Combined effect of protein fusion and signal sequence greatly enhances the production of recombinant human GM-CSF in Escherichia coli

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor, that has been used as a therapeutic agent in facilitating bone marrow and stem cell transplantation and in other clinical cases like neutropenia. Although biologically active recombinant GM-CSF has been succe...

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Bibliographic Details
Published in:Molecular biotechnology 2005-06, Vol.30 (2), p.103-115
Main Authors: BHATTACHARYA, Palash, PANDEY, Gaurav, SRIVASTAVA, Poonam, KRISHNA JYOTI MUKHERJEE
Format: Article
Language:English
Subjects:
Biological and medical sciences
Biotechnology
Bone marrow
Carrier Proteins - genetics
Codon - genetics
E coli
Escherichia coli
Escherichia coli - genetics
Escherichia coli - metabolism
Fundamental and applied biological sciences. Psychology
Granulocyte-Macrophage Colony-Stimulating Factor - biosynthesis
Granulocyte-Macrophage Colony-Stimulating Factor - genetics
Humans
Industrial Microbiology
Maltose-Binding Proteins
Periplasm - chemistry
Periplasm - metabolism
Plasmids - genetics
Protein Sorting Signals
Proteins
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - genetics
Recombinant Proteins
RNA Stability
RNA, Messenger - metabolism
Stem cells
Studies
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Summary:Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor, that has been used as a therapeutic agent in facilitating bone marrow and stem cell transplantation and in other clinical cases like neutropenia. Although biologically active recombinant GM-CSF has been successfully produced in Escherichia coli, the reported levels are extremely poor. In this study we looked into the possible reasons for poor expression and found that protein toxicity coupled with protease-based degradation was the principal reason for low productivity. To overcome this problem we attached a signal sequence, as well as an amino-terminal His-tag fusion to the GM-CSF gene. This combination had a dramatic effect on expression levels, which increased from 0.8 microg/mL in the control to 40 microg/mL. When a larger fusion partner, such as the Maltose-binding protein (MBP-tag), was used the expression levels increased further to 69.5 microg/mL, which along with the MBP-tag represented approx 12% of the total cellular protein.
ISSN:1073-6085
1559-0305
1073-6085
DOI:10.1385/mb:30:2:103