Large-scale purification of human granulocyte-macrophage colony-stimulating factor expressed in Bombyx mori pupae

Area of Science:

• Biotechnology
• Protein Purification
• Recombinant Protein Expression

Background:

• Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is crucial for various immune cells and stem cells.
• Large-scale purification of recombinant proteins from silkworm pupae presents significant challenges.

Purpose of the Study:

• To establish efficient purification methods for recombinant hGM-CSF produced in silkworm pupae.
• To optimize large-scale production of biologically active hGM-CSF.

Main Methods:

• Recombinant hGM-CSF was expressed using the Bombyx mori nucleopolyhedrovirus system.
• Two crude preparation methods were compared: (NH4)2SO4 fractional precipitation and isoelectric precipitation.
• Purification involved gel filtration and ion-exchange chromatography.

Main Results:

• Isoelectric precipitation combined with chromatography yielded approximately 11.7 mg of 95% pure hGM-CSF per 1000 g of pupae.
• This method increased protein recovery by approximately 40% compared to fractional precipitation.
• The purified hGM-CSF exhibited a biologic activity of up to 9.0 x 10^6 colony-forming units/mg.

Conclusions:

• Isoelectric precipitation is a more efficient method for purifying recombinant hGM-CSF from silkworm pupae.
• The developed purification strategy enables scalable production of active hGM-CSF.
• This facilitates further exploration of hGM-CSF's pharmaceutical actions.