Proteolytic selection for protein folding using filamentous bacteriophages

被引:228
作者
Kristensen, P [1 ]
Winter, G [1 ]
机构
[1] MRC, Ctr Prot Engn, Cambridge CB2 2QH, England
来源
FOLDING & DESIGN | 1998年 / 3卷 / 05期
关键词
phage display; protein folding; proteolytic cleavage;
D O I
10.1016/S1359-0278(98)00044-3
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: Filamentous bacteriophages have been used for the selection of folded peptide and protein 'ligands' by binding the phage to 'receptor'-coated solid phase. Here, using proteolysis, we have developed a technique for the selection of folded and stable proteins that is independent of their binding activities. Results: When a 21-residue peptide comprising a protease cleavage site was introduced into the flexible linker between the second and third domains of the minor coat protein p3 of filamentous bacteriophage, the phages could be cleaved by trypsin and were rendered non-infective. By contrast, phages displaying mutant barnases at this site were resistant to proteolysis, but were cleaved and their infectivity was destroyed as the temperature was raised. By mixing phages bearing two barnase mutants of differing stability, and adding protease at a temperature at which one mutant was resistant and the other was sensitive, we were able to enrich by 1.6 x 10(4)-fold for phages bearing the more stable barnase. Conclusions: The approach provides a means for the selection of folded and stable proteins, and may be applicable to the selection of de novo proteins.
引用
收藏
页码:321 / 328
页数:8
相关论文
共 50 条
[1]   Active barnase variants with completely random hydrophobic cores [J].
Axe, DD ;
Foster, NW ;
Fersht, AR .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1996, 93 (11) :5590-5594
[2]   HORMONE PHAGE - AN ENRICHMENT METHOD FOR VARIANT PROTEINS WITH ALTERED BINDING-PROPERTIES [J].
BASS, S ;
GREENE, R ;
WELLS, JA .
PROTEINS-STRUCTURE FUNCTION AND GENETICS, 1990, 8 (04) :309-314
[3]   Minimizing a binding domain from protein A [J].
Braisted, AC ;
Wells, JA .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1996, 93 (12) :5688-5692
[4]   MAKING ANTIBODY FRAGMENTS USING PHAGE DISPLAY LIBRARIES [J].
CLACKSON, T ;
HOOGENBOOM, HR ;
GRIFFITHS, AD ;
WINTER, G .
NATURE, 1991, 352 (6336) :624-628
[5]   De novo protein design: Towards fully automated sequence selection [J].
Dahiyat, BI ;
Sarisky, CA ;
Mayo, SL .
JOURNAL OF MOLECULAR BIOLOGY, 1997, 273 (04) :789-796
[6]   FOLDED PROTEINS OCCUR FREQUENTLY IN LIBRARIES OF RANDOM AMINO-ACID-SEQUENCES [J].
DAVIDSON, AR ;
SAUER, RT .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1994, 91 (06) :2146-2150
[7]   COOPERATIVELY FOLDED PROTEINS IN RANDOM SEQUENCE LIBRARIES [J].
DAVIDSON, AR ;
LUMB, KJ ;
SAUER, RT .
NATURE STRUCTURAL BIOLOGY, 1995, 2 (10) :856-864
[8]   CLONAL SELECTION AND AMPLIFICATION OF PHAGE DISPLAYED ANTIBODIES BY LINKING ANTIGEN RECOGNITION AND PHAGE REPLICATION [J].
DUENAS, M ;
BORREBAECK, CAK .
BIO-TECHNOLOGY, 1994, 12 (10) :999-1002
[9]   PROTEIN-FOLDING AND STABILITY - THE PATHWAY OF FOLDING OF BARNASE [J].
FERSHT, AR .
FEBS LETTERS, 1993, 325 (1-2) :5-16
[10]   A strategy of exon shuffling for making large peptide repertoires displayed on filamentous bacteriophage [J].
Fisch, I ;
Kontermann, RE ;
Finnern, R ;
Hartley, O ;
SolerGonzalez, AS ;
Griffiths, AD ;
Winter, G .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1996, 93 (15) :7761-7766