An efficient trypsin digestion strategy for improving desB30 productivity from recombinant human insulin precursor fusion protein

Abstract

The insulin precursor (IP) 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 threonineB30. The three restriction sites on IP 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 IP fusion protein to desB30 products, a new digestion method was established. The IP 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%.