Heterologous expression of secreted biologically active human interleukin-10 in Bifidobacterium breve.
Construction of Bifidobacterium breve capable of production of secreted biologically active human interleukin-10 (hIL-10) is described. ORF coding for full-length mature human interleukin-10 was cloned into a series of expression vectors. This resulted in generation of translational fusions between hIL-10 and signal peptides sequences derived from Bifidobacterium breve genes sec2, apuB and B. adolescentis gene amyB under the control of constitutively active bifidobacterial promoter. We have shown that fusion to amyB signal peptide resulted in highest expression level of hIL-10 at the mRNA and protein level. Secreted hIL-10 was highly unstable in bifidobacterial culture supernatants in standard growth conditions. However, incubation of stationary cultures in buffered tissue culture medium resulted in production of stable biologically active hIL-10, albeit in low amounts (1.9 ng/ml).
Khokhlova
E V
EV
Department of Microbiology and Virology, Russian State Medical University, Moscow, Russian Federation.
Efimov
B A
BA
Kafarskaia
L I
LI
Shkoporov
A N
AN
D001644:Bifidobacterium / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003470:Culture Media; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D019073:HT29 Cells; D006801:Humans; D007218:Industrial Microbiology; D016753:Interleukin-10 / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D003470:Culture Media; C508609:IL10 protein, human; D011993:Recombinant Fusion Proteins; D016753:Interleukin-10