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Preparation and characterization of a novel exendin‐4 human serum albumin fusion protein expressed in Pichia pastoris
Author(s) -
Huang YanShan,
Chen Zhi,
Chen YiQiong,
MA GuoChang,
Shan JianFeng,
Liu Wei,
Zhou LinFu
Publication year - 2008
Publication title -
journal of peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 66
eISSN - 1099-1387
pISSN - 1075-2617
DOI - 10.1002/psc.942
Subject(s) - pichia pastoris , fusion protein , human serum albumin , ex vivo , recombinant dna , peptide , microbiology and biotechnology , biochemistry , yeast , biology , albumin , in vitro , chemistry , gene
A novel recombinant exendin‐4 human serum albumin fusion protein (rEx‐4/HSA) expressed in Pichia pastoris was prepared and characterized. Ex‐4 is a 39‐amino 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 half‐life ( t 1/2 = 2.4 h). To extend the half‐life of Ex‐4 molecule in vivo , we designed a genetically engineered Ex‐4/HSA fusion protein. Between Ex‐4 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 three‐step purification procedure. cAMP assay demonstrated that the fusion protein had a bioactivity similar to Ex‐4 for interaction with GLP‐1 receptors in vitro . Results from oral glucose tolerance test indicated that rEx‐4/HSA could effectively improve glucose tolerance in diabetic db/db mice. Pharmacokinetics studies in cynomologus monkeys also showed that rEx‐4/HSA had a much longer plasma half‐life. Therefore, rEx‐4/HSA fusion protein could potentially be used as a new recombinant biodrug for type 2 diabetes therapy. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.