Improving Soluble Expression of β-Galactosidase in Escherichia coli by Fusion with Thioredoxin*

Recombinant heterologous proteins can be produced as insoluble aggregates partially or perfectly inactive in Escherichia coli. One of the strateges to improve the solubility of recombinant proteins is fusion with a partner that is excellent in producing soluble fusion proteins. To improve the production of soluble β-galactosidase, the gene of Thermus thermophilus KNOUC112 β-galactosidase (KNOUC112 β-gal) was fused with thioredoxin gene, and optimization of its expression in E. coli TOP10 was performed. KNOUC112 β-gal in pET-5b was isolated out, fused with thioredoxin gene in pThioHis C, and transformed to E. coli TOP10. The β-galactosidase fused with thioredoxin was produced in E. coli TOP10 as dimer and trimer. The productivity of fusion βgalactosidase expressed via pThioHis C at 37°C was about 5 times higher than that of unfused β-galactosidase expressed via pET-5b at 37°C. Inclusion body of β-galactosidase was formed highly, regardless of the induction by IPTG when KNOUC112 β-gal was expressed via pET-5b at 37°C. Fusion β-galactosidase expressed at 37°C via pThioHis C without the induction by IPTG was soluble, but the induction by IPTG promoted the formation of inclusion body. Lowering the incubation temperature for the expression of fusion gene under 25°C prevented the formation of inclusion body, optimally at 25°C. 0.07 mM of IPTG was sufficient for the soluble expression of fusion gene at 25°C. The soluble production of Thermus thermophilus KNOUC112 β-galactosidase could be increased about 10 times by fusion with thioredoxin, and optimization of incubation temperature and IPTG concentration for induction. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 12 : 1751-1757)