title: High cell density cultivation of recombinant Escherichia coli for prodrug of recombinant human GLPs production.
abstract: Glucagon-like peptide-1 (GLP-1)(2) has been attracting increasing interest on account of its prominent benefits in type 2 diabetes. However, its clinical applications are limited by the short half-life in vivo. To overcome this limitation, a new polymer of GLP-1 was developed by prodrug strategy. In this study a recombinant protein, rhGLPs, was successfully constructed, cloned into plasmid pET30a (+) and expressed in Escherichia coli ArcticExpress(DE3)RP in the form of inclusion body. The recombinant fusion protein productivity could be enhanced by high cell density culture of the recombinant strain. As a result, about 40 g wet weight cells per liter were obtained. The protein was purified by size-exclusion chromatography on a Superdex 75 column and refolded using reverse dilution and dialysis methods. SDS-PAGE, HPLC and MALDI-TOF mass spectrometry were undertaken to determine the purity and molecular weight of rhGLPs. Bioactivity assay revealed that it had glucose-lowering and insulin-releasing action in vivo.
authors: Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi'an, Shaanxi 710032, China.
authors: Ying
authors: Y
authors: Zhou
authors: Xue
authors: X
authors: Ma
authors: Zheng
authors: Z
authors: Hou
authors: Xiaoyan
authors: X
authors: Xue
authors: Jingru
authors: J
authors: Meng
authors: Mingkai
authors: M
authors: Li
authors: Min
authors: M
authors: Jia
authors: Xiaoxing
authors: X
authors: Luo
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics*; D005260:Female; D052216:Glucagon-Like Peptide 1 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007004:Hypoglycemic Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D002479:Inclusion Bodies / Q000235:genetics; D007328:Insulin / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D007004:Hypoglycemic Agents; D007328:Insulin; D011993:Recombinant Fusion Proteins; D052216:Glucagon-Like Peptide 1; D005947:Glucose