The methylotrophic yeasts Pichia pastoris and Hansenula polymorpha have been used for the production of recombinant monomeric insulin precursor (MIP). Recombinant plasmids with one, two and four cassettes of the MIP gene have been successfully constructed in the pPICZ alpha A expression vector to study the effects of gene copy number on MIP production. The MIP protein can be detected by dot-blot analysis from the culture broth of P. pastoris KM71H 24 h after placement in MMH induction medium. The secretion levels of MIP protein in culture broth at 72 h after induction indicated that P. pastoris KM71H with one cassette of the MIP gene had highest MIP protein levels (4.19 +/- 0.96 mg L-1). The transcription levels of the MIP gene increased proportionately with copy number. However, the amount of secreted MIP protein showed no correlation. The MIP molecular mass was 5756.951 Da, as confirmed by typical MALDI-TOF mass spectrometry. The MIP protein in culture broth was purified by two steps purification including SP Sepharose Fast Flow chromatography followed by ultrafiltration (10 kDa MW cutoff). The percentage of MIP recovery after the two-step purification was 70%, with a single band in a native-PAGE. The biological activity of tryptic hydrolyzed MIP was determined via the expression of the glucose transporter 4 gene (GLUT4) in H9c2 (2-1) cell line by RT-qPCR, and the results demonstrated that the MIP protein can induce glucose uptake and upregulation of GLUT4 mRNA transcription at 3 h and that this activity was related to Humalog(center dot) insulin.