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Complete solubilization and purification of recombinant human growth hormone produced in Escherichia coli.

ABSTRACT: High-level expression of recombinant human growth hormone (hGH) in Escherichia coli (E. coli) leads to the formation of insoluble aggregates as inclusion bodies devoid of biological activity. Until recently, significant efforts have been made to improve the recovery of active hGH from inclusion bodies. Here, we developed an efficient procedure for the production of completely soluble hGH by minimizing the formation of inclusion bodies and optimizing protein purification conditions. Under the newly established conditions we were able to obtain most of the total hGH in the soluble fraction. We show that the soluble protein can be efficiently purified in high yield by a series of chromatographic procedures. We analyzed the resulting hGH using various analytical techniques such as reversed-phase high-performance liquid chromatography (RP-HPLC), size-exclusion chromatography (SEC), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and circular dichroism (CD). These multiple analyses support the conclusion that we obtained highly pure hGH with the expected molecular mass and intact secondary structure. The biological activity of purified hGH was also confirmed by evaluating its growth-promoting effect using a cell proliferation assay. Taken together, we describe a straightforward strategy for the production of completely soluble and biologically active hGH in E. coli.

SUBMITTER: Kim MJ

PROVIDER: S-EPMC3567055 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Complete solubilization and purification of recombinant human growth hormone produced in Escherichia coli.

Kim Min-Ji MJ Park Hyun Soo HS Seo Kyung Hye KH Yang Hyo-Jin HJ Kim Sook-Kyung SK Choi Jun-Hyuk JH

PloS one 20130207 2

High-level expression of recombinant human growth hormone (hGH) in Escherichia coli (E. coli) leads to the formation of insoluble aggregates as inclusion bodies devoid of biological activity. Until recently, significant efforts have been made to improve the recovery of active hGH from inclusion bodies. Here, we developed an efficient procedure for the production of completely soluble hGH by minimizing the formation of inclusion bodies and optimizing protein purification conditions. Under the new ...[more]

PMID: 23409149

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Project description:BackgroundHuman Growth Hormone (hGH) is a glycoprotein released from the pituitary gland. Due to the wide range of effects in humans, any disruption in hGH secretion could have serious consequences. This highlights the clinical importance of hGH production in the treatment of different diseases associated with a deficiency of this hormone. The production of recombinant mature hormone in suitable hosts and secretion of this therapeutic protein into the extracellular space can be considered as one of the best cost-effective approaches not only to obtain the active form of the protein but also endotoxin-free preparation. Since the natural growth hormone signal peptide is of eukaryotic origin and is not detectable by any of the Escherichia coli secretory systems, including Sec and Tat, and is therefore unable to secrete hGH in the prokaryotic systems, designing a new and efficient signal peptide is essential to direct hGh to the extracellular space.ResultsIn this study, using a combination of the bioinformatics design and molecular genetics, the protein A signal peptide from Staphylococcus aureus was modified, redesigned and then fused to the mature hGH coding region. The recombinant hGH was then expressed in E. coli and successfully secreted to the medium through the Sec pathway. Secretion of the hGH into the medium was verified using SDS-PAGE and western blot analysis. Recombinant hGH was then expressed in E. coli and successfully secreted into cell culture medium via the Sec pathway. The secretion of hGH into the extracellular medium was confirmed by SDS-PAGE and Western blot analysis. Furthermore, the addition of glycine was shown to improve hGH secretion onto the culture medium. Equations for determining the optimal conditions were also determined. Functional hGH analysis using an ELISA-based method confirmed that the ratio of the active form of secreted hGH to the inactive form in the periplasm is higher than this ratio in the cytoplasm.ConclusionsSince the native signal protein peptide of S. aureus protein A was not able to deliver hGH to the extracellular space, it was modified using bioinformatics tools and fused to the n-terminal region of hGh to show that the redesigned signal peptide was functional.

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Complete solubilization and purification of recombinant human growth hormone produced in Escherichia coli.

Publications

Complete solubilization and purification of recombinant human growth hormone produced in Escherichia coli.

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