Abstract
A novel recombinant exendinβ4 human serum albumin fusion protein (rExβ4/HSA) expressed in Pichia pastoris was prepared and characterized. Exβ4 is a 39βamino acid peptide isolated from the salivary gland of the lizard Heloderma suspectum and is thought to be a novel therapeutic agent for type 2 diabetes. But to gain a continued effect, the peptide has to be injected twice a day owing to its short plasma halfβlife (t~1/2~ = 2.4 h). To extend the halfβlife of Exβ4 molecule in vivo, we designed a genetically engineered Exβ4/HSA fusion protein. Between Exβ4 and HSA, a peptide linker GGGGS was inserted and the fusion protein was expressed in methylotrophic yeast P. pastoris with native HSA secretion signal sequence. The recombinant protein was secreted correctly and was obtained with high purity (typically > 98%) by a threeβstep purification procedure. cAMP assay demonstrated that the fusion protein had a bioactivity similar to Exβ4 for interaction with GLPβ1 receptors in vitro. Results from oral glucose tolerance test indicated that rExβ4/HSA could effectively improve glucose tolerance in diabetic db/db mice. Pharmacokinetics studies in cynomologus monkeys also showed that rExβ4/HSA had a much longer plasma halfβlife. Therefore, rExβ4/HSA fusion protein could potentially be used as a new recombinant biodrug for type 2 diabetes therapy. Copyright Β© 2007 European Peptide Society and John Wiley & Sons, Ltd.