Expression, purification and activity identification of rat uncarboxylated osteocalcin fusion protein
10.13699/j.cnki.1001-6821.2017.09.010
- VernacularTitle:大鼠未羧化骨钙素融合蛋白的表达纯化及活性鉴定
- Author:
Jing-Ying GAO
1
;
Le-Le REN
;
Ya-Qin DING
;
Xiang-Qin ZHONG
;
Tao BAI
;
Yun-Feng LIU
;
Yi ZHANG
Author Information
1. 山西医科大学药理教研室
- Keywords:
small ubiquitin related modifier;
bone gamma-carboxyglutamate protein;
prokaryotic expression;
insulin secretion
- From:
The Chinese Journal of Clinical Pharmacology
2017;33(9):806-809
- CountryChina
- Language:Chinese
-
Abstract:
Objective To further study the regulatory mechanisms of uncarboxylated osteocalcin on the glucose metabolism,rat uncarboxylated osteocalcin fusion protein was expressed,purified and identified.Methods The rat osteocalcin gene (BGLAP) was cloned into pSumo-Mut expression vector.The pSumo-BGLAP plasmid was obtained and identified.Through transformed in prokaryotic host bacterium and induced by isopropyl 3-D-thiogalactoside (IPTG) and 11 ℃ low temperature,the Sumo-BGLAP fusion protein was expressed.The purification and activity identification of the fusion protein was performed by Ni2+ affinity chromatography and insulin secretion experiments,respectively.Results The enzyme digestion and sequencing results of the plasmid showed that the recombinant plasmid pSumo-BGLAP was constructed successfully,which matched the target gene and protein.After the transformation,induction and purification,the high purity Sumo-BGLAP fusion protein was obtained.The data from insulin secretion experiments show that the fusion protein can promote insulin secretion under 16.7 mmol · L-1 glucose conditions [(37.64 ± 3.80)μU· L-1at 16.7 mmol · L-1glucose,(63.91 ±4.67) μU · L-1 at 16.7 mmol · L-1 glucose +0.03 ng· mL-1 Sumo-BGLAP,(68.47 ±5.83) μU · L-1 at 16.7 mmol · L-1 glucose +0.3 ng· mL-1 Sumo-BGLAP,P ＜ 0.01)].Conclusion The rat uncarboxylated osteocalcin fusion protein is successfully achieved and purified.The fusion protein with biological activity can be used to further study the regulatory mechanisms of uncarboxylated osteocalcin on the glucose metabolism.
