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Animal Breeding and Genetics
Asian-Australasian Journal of Animal Sciences 2004;17(12): 1751-1757.
DOI: https://doi.org/10.5713/ajas.2004.1751    Published online January 1, 2004.
Improving Soluble Expression of β-Galactosidase in Escherichia coli by Fusion with Thioredoxin
E. S. Nam, H. J. Jung, J. K. Ahn
Abstract
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 棺-galactosidase, the gene of Thermus thermophilus KNOUC112 棺-galactosidase (KNOUC112 棺-gal) was fused with thioredoxin gene, and optimization of its expression in E. coli TOP10 was performed. KNOUC112 棺-gal in pET-5b was isolated out, fused with thioredoxin gene in pThioHis C, and transformed to E. coli TOP10. The 棺-galactosidase fused with thioredoxin was produced in E. coli TOP10 as dimer and trimer. The productivity of fusion 棺-galactosidase expressed via pThioHis C at 37째C was about 5 times higher than that of unfused 棺-galactosidase expressed via pET-5b at 37째C. Inclusion body of 棺-galactosidase was formed highly, regardless of the induction by IPTG when KNOUC112 棺-gal was expressed via pET-5b at 37째C. Fusion 棺-galactosidase expressed at 37째C 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 25째C prevented the formation of inclusion body, optimally at 25째C. 0.07 mM of IPTG was sufficient for the soluble expression of fusion gene at 25째C. The soluble production of Thermus thermophilus KNOUC112 棺-galactosidase could be increased about 10 times by fusion with thioredoxin, and optimization of incubation temperature and IPTG concentration for induction.
Keywords: 棺-Galactosidase; Thioredoxin; Inclusion Body; IPTG
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