Dynamic combinatorial libraries of artificial repeat proteins

被引:10
作者
Eisenberg, Margarita [1 ]
Shumacher, Inbal [1 ]
Cohen-Luria, Rivka [1 ]
Ashkenasy, Gonen [1 ]
机构
[1] Ben Gurion Univ Negev, Dept Chem, IL-84105 Beer Sheva, Israel
基金
欧洲研究理事会;
关键词
Repeat proteins; Dynamic combinatorial chemistry; Dynamic combinatorial library; Native chemical ligation; NATIVE CHEMICAL LIGATION; TPR DOMAINS; THIOESTER EXCHANGE; CONSENSUS DESIGN; LIGAND-BINDING; STABILITY; COMPLEXES; MOTIF; POLYMERIZATION; RECOGNITION;
D O I
10.1016/j.bmc.2013.03.007
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
Repeat proteins are found in almost all cellular systems, where they are involved in diverse molecular recognition processes. Recent studies have suggested that de novo designed repeat proteins may serve as universal binders, and might potentially be used as practical alternative to antibodies. We describe here a novel chemical methodology for producing small libraries of repeat proteins, and screening in parallel the ligand binding of library members. The first stage of this research involved the total synthesis of a consensus-based three-repeat tetratricopeptide (TPR) protein (similar to 14 kDa), via sequential attachment of the respective peptides. Despite the effectiveness of the synthesis and ligation steps, this method was found to be too demanding for the production of proteins containing variable number of repeats. Additionally, the analysis of binding of the individual proteins was time consuming. Therefore, we designed and prepared novel dynamic combinatorial libraries (DCLs), and show that their equilibration can facilitate the formation of TPR proteins containing up to eight repeating units. Interestingly, equilibration of the library building blocks in the presence of the biologically relevant ligands, Hsp90 and Hsp70, induced their oligomerization into forming more of the proteins with large recognition surfaces. We suggest that this work presents a novel simple and rapid tool for the simultaneous screening of protein mixtures with variable binding surfaces, and for identifying new binders for ligands of interest. (C) 2013 Published by Elsevier Ltd.
引用
收藏
页码:3450 / 3457
页数:8
相关论文
共 42 条
[1]   Protein repeats: Structures, functions, and evolution [J].
Andrade, MA ;
Perez-Iratxeta, C ;
Ponting, CP .
JOURNAL OF STRUCTURAL BIOLOGY, 2001, 134 (2-3) :117-131
[2]   The emergence of halophilic evolutionary patterns from a dynamic combinatorial library of macrocyclic pseudopeptides [J].
Atcher, Joan ;
Moure, Alejandra ;
Alfonso, Ignacio .
CHEMICAL COMMUNICATIONS, 2013, 49 (05) :487-489
[3]   Artificial leucine rich repeats as new scaffolds for protein design [J].
Baabur-Cohen, Hemda ;
Dayalan, Subashini ;
Shumacher, Inbal ;
Cohen-Luria, Rivka ;
Ashkenasy, Gonen .
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 2011, 21 (08) :2372-2375
[4]  
Blatch GL, 1999, BIOESSAYS, V21, P932, DOI 10.1002/(SICI)1521-1878(199911)21:11<932::AID-BIES5>3.0.CO
[5]  
2-N
[6]   Ligand discrimination by TPR domains -: Relevance and selectivity of EEVD-recognition in Hsp70•Hop•Hsp90 complexes [J].
Brinker, A ;
Scheufler, C ;
von der Mülbe, F ;
Fleckenstein, B ;
Herrmann, C ;
Jung, G ;
Moarefi, I ;
Hartl, FU .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2002, 277 (22) :19265-19275
[7]   Mechanosensitive Self-Replication Driven by Self-Organization [J].
Carnall, Jacqui M. A. ;
Waudby, Christopher A. ;
Belenguer, Ana M. ;
Stuart, Marc C. A. ;
Peyralans, Jerome J. -P. ;
Otto, Sijbren .
SCIENCE, 2010, 327 (5972) :1502-1506
[8]   Molecular recognition via coupled folding and binding in a TPR domain [J].
Cliff, MJ ;
Williams, MA ;
Brooke-Smith, J ;
Barford, D ;
Ladbury, JE .
JOURNAL OF MOLECULAR BIOLOGY, 2005, 346 (03) :717-732
[9]   Dynamic combinatorial chemistry [J].
Corbett, Peter T. ;
Leclaire, Julien ;
Vial, Laurent ;
West, Kevin R. ;
Wietor, Jean-Luc ;
Sanders, Jeremy K. M. ;
Otto, Sijbren .
CHEMICAL REVIEWS, 2006, 106 (09) :3652-3711
[10]   Designed TPR modules as novel anticancer agents [J].
Cortajarena, Aitziber L. ;
Yi, Fang ;
Regan, Lynne .
ACS CHEMICAL BIOLOGY, 2008, 3 (03) :161-166