Identification of an intestine-specific promoter and inducible expression of bacterial α-galactosidase in mammalian cells by a <it>lac</it> operon system

• Main Authors: Ya-Feng Zhai, Gang Shu, Xiao-Tong Zhu, Zhi-Qi Zhang, Xia-Jing Lin, Song-Bo Wang, Li-Na Wang, Yong-Liang Zhang, Qing-Yan Jiang
• Format: Article
• Language: English
• Published: BMC 2012-10-01
• Series: Journal of Animal Science and Biotechnology
• Subjects: α-galactosidase; Inducible expression; Intestine-specific promoters; <it>Lac</it> operon
• Online Access: http://www.jasbsci.com/content/3/1/32
• ISSN: 1674-9782; 2049-1891

<p>Abstract</p> <p>Background</p> <p>α-galactosidase has been widely used in animal husbandry to reduce anti-nutritional factors (such as α-galactoside) in feed. Intestine-specific and substrate inducible expression of α-galactosidase would be highly beneficial for transgenic animal production.</p> <p>Methods</p> <p>To achieve the intestine-specific and substrate inducible expression of α-galactosidase, we first identified intestine-specific promoters by comparing the transcriptional activity and tissue specificity of four intestine-specific promoters from human intestinal fatty acid binding protein, rat intestinal fatty acid binding protein, human mucin-2 and human lysozyme. We made two chimeric constructs combining the promoter and enhancer of human mucin-2, rat intestinal trefoil factor and human sucrase-isomaltase. Then a modified <it>lac</it> operon system was constructed to investigate the induction of α-galactosidase expression and enzyme activity by isopropyl β-D-1-thiogalactopyranoside (IPTG) and an α-galactosidase substrate, α-lactose.</p> <p>We declared that the research carried out on human (Zhai Yafeng) was in compliance with the Helsinki Declaration, and experimental research on animals also followed internationally recognized guidelines.</p> <p>Results</p> <p>The activity of the human mucin-2 promoter was about 2 to 3 times higher than that of other intestine-specific promoters. In the <it>lac</it> operon system, the repressor significantly decreased (<it>P</it> < 0.05) luciferase activity by approximately 6.5-fold and reduced the percentage of cells expressing green fluorescent protein (GFP) by approximately 2-fold. In addition, the expression level of α-galactosidase mRNA was decreased by 6-fold and α-galactosidase activity was reduced by 8-fold. In line with our expectations, IPTG and α-lactose supplementation reversed (<it>P</it> < 0.05) the inhibition and produced a 5-fold increase of luciferase activity, an 11-fold enhancement in the percentage of cells with GFP expression and an increase in α-galactosidase mRNA abundance (by about 5-fold) and α-galactosidase activity (by about 7-fold).</p> <p>Conclusions</p> <p>We have successfully constructed a high specificity inducible <it>lac</it> operon system in an intestine-derived cell line, which could be of great value for gene therapy applications and transgenic animal production.</p>