Selection and characterisation of affibody molecules inhibiting the interaction between Ras and Raf in vitro

被引:12
作者
Grimm, Sebastian [1 ]
Lundberg, Emma [1 ]
Yu, Feifan [1 ]
Shibasaki, Seiji [1 ]
Vernet, Erik [1 ]
Skogs, Marie [1 ]
Nygren, Per-Ake [1 ]
Graslund, Torbjorn [1 ]
机构
[1] Royal Inst Technol, Div Mol Biotechnol, SE-10691 Stockholm, Sweden
基金
瑞典研究理事会;
关键词
PROTEIN-PROTEIN INTERACTIONS; BINDING-PROTEINS; INTRACELLULAR ANTIBODIES; AFFINITY; INTRABODIES; RECEPTOR; RECOGNITION; LIGANDS; TARGETS;
D O I
10.1016/j.nbt.2010.07.016
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Development of molecules with the ability to selectively inhibit particular protein-protein interactions is important in providing tools for understanding cell biology In this work, we describe efforts to select small Ras- and Raf-specific three-helix bundle affibody binding proteins capable of inhibiting the interaction between H-Ras and Raf-1, from a combinatorial library displayed on bacteriophage Target-specific variants with typically high nanomolar or low micromolar affinities (K-D) could be selected successfully against both proteins, as shown by dot blot, ELISA and real-time biospecific interaction analyses Affibody molecule variants selected against H-Ras were shown to bind epitopes overlapping each other at a site that differed from that at which H-Ras interacts with Raf-1 In contrast, an affibody molecule isolated during selection against Raf-1 was shown to effectively inhibit the interaction between H-Ras and Raf-1 in a dose-dependent manner Possible intracellular applications of the selected affibody molecules are discussed
引用
收藏
页码:766 / 773
页数:8
相关论文
共 25 条
[1]   The selection of intracellular antibodies [J].
Cattaneo, A ;
Biocca, S .
TRENDS IN BIOTECHNOLOGY, 1999, 17 (03) :115-121
[2]  
Emerson S D, 1996, Drug Des Discov, V13, P83
[3]   Selection and characterization of affibody ligands binding to Alzheimer amyloid β peptides [J].
Gronwall, Caroline ;
Jonsson, Andreas ;
Lindstrom, Sara ;
Gunneriusson, Elin ;
Stahl, Stefan ;
Herne, Nina .
JOURNAL OF BIOTECHNOLOGY, 2007, 128 (01) :162-183
[4]   Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling [J].
Gunneriusson, E ;
Nord, K ;
Uhlén, M ;
Nygren, PÅ .
PROTEIN ENGINEERING, 1999, 12 (10) :873-878
[5]   An in vitro selected binding protein (affibody) shows conformation-dependent recognition of the respiratory syncytial virus (RSV) G protein [J].
Hansson, M ;
Ringdahl, J ;
Robert, A ;
Power, U ;
Goetsch, L ;
Nguyen, TN ;
Uhlén, M ;
Ståhl, S ;
Nygren, PÅ .
IMMUNOTECHNOLOGY, 1999, 4 (3-4) :237-252
[6]   Structural basis for recognition by an in vitro evolved affibody [J].
Högbom, M ;
Eklund, M ;
Nygren, PÅ ;
Nordlund, P .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2003, 100 (06) :3191-3196
[7]   High-throughput protein expression of cDNA products as a tool in functional genomics [J].
Larsson, M ;
Gräslund, S ;
Li, YB ;
Brundell, E ;
Uhlén, M ;
Höög, C ;
Stähl, S .
JOURNAL OF BIOTECHNOLOGY, 2000, 80 (02) :143-157
[8]   Diverting a protein from its cellular location by intracellular antibodies - The case of p21Ras [J].
Lener, M ;
Horn, IR ;
Cardinale, A ;
Messina, S ;
Nielsen, UB ;
Rybak, SM ;
Hoogenboom, HR ;
Cattaneo, A ;
Biocca, S .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 2000, 267 (04) :1196-1205
[9]   Selection and characterization of Affibody® ligands to the transcription factor c-Jun [J].
Lundberg, Emma ;
Brismar, Hjalmar ;
Graslund, Torbjorn .
BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY, 2009, 52 :17-27
[10]   A protein silencing switch by ligand-induced proteasome-targeting intrabodies [J].
Melchionna, Teresa ;
Cattaneo, Antonino .
JOURNAL OF MOLECULAR BIOLOGY, 2007, 374 (03) :641-654