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 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.
Bhattacharya
Palash
P
Center for Biotechnology, Jawaharlal Nehru University, New Dehli, Delhi, 110067, India.
Pandey
Gaurav
G
Srivastava
Poonam
P
Mukherjee
Krishna Jyoti
KJ
D002352:Carrier Proteins / Q000235:genetics; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007218:Industrial Microbiology; D058619:Maltose-Binding Proteins; D019897:Periplasm / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D021382:Protein Sorting Signals; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins
D002352:Carrier Proteins; D003062:Codon; D058619:Maltose-Binding Proteins; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor