The B27K-DTrI insulin (human insulin with 828-30 removed and B27 Thr replaced by Lys) was reported to have superior monomeric property with 80% insulin activity in vivo. It has potential use as a new fast-acting analog of insulin. We cloned the monomeric insulin B27 DTrI precursor (MIP) into the pTWIN1 vector, and prepared by intein mediated expression in E. coli. After tryptic digestion, the MIP was converted to B27K-DTrI insulin. The product was purified by HPLC. The mass spectrometry showed that the molecular mass of purified B27K-DTrI was consistent with the theoretical value. (C) 2011 Elsevier Inc. All rights reserved.