MS data was performed by the Solver add in of Microsoft Excel based on statistical considerations [73,74].…”

Ni-Nitrilotriacetic Acid Affinity SELEX Method for Selection of DNA Aptamers Specific to the N-Cadherin Protein

“…Note that immobilized metal ion affinity chromatography is a conventional technique for protein purification whose best known application is purification of histidine-tagged fusion proteins by transition metal ions. − Thus, in this work, we employed the high-affinity interactions between histidine and Ni­(II) to immobilize a C-terminal polyhistidine-tagged N-cadherin derived from the human protein (Met1-Ala724) in a Ni-NTA (nicke-charged nitrilotriacetic acid) Sepharose affinity column . Compared to standard SELEX, a modified Ni-NTA affinity-column SELEX platform was used to identify high affinity binding aptamers against N-cadherin from a nucleic acid library.…”