Although the junctional sequences recognized by NE, PR3, and CG in His 6 -proHNP1 are identical to those in the natural precursor, we questioned whether the 41-residue extension of the proHNP-1 sequence resulting from the hexa-His and DDDDK tags and spacer sequences might alter protease interactions with the target. To test this, we produced proHNP1 without the Nterminal tag by incubating proHNP1A with enterokinase, thereby releasing the 41-residue tag sequence. This peptide was then treated with NE, PR3, or CG which liberated peptides with monoisotopic masses of 3439.7, 3439.7 and 3684.5 A.M.U., respectively, products equivalent to those generated from His 6 -proHNP1A. Thus the 41-residue N-terminal fusion tag did not alter the specificity of cleavage of proHNP1A by NE, PR3, or CG.