Preparation and characterization of a novel exendin-4 human serum albumin fusion protein expressed in Pichia pastoris.
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.
Huang
Yan-Shan
YS
Department of Cell Biology, School of Medicine, Zhejiang University, Hangzhou, China.
Chen
Zhi
Z
Chen
Yi-Qiong
YQ
Ma
Guo-Chang
GC
Shan
Jian-Feng
JF
Liu
Wei
W
Zhou
Lin-Fu
LF
D000818:Animals; D001483:Base Sequence; D001786:Blood Glucose / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D017931:DNA Primers / Q000235:genetics; D000077270:Exenatide; D005260:Female; D005951:Glucose Tolerance Test; D006801:Humans; D007004:Hypoglycemic Agents / Q000302:isolation & purification / Q000493:pharmacokinetics / Q000494:pharmacology; D008116:Lizards / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D010455:Peptides / Q000235:genetics* / Q000302:isolation & purification* / Q000493:pharmacokinetics / Q000494:pharmacology; D010843:Pichia / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000493:pharmacokinetics / Q000494:pharmacology; D012709:Serum Albumin / Q000235:genetics* / Q000302:isolation & purification* / Q000493:pharmacokinetics / Q000494:pharmacology; D014162:Transfection; D014688:Venoms / Q000235:genetics* / Q000302:isolation & purification* / Q000493:pharmacokinetics / Q000494:pharmacology
D001786:Blood Glucose; D017931:DNA Primers; D007004:Hypoglycemic Agents; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D014688:Venoms; D000077270:Exenatide; D000242:Cyclic AMP