Abstract
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.
Highlights
Current techniques of protein science often require significant amounts of pure recombinant proteins
The SRHWAP peptide was cloned between the SPI2 protein and the C-terminal hexahistidine affinity tag
The SPI2SRHWAP-His6 fusion protein secreted to the medium was purified by affinity chromatography on Ni-NTA agarose
Summary
Current techniques of protein science often require significant amounts of pure recombinant proteins. The development of self-cleaving affinity tags helped overcome some of these limitations These tags consist of an autoprocessing domain fused to the affinity tag, enabling onestep purification. Those most commonly utilized for recombinant protein purification include inteins [2], the catalytic core of sortase A [3], and FrpC protein [4]. In this case, the cleavage process is strictly dependent on the preservation of the native self-cleaving domain conformation, which narrows requirements for reaction conditions. Large sizes of these moieties constitute disadvantage, diminishing the solubility and purification efficiency
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