Production processes of recombinant IL-1 beta from Bacillus subtilis: comparison between intracellular and exocellular expression.
The human IL-1 beta coding sequence derived from a cDNA library was inserted into two different plasmid expression vectors, pSM214 and pSM308, which promote the synthesis of recombinant products intracellularly and exocellularly, respectively. The hybrid constructs pSM261 and pSM320 were obtained. Bacillus subtilis SMS118 was transformed with these plasmids and the recombinant strains were grown in 1 litre bioreactors. Different growth conditions were analyzed to optimize yields both in terms of biomass and IL-1 beta production. In the pSM261-harbouring strain, IL-1 beta was synthesized in the cytoplasm to levels ranging from 1 to 2.7 mg g-1 of cells, corresponding to up to 40 mg l-1 of the culture. In contrast, SMS118(pSM320) was able to secrete 0.27 mg of natural IL-1 beta per g of cells (6.7 mg l-1 of culture). Processes for the purification of IL-1 beta from the supernatant and the biomass of the two cultures were also developed and compared in terms of yield and simplicity of the purification schemes. From our data it turns out that the route of intracellular expression is very efficient and superior to the one which results in secretion of IL-1 beta. This indicates that the use of B. subtilis as a recombinant host in biotechnology is not strictly dependent on its ability to secrete proteins into the culture medium.
Eniricerche S.p.A., Milan, Italy.
A V
AV
Bellini
G
G
Galli
E
E
Fascetti
G
G
Frascotti
P
P
Branduzzi
G
G
Lucchese
G
G
Grandi
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D003470:Culture Media / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000138:chemical synthesis; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D014169:Transformation, Bacterial
D003470:Culture Media; D007375:Interleukin-1; D011994:Recombinant Proteins; D004247:DNA