Secretion of functional human interleukin-3 from Bacillus subtilis
The Gram-positive bacterium Bacillus subtilis is well-known for its huge capacity to produce secreted bacterial enzymes. Nevertheless, the secretion of pharmaceutically interesting recombinant proteins by this organism is frequently inefficient. This paper documents for the first time on the optimisation of B. subtilis for the production of human interleukin-3 (hIL-3), a four-helix bundle cytokine, which stimulates the proliferation and differentiation of a broad range of blood cells. By developing a host-vector system on the basis of the multiple protease-deficient B. subtilis strain WB700 and a multicopy plasmid containing two tandemly positioned strong promoters plus an efficient signal sequence, the hIL-3 protein was efficiently produced and secreted into the growth medium. As verified by SDS-PAGE, mass spectrometry and cross-linking experiments with a thiol-specific reagent, intact and properly folded hIL-3 was purified from the B. subtilis growth medium. Bioactivity tests showed that the isolated hIL-3 was able to specifically induce proliferation of the hIL-3-dependent leukaemia cell line MO7e. Using the eight-fold protease-deficient strain WB800 the hIL-3 accumulation in the growth medium was increased to levels up to 100 mg l−1.
Journal: Journal of Biotechnology - Volume 123, Issue 2, 17 May 2006, Pages 211–224