Selection for improved protein stability by phage display

Citation
S. Jung et al., Selection for improved protein stability by phage display, J MOL BIOL, 294(1), 1999, pp. 163-180
Citations number
48
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Molecular Biology & Genetics
Journal title
JOURNAL OF MOLECULAR BIOLOGY
ISSN journal
0022-2836 → ACNP
Volume
294
Issue
1
Year of publication
1999
Pages
163 - 180
Database
ISI
SICI code
0022-2836(19991119)294:1<163:SFIPSB>2.0.ZU;2-Z
Abstract
A library of mutants of a single-chain Fv fragment (scFv) was generated by a combination of directed and random mutagenesis, using oligonucleotides ra ndomized at defined positions and two rounds of DNA shuffling. The library was based on the already well folding and stable scFv fragment 4D5Flu. Ln o rder to further improve this framework and test the efficiency of various s election strategies, phage display selection was carried out under differen t selective pressures for higher thermodynamic stability. Incubation of the display phages at elevated temperatures was compared to exposure of the ph ages to high concentrations of guanidinium chloride. Temperature stress-gui ded selection yielded the most stable scFv mutant after two rounds of mutag enesis and selection, due to the irreversibility of the unfolding process. It possessed only two mutations (His(L27d)Asn and Phe(L55)Val) and showed a thermodynamic stability improved by roughly 4 kcal/mol, threefold better e xpression yields in Escherichia coli as well as a 20-fold better binding co nstant than the 4D5Flu wild-type. The selection results obtained in this st udy delineate the advantages, disadvantages and limitations of different st ability stress selection methods in phage display. (C) 1999 Academic Press.