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Soluble Expression, One-Step Purification and Characterization of Recombinant Human Growth Hormone Fused with ompA3 in Escherichia coli

[ Vol. 28 , Issue. 5 ]

Author(s):

Zhen-Ru Zhou, Wei Huang, Kang-Jia Liu, Fo-Lan Lin, Xiao-Lu Wang, Feng Wang and Ren-Wang Jiang*   Pages 533 - 542 ( 10 )

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 μg/mL) was achieved by induction with 0.5 mM IPTG at 30°C 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.

Keywords:

Human growth hormone, ompA3, soluble expression, Escherichia coli, purification, growth-promoting effect.

Affiliation:

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, 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, 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, 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, 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, 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, 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



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