Tuning ligand affinity, specificity, and folding stability of an engineered lipocalin variant - a so-called 'anticalin' - using a molecular random approach

被引:55
作者
Schlehuber, S [1 ]
Skerra, A [1 ]
机构
[1] Tech Univ Munich, Lehrstuhl Biol Chem, D-8050 Freising Weihenstephan, Germany
关键词
anticalin; bilin-binding protein; digoxigenin; ligand binding; thermal stability;
D O I
10.1016/S0301-4622(02)00026-1
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Anticalins are prepared by reshaping the ligand pocket of a natural lipocalin via protein engineering in order to recognize a prescribed ligand. In this manner, the anticalin DigA with specificity for digoxigenin was previously derived from the bilin-binding protein (BBP), a natural lipocalin from Pieris brassicae. The four peptide loops that form its ligand-binding site were randomized and a cognate variant was selected from the resulting library. Here, we propose a concept for improving the ligand-binding properties of this anticalin in an in vitro affinity maturation process by step-wise randomization of restricted areas of the loop region. Following selection on digoxigenin-binding activity via phage display and colony screening, several DigA variants were thus obtained. The recombinant proteins were thoroughly characterized in terms of ligand affinity and specificity, secondary structure and thermal stability against unfolding. The variant DigA16/19, which carries several new mutations, exhibits clearly improved affinity for digoxigenin, with K-D = 12.4 nM. Hence, it is suitable as a sensitive reagent in biochemical detection experiments, especially when produced as a functional fusion protein with alkaline phosphatase as reporter enzyme. In addition, DigA16/19 possesses enhanced ligand specificity and recognizes part of the linker that was used for fixing the steroid group to a carrier protein. Finally, the digoxigenin-binding anticalins appear to have high physico-chemical stability, with T-m values in the 70 degreesC range. Our present findings support the notion that anticalins provide a useful class of compact and robust ligand-receptor proteins that can be tailored for practical demands. (C) 2002 Elsevier Science B.V. All fights reserved.
引用
收藏
页码:213 / 228
页数:16
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