Enhancing the specificity of the enterokinase cleavage reaction to promote efficient cleavage of a fusion tag

Abstract

In our work with designed minimalist proteins based on the bZIP motif, we have found our Histagged proteins to be prone to inclusion body formation and aggregation; we suspect this problem is largely due to the His tag, known to promote aggregation. Using AhR6-C/EBP, a hybrid of the AhR basic region and C/EBP leucine zipper, as representative of our bZIP-like protein family, we attempted removal of the His tag with enterokinase (EK) but obtained the desired cleavage product in very small yield. EK is known for proteolysis at noncanonical sites, and most cleavage occurred at unintended sites. We manipulated experimental conditions to improve specificity of proteolysis and analyzed the cleavage products; no effect was observed after changing pH, temperature, or the amount of EK. We then suspected the accessibility of the EK site was impeded due to protein aggregation. We found that the easily implemented strategy of addition of urea (1-4 M) greatly improved EK cleavage specificity at the canonical site and reduced adventitious cleavage. We believe that this enhancement in specificity is due to a more "open" protein structure, in which the now accessible canonical target can compete effectively with adventitious cleavage sites of related sequence.

Construction of novel chimeric proteins through the truncation of SEC2 and Sak from Staphylococcus aureus

“…11) N-terminus His-tag of proteins was removed with EK, 19) and the purity of the eluted protein was determined by SDS-PAGE.…”

Exploiting the interactions between poly-histidine fusion tags and immobilized metal ions

“…In some cases, the problem of unspecific internal cleavage stems from steric hindrance arising from the target protein structure that prevents the protease from access to the intended cleavage site Shahravan, 2008). Certain concentrations of denaturant were added in an attempt to improve the specificity and yield of the cleavage reactions (Shahravan et al, 2008).…”

“…Certain concentrations of denaturant were added in an attempt to improve the specificity and yield of the cleavage reactions (Shahravan et al, 2008). The addition of 1-4 M urea successfully improved the cleavage specificity and yield by making the protein molecules assume a more open structure.…”

“…Nevertheless, to make this a general approach to improve protease specificity and yield applicable to other proteins, the tolerance of the chosen protease and target protein to denaturant concentration is important. For example, urea at 5 mM began to inhibit enterokinase activity (Shahravan et al, 2008).…”

Efficient Expression and Purification of Recombinant Human Enteropeptidase Light Chain in Esherichia coli

“…At present, commonly used proteases include thrombin, enterokinase, and factor Xa etc, among which enterokinase is regarded as an ideal enzyme to prepare target protein by sequence specific cleavage from fusion protein (Shahravan et al 2008;Mikhailova et al 1998). Enterokinase, which consists of two chains connected by a disulfide bond, is a heterodimeric serine protease from the mammalian duodenum.…”