Secretion of functional human interleukin-3 from Bacillus subtilis

Area of Science:

• Microbiology
• Biotechnology
• Protein Engineering

Background:

• Bacillus subtilis is a robust producer of secreted enzymes, but recombinant protein production can be inefficient.
• Human interleukin-3 (hIL-3) is a crucial cytokine for blood cell proliferation and differentiation, with therapeutic potential.
• Optimizing secretion pathways in B. subtilis is essential for efficient pharmaceutical protein production.

Purpose of the Study:

• To optimize the production and secretion of human interleukin-3 (hIL-3) using Bacillus subtilis.
• To develop a host-vector system for enhanced recombinant protein expression in B. subtilis.
• To validate the functionality and purity of the secreted hIL-3.

Main Methods:

• Utilized a multiple protease-deficient B. subtilis strain (WB700) and a multicopy plasmid with strong promoters and an efficient signal sequence.
• Employed SDS-PAGE, mass spectrometry, and cross-linking experiments for protein verification and purification.
• Conducted bioactivity assays using the hIL-3-dependent leukaemia cell line MO7e.

Main Results:

• Successfully produced and secreted intact, properly folded hIL-3 into the growth medium.
• Purified hIL-3 demonstrated specific bioactivity, inducing proliferation of MO7e cells.
• Achieved a significant increase in hIL-3 accumulation (up to 100 mg/L) using an eight-fold protease-deficient strain (WB800).

Conclusions:

• Developed an optimized B. subtilis system for efficient production of biologically active human interleukin-3.
• Demonstrated the potential of protease-deficient B. subtilis strains for high-yield recombinant protein secretion.
• This optimized system offers a promising platform for the large-scale production of therapeutic proteins like hIL-3.