Enhanced periplasmic expression of human Granulocyte Macrophage Colony Stimulating Factor in Escherichia coli by the modification of the signal peptide cleavage site

Human ganulocyte macrophage colony stimulating factor (hGM-CSF) is one of the hematopoetic growth factors and regulator of hematopoesis that has been used as therapeutic agent in treatment of various ailments, such as myelodysplastic syndrome, neutrogena and chemotherapy induced myleosuppression. With the aim of the large-scale production of recombinant hGM-CSF in the periplasmic space of E. coli a fusion fragment coding for pelB::hGM-CSF, constructed by splice overlap extension PCR (SOE-PCR) technique, was inserted in a T7-based expression plasmid. The expression analysis was performed on the IPTG (or lactose)-induced BL21 (DE3) strain of E. coli, containing the recombinant plasmid. Using SDS-PAGE and densitometric assay of protein bands corresponding to the recombinant hGM-CSF, we analysed periplasmic expression of hGM-CSF after induction with either 1mM IPTG or various concentrations of lactose. Both the processing of signal peptide and the translocation of the mature hGM-CSF took place more efficiently when lactose was used as inducer, comparing to the results obtained from the IPTG induction. The optimum concentration of lactose was found to be 0.01% after 20 hours, lead to an over-expression of pelB::hGM-CSF followed by nearly complete processing and transport of the mature protein into in the periplasmic space. The mature hGM-CSF expressed in the present construct was estimated around 60% of the bacterial periplasmic proteins. Comparing to a previously-made hGM-CSF expressing plasmid (K1), which carries six extra-nucleotides, coding for methionine and alanine, in the signal peptide C-terminal, the periplasmic expression efficiency of the newly made plasmid is higher both at processing level and the translocation of mature protein into the periplasmic space. These data support the idea that amino acid context in the cleavage site region play a key-important role in the expression efficiency of secretory proteins. Moreover it was documented that a combination of the improved signal peptide cleavage site and the use of an optimized lactose concentration can lead to a highest of production level of mature hGM-CSF.

Keywords:

human GM-CSF , lactose , IPTG , T7/lac over- expression system , induction , Escherichia coli and recombinant protein , signal peptide & processing