Tài liệu Báo cáo khoa học: Phage-display as a tool for quantifying protein stability determinants

MINIREVIEW

Phage-display as a tool for quantifying protein stability

determinants

Joanne D. Kotz1

, Christopher J. Bond2 and Andrea G. Cochran1

1

Department of Protein Engineering and 2

Medicinal Chemistry, Genentech, Inc., South San Francisco, CA, USA

To address questions of protein stability, researchers have

increasingly turned to combinatorial approaches that permit

the rapid analysis of libraries of protein variants. Phage￾display has proved to be a powerful tool for analyzing protein

stability due to the large library size and the robustness of the

phage particle to a variety of denaturing conditions.With the

B1 domain of protein G (GB1) and a camelid heavy chain

antibody as model systems, we are using phage-display lib￾raries to experimentally address questions that have generally

been addressed in silico, either through computational stud￾ies or statistical analysis of known protein structures. One

effort has focused on identifying novel solutions to repacking

the hydrophobic core of GB1, while maintaining stability

comparable to the wild type protein. In a second study,

a small set of substitutions in complimentarity-determining

region 3 was found to stabilize the framework of the camelid

antibody. Another major focus has been to obtain quanti￾tative data on b-sheet stability determinants. We have suc￾cessfully adapted a phage-display method for quantitating

affinities of protein variants (shotgun alanine scanning) to

analysis of GB1 stability. Using this method, we have ana￾lyzed the energetic contributions of cross-strand side chain–

side chain interactions. Finally, we discuss parameters to

consider in using phage-display to discriminate subtle sta￾bility differences among fully folded variants. Overall, this

method provides a fast approach for quantitatively addres￾sing biophysical questions.

Keywords: beta sheet; hydrophobic core; phage-display;

protein G; protein stability.

Introduction

Understanding determinants of protein stability is critical

both for predicting the tertiary structure of a protein from

an amino acid sequence, as well as for protein design.

Rather than characterizing individual proteins with single

mutations, or defined combinations of mutations, research￾ers have increasingly been using selection and screening

methods to investigate protein stability. In comparison to

the labor-intensive process of generating and characterizing

individual mutant proteins, these combinatorial approaches

offer the important advantage of simultaneously generating

libraries of protein variants, thus allowing a much larger

number of mutations to be investigated. However, inter￾preting the results fromcombinatorial experiments is not as

straightforward as characterizing individual proteins. Con￾sequently, results must be carefully assessed in light of the

library design and selection pressure applied. Each screening

or selection method, a number of which are discussed in this

review series, will have inherent advantages and limitations

that should be considered in addressing specific questions

of protein structure.

Phage-display is one selection technique that has been

successfully applied to investigating protein stability [1,2]. In

adapting phage-display from the more common selection

for binding affinity, investigators have focused on mutating

residues affecting protein stability, but not directly involved

in ligand binding (Fig. 1). Proteins are selected that retain

binding capacity, with the implicit assumption that a

properly folded protein is required for an intact binding

interface [3,4].

As a protein mutagenesis strategy, phage-display offers

a number of important advantages. The technology for

generating large libraries (
1010 members) has been well

developed [5], permitting the simultaneous characterization

of a relatively large number of mutants. In addition, the

high in vitro stability of the phage particle [6] permits the

use of a wide range of selection conditions. For example,

investigators have used high temperature [7–9] and denat￾urants [8,9] to increase selective pressure. Varying the

stringency of selection conditions by these methods allows

greater flexibility in experimental design and is particularly

relevant to questions of protein stability.

One limitation of the above approach is the requirement

for a known binding partner with a binding interface that

is unaffected by the mutations introduced. A number of

researchers have developed strategies for circumventing this

coupling of protein stability and function, relying on the

greater susceptibility to proteolysis of unfolded proteins. An

Correspondence to A. G. Cochran, Department of Protein

Engineering, Genentech, Inc., 1 DNA Way, South San Francisco,

CA, 94080, USA. Fax: + 1 650 225 3734, Tel.: + 1 650 225 5943,

E-mail: [email protected]

Abbreviations: CDR3, complimentarity-determining region 3; GB1,

B1 domain of protein G; scFv, single chain variable fragment;

VH, variable heavy chain.

(Received 5 January 2004, revised 18 February 2004,

accepted 5 March 2004)

Eur. J. Biochem. 271, 1623–1629 (2004)
FEBS 2004 doi:10.1111/j.1432-1033.2004.04076.x