Affiliation:
1. Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development of Chines Ministry of Education, Jinan University, Guangzhou 510632, China
Abstract
Background:
Human growth hormone (hGH) is the first recombinant protein approved for the treatment of human growth hormone deficiency. However, expression in inclusion bodies and low expression levels are enormous challenges for heterologous expression of hGH in Escherichia coli.
Objective:
To increase the soluble expression of recombinant hGH with correct folding in E. coli.
Methods:
We constructed a new recombinant expression plasmid containing the coding sequence
of the outer membrane protein A (ompA3) which was used for the expression in Transetta (DE3)
E. coli. In order to simplify the purification process and cleavage of recombinant proteins, the fusion
sequence should contain hexahistidine-tag (His6) and enterokinase recognition sites (D4K).
The effect of different expression conditions on recombinant hGH expression was optimized in
flask cultivations. Furthermore, the periplasmic solution containing soluble hGH was purified by
Ni-NTA affinity chromatography. Circular dichroism (CD), western blot and mass spectrometry
analyses were used to characterize the protein. Moreover, the growth-promoting effect of the purified
hGH was also evaluated by cell proliferation assay.
Results:
High-level expression (800 g/mL) was achieved by induction with 0.5 mM IPTG at 30 ºC for 10 hours. The purity
of hGH was over 90%. The immunological activity, secondary structure and molecular weight of the purified hGH were
consistent with native hGH. The purified hGH was found to promote the growth of MC3T3-E1 cells, and was found to show
the highest activity at a concentration of 100 ng/mL.
Conclusion:
Our research provides a feasible and convenient method for the soluble expression of recombinant hGH in E.
coli, and may lay a foundation for the production and application of hGH in the industry.
Funder
National Natural Science Foundation of China
Publisher
Bentham Science Publishers Ltd.
Subject
Biochemistry,General Medicine,Structural Biology
Cited by
3 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献