The insulin precursor (IF) expressed in Pichia pastoris is a single-chain peptide fused with a spacer peptide (EEAEAEAEPK) localized at its N-terminus and containing three trypsin cleavage sites in the polypeptide chain. The IP fusion protein is trypsinized to generate the insulin product desB30, which has a deletion of threonine(B30). The three restriction sites on IF fusion protein had different affinities for trypsin and were digested in sequential order. Further analysis showed that approximately 20% of the IP digestion intermediates could not be converted into the final desB30 product if the IP fusion protein was digested in an aqueous phase. This result can be attributed to the formation of IP dimers or hexamers, which could restrict enzyme reactivity in the aqueous phase. To enhance the conversion yield of the IF fusion protein to desB30 products, a new digestion method was established. The IF was digested in the eluent that resulted from reverse phase chromatography during the purification process, which improved the yield of digestion from 80.2% to 95.6%. (C) 2013 Elsevier Ltd. All rights reserved.