C-type natriuretic peptide (CNP) acts as a paracrine hormone to dilate blood vessels and is also required for the growth of long bones. In vivo, CNP is produced by cleavage from the C-terminal end of a larger proCNP peptide. The remaining N-terminal proCNP fragment (NT-proCNP) escapes into the circulation where its concentration is much higher than that of CNP due presumably to a lower clearance rate. Our strategy to obtain large quantities of pure NT-proCNP for further physiological investigations was to express it as a fusion protein with His(6)-tagged thioredoxin followed by cleavage using enterokinase to yield NT-proCNP alone. We have successfully designed and artificially synthesized the coding sequence specifying both mouse and human NT-proCNP with built-in codon bias towards Escherichia coli codon preference. An enterokinase recognition sequence was incorporated immediately upstream of the NT-proCNP coding sequence to allow the fusion protein to be cleaved without leaving any extra residues on the NT-proCNP peptide. High levels of fusion proteins were obtained, constituting 50-58 % of total bacterial proteins. Greater than 90 % of recombinant thioredoxin/NT-proCNP was expressed in the soluble form and purified to near homogeneity in a single chromatographic step using nickel as the metal ion in IMAC. A time course analysis of the products released from enterokinase cleavage of the recombinant proteins by ESI-NIS revealed three sensitive secondary cleavage sites: two were located on vector-associated sequences linking the thioredoxin moiety and NT-proCNP, and one at the C-terminal end of NT-proCNP. Clearly, substrate specificity of both the native and recombinant forms of enterokinase for the recognition sequence DDDDK was by no means exclusive. Hydrolysis at the unexpected LKGDR site located towards the carboxyl end on NT-proCNP was significantly more efficient than at the internally sited DDDDK target sequence. However, when this same sequence was sited internally replacing the DDDDK in another construct of thioredoxin/mouse NT-proCNP, it was found to be poorly processed by enterokinase. Our results showed that non-target sequences can be preferentially recognized over the canonical DDDDK sequence when located accessibly at the ends of proteins. (c) 2005 Elsevier Inc. All rights reserved.