Characterization of GST–ARTS-1 aminopeptidase activity. (a) Generation of GST–ARTS-1. Soluble and insoluble protein fractions were isolated from BL21 E. coli transformed with empty pGEX-6P-1 (Lane 1, soluble fraction; Lane 2, insoluble fraction) or ARTS-1 pGEX-6P-1 (Lane 3, soluble fraction; Lane 4, insoluble fraction). Proteins were subjected to SDS-PAGE and stained with Coomassie brilliant blue. Purified GST–ARTS-1 fusion protein from the insoluble fraction is shown as a predominant 130-kDa band in Lane 5, and the 26-kDa purified control GST tag is shown in Lane 6. (b) FPLC analysis of purified recombinant GST–ARTS-1 fusion protein revealed a major peak that eluted at approximately 40 minutes. (c) Assay of GST–ARTS-1 aminopeptidase activity. FPLC fractions were assessed for aminopeptidase activity using a phenylalanine–p-nitroanilide (Phe-pNA) substrate. Phenylalanine aminopeptidase activity was present in pooled fractions eluting from 38 to 44 minutes, which correlated with the major FPLC peak.