The production of proteins containing native disulfide bonds in Escherichia coli is a challenging task of research. Secretion of such proteins into the oxidizing periplasm of E. coli gives a chance of proper folding. In this study, human pepsinogen and human proinsulin were used as model proteins. As a new approach, human pepsinogen was fused to the C-terminus of ecotin, E. coli trypsin inhibitor, which is a highly stable homodimeric periplasmic protein (16 kDa). After production of ecotin-pepsinogen under control of a trc promoter, ecotin-pepsinogen was purified to homogeneity. The yield of purified native pepsinogen was 7.6 mg per liter fermentation broth. For the quantification of pepsinogen, a fluorogenic assay was developed using EGFP, enhanced green fluorescent protein, as a substrate. To evaluate the applicability of ecotin as a periplasmic fusion tag, human proinsulin was chosen as second model protein because it differs from pepsinogen in size, fold, pattern of disulfide bonds, and function. Proinsulin was fused to the C-terminus of ecotin and was expressed under control of the trc promoter. In high cell density cultivation, 153 mg ecotin-proinsulin per liter broth was produced. Downstream processing of ecotin-proinsulin was done in one step using a newly established affinity purification method based on the strong binding affinity of ecotin for trypsinogen.