Generation of a dual-labeled fluorescence biosensor for Crk-II phosphorylation using solid-phase expressed protein ligation

被引:82
作者
Cotton, GJ [1 ]
Muir, TW [1 ]
机构
[1] Rockefeller Univ, Lab Synthet Prot Chem, New York, NY 10021 USA
来源
CHEMISTRY & BIOLOGY | 2000年 / 7卷 / 04期
关键词
Crk-II phosphorylation; fluorescent biosensor; protein engineering; site-specific labeling;
D O I
10.1016/S1074-5521(00)00100-9
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
Background: The site-specific chemical modification of proteins has proved to be extremely powerful for generating tools for the investigation of biological processes. Although a few elegant methods exist for engineering a recombinant protein at a unique position, these techniques cannot be easily extended to allow several different chemical probes to be specifically introduced into a target sequence, As such multiply labeled proteins could be used to study many biological processes, and in particular biomolecular interactions, we decided to investigate whether such protein reagents could be generated using an extension of the semisynthesis technique known as expressed protein ligation. Results: A solid-phase expressed protein ligation (SPPL) technology is described that enables large semisynthetic proteins to be assembled on a solid support by the controlled sequential ligation of a series of recombinant and synthetic polypeptide building blocks. This modular approach allows multiple, different chemical modifications to be introduced site-specifically into a target protein. This process, which is analogous to solid-phase peptide synthesis, was used to dual-label the amino and carboxyl termini of the Crk-II adapter protein with the fluorescence resonance energy transfer pair tetramethylrhodamine and fluorescein, respectively. The resulting construct reports (through a fluorescence change) the phosphorylation of Crk-II by the nonreceptor protein tyrosine kinase, c-Abl, and was used to probe the protein-protein interactions that regulate this important post-translational process. Conclusions: SPPL provides a powerful method for specifically modifying proteins at multiple sites, as was demonstrated by generating a protein-based biosensor for Crk-II phosphorylation. Such protein derivatives are extremely useful for investigating protein function in vitro and potentially in vivo. This modular approach should be applicable to many different protein systems.
引用
收藏
页码:253 / 261
页数:9
相关论文
共 36 条
[1]   A potential SH3 domain-binding site in the Crk SH2 domain [J].
Anafi, M ;
Rosen, MK ;
Gish, GD ;
Kay, LE ;
Pawson, T .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1996, 271 (35) :21365-21374
[2]  
Ayers B, 1999, BIOPOLYMERS, V51, P343, DOI 10.1002/(SICI)1097-0282(1999)51:5<343::AID-BIP4>3.0.CO
[3]  
2-W
[4]   SH2 and SH3-containing adaptor proteins: Redundant or Independent mediators of intracellular signal transduction [J].
Birge, RB ;
Knudsen, BS ;
Besser, D ;
Hanafusa, H .
GENES TO CELLS, 1996, 1 (07) :595-613
[5]   Membrane-targeting of signalling molecules by SH2/SH3 domain-containing adaptor proteins [J].
Buday, L .
BIOCHIMICA ET BIOPHYSICA ACTA-REVIEWS ON BIOMEMBRANES, 1999, 1422 (02) :187-204
[6]  
Camarero JA, 1998, J PEPT RES, V51, P303
[7]   Chemical protein synthesis by solid phase ligation of unprotected peptide segments [J].
Canne, LE ;
Botti, P ;
Simon, RJ ;
Chen, YJ ;
Dennis, EA ;
Kent, SBH .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1999, 121 (38) :8720-8727
[8]   IDENTIFICATION OF THE TARGET OF A TRANSCRIPTION ACTIVATOR PROTEIN BY PROTEIN-PROTEIN PHOTO-CROSS-LINKING [J].
CHEN, Y ;
EBRIGHT, YW ;
EBRIGHT, RH .
SCIENCE, 1994, 265 (5168) :90-92
[9]   Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element [J].
Chong, SR ;
Mersha, FB ;
Comb, DG ;
Scott, ME ;
Landry, D ;
Vence, LM ;
Perler, FB ;
Benner, J ;
Kucera, RB ;
Hirvonen, CA ;
Pelletier, JJ ;
Paulus, H ;
Xu, MQ .
GENE, 1997, 192 (02) :271-281
[10]   The development and therapeutic potential of protein kinase inhibitors [J].
Cohen, P .
CURRENT OPINION IN CHEMICAL BIOLOGY, 1999, 3 (04) :459-465