Enhancing the specificity of the enterokinase cleavage reaction to promote efficient cleavage of a fusion tag.
In our work with designed minimalist proteins based on the bZIP motif, we have found our His-tagged 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.
Shahravan
S Hesam
SH
Department of Chemistry, University of Toronto, Mississauga, Ont., Canada L5L 1C6.
Qu
Xuanlu
X
Chan
I-San
IS
Shin
Jumi A
JA
D000595:Amino Acid Sequence; D022762:CCAAT-Enhancer-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004765:Enteropeptidase / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D018336:Receptors, Aryl Hydrocarbon / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D013696:Temperature; D014508:Urea / Q000737:chemistry
D022762:CCAAT-Enhancer-Binding Proteins; D018336:Receptors, Aryl Hydrocarbon; D011993:Recombinant Fusion Proteins; D014508:Urea; D004765:Enteropeptidase