Introduction Heloderma suspectum 1992 2004 2005 2004 2001 2005 2003 2006 E. coli 2006 2005 Pichia pastoris 2007 P. pastoris Materials and methods Microorganism and expression of exendin-4 fusion protein Pichia pastoris 2007 P. pastoris g Purification of fusion protein 1 Table 1 Summary of exendin-4 purification  Purification step Total protein (mg) a  Recovery (%) Fusion exendin-4 Broth supernatant 1,640 580 100 DEAE A-25 1,200 390 67 Phenyl FF 760 285 49 G-75 410 214 37 Recombinant exendin-4 G-25 214 26 4.5 4 2 4 4 2 4 a Cleavage of the fusion protein by enterokinase After desalting, the fusion protein was incubated with enterokinase (1 U/1 mg fusion protein) at 37°C for 8 h to obtain the recombinant exendin-4. The reaction mixture was analyzed by Tricine SDS-PAGE. The recombinant exendin-4 was purified by Sepharose G-25 column and its purity was assayed by HPLC. SDS-PAGE, Tricine SDS-PAGE, immunoblotting and protein determination Proteins were analyzed by SDS-PAGE under reducing conditions using 12% (v/v) gels. Tricine SDS-PAGE was carried out with the supplier’s protocol (Amersham Bioscience). The separated proteins were stained with Coomassie Brilliant Blue R-250 or electroblotted to nitrocellulose membrane for western blotting. After blocking with 3% (v/v) non-fat milk in 0.05% Tween/PBS (PBST), the membrane was washed 3 times with PBST for 10 min and incubated further with a 1:2,000 dilution of polyclonal rat anti exendin-4 antibody for 1 h, followed by washing as was described above. The membrane was incubated with a 1:1,000 dilution of HRP-conjugated goat anti-rat IgG as the second antibody, washed with the same procedure as above and revealed with DAB substrate (Sigma). Protein concentration was determined by the method of Bradford using bovine serum albumin as a standard. Biological activity assay 2000 P Results Expression and detection 1 Fig. 1 a b P. pastoris  Purifying the fusion protein and obtaining the recombinant exendin-4 by enterokinase cleave 1 2 Fig. 2 a b c 5  Bioactivity of exendin-4 in vivo 3 P 3 P 3 Fig. 3 a b n P  Discussion 2006 2005 E. coli E. coli E. coli P. pastoris P. pastoris 2005 E. coli Pichia pastoris P. pastoris E. coli P. pastoris 3 3 2007 2003 2004 2006 3 1995 2000 It is important to note that in the three independent experiments, the recombinant exendin-4 did not have obvious effects on one or two rats of the six. It was probably because of individual differences. The results of the three independent experiments were comparable. According to the research, the recombinant exendin-4 may also have effects at smaller doses but remains to be proved. In summary P. pastoris P. pastoris