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Engineering of ligand specificity of periplasmic binding protein for glucose sensing.

Biotechnol. Lett.2008 Aug;30(8):1453-60. doi:10.1007/s10529-008-9712-7. Epub 2008 Apr 15
Akane Sakaguchi-Mikami 1 , Atsushi Taneoka , Rie Yamoto , Stefano Ferri , Koji Sode
Akane Sakaguchi-Mikami 1 , Atsushi Taneoka , Rie Yamoto , Stefano Ferri , Koji Sode

[No authors listed]

Author information
  • 1 Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.

摘要


A novel glucose-sensing molecule was created based on galactose/glucose-binding protein (GGBP). GGBP mutants at Asp14, a residue interacting with the 4th hydroxyl group of the sugar molecule, were constructed by mutagenesis to improve the ligand specificity of GGBP. The autofluorescence-based analysis of the binding abilities of these engineered GGBPs showed that the GGBP mutants Asp14Asn and Asp14Glu bound only to glucose in a concentration-dependent manner, without being affected by the presence of galactose. The Phe16Ala mutation, which leads to an increase in the K (d) value toward glucose, was then introduced into these two glucose-specific mutant GGBPs. One of the constructed GGBP double-mutants, Asp14Glu/Phe16Ala, had a glucose specificity with a K(d) value of 3.9 mM, which makes it suitable for use in the measurement of the physiological glucose concentration. Our results demonstrate that it is possible to construct a GGBP which specifically recognizes glucose and has a higher K(d) value and use it as a molecular recognition element of blood glucose monitoring systems by combining two different mutations based on the 3D structure of GGBP.

基因