A variant form of Keratinocyte growth factor-2 (KGF-2) spanning amino acids A63-S208 was produced in the Escherichia coli K-12 host W3110. When the protein was purified using a standard process, the first six N-terminal amino acids were rapidly and specifically removed from the protein. This cleavage resulted in a truncated KGF-2 species (S69-S208). To circumvent this problem, guanidine-HCl was used to inhibit the putative proteolytic activity. This modified process resulted in a massive loss of protein product due to precipitation, in addition to the cost and corrosiveness of guanidine-HCl. To develop an economically feasible, scaleable, and robust process for KGF-2 production, we were tasked with identifying the protease(s) responsible for the N-terminal degradation. Experimental evidence revealed that OmpT (outer membrane protein T) was the primary protease involved in the N-terminal cleavage of the A63-S208 KGF-2 protein. Moreover, the OmpT-mediated cleavage occurred at a novel site (Arg-Ser). From this work, we show that production of the A63-S208 form of KGF-2 in an ompT-deleted E. coli host nearly abolished the N-terminal cleavage issue.