In vitro display technologies: novel developments and applications

doi:10.1016/S0958-1669(00)00234-2

Current Opinion in Biotechnology

Volume 12, Issue 4, 1 August 2001, Pages 400-405

https://doi.org/10.1016/S0958-1669(00)00234-2 Get rights and content

Abstract

In vitro display techniques are powerful tools to select polypeptide binders against various target molecules. Novel applications include maturation of protein affinity and stability, selection for enzymatic activity, and the display of cDNA and random polypeptide libraries. Taken together, these display techniques have great potential for biotechnological, medical and proteomic applications.

Introduction

The growing interest of the research community and pharmaceutical companies in protein–protein interactions has led to an increasing demand for sophisticated methods for the rapid identification, characterization, and potential improvement of interaction partners. The most popular of these methods, namely the yeast two-hybrid system [1] and phage display [2] (see the article by Sidhu and Pelletier in this issue pp 340–347), are limited by the involvement of living cells in the process of library generation or screening. This is not the case for in vitro selection technologies. In these techniques the number of molecules that can be handled is not limited by cellular transformation efficiencies; thus, very large libraries of up to 10¹⁴ members can be built. This feature also facilitates directed evolution experiments, in which rounds of randomization and selection alternate, as transformation can be avoided between rounds. Furthermore, special reagents such as the reducing agent dithiothreitol or detergents can be added to select binders under conditions chosen by the experimentor.

Two main groups of in vitro selection technologies can be distinguished. The first group imitates the compartmentalization of living cells by performing translation and selection within a water-in-oil emulsion 3., 4., 5.; this method was recently summarized in an excellent review [6]. This compartmentalization ensures the coupling of genotype and phenotype — a prerequisite for any selection method. The second group, the in vitro display technologies, makes use of a physical link between messenger RNA (mRNA) and nascent polypeptide during translation to couple genotype and phenotype. The most popular in vitro display technologies are ribosome display and mRNA display (reviewed in 7., 8., 9., 10., 11., 12.). This review focuses on the recent advances in the field of in vitro display methods and discusses the potential of these technologies for future applications in basic and applied research.

Section snippets

In vitro display technologies

Ribosome display (Fig. 1a) was first developed by Mattheakis et al. [13] for the selection of peptides and further improved for the selection of folded proteins by Hanes and Plückthun [14] and He and Taussig [15]. This method relies on non-covalent ternary complexes of mRNA, ribosome and nascent polypeptide, ensuring the coupling of genotype and phenotype. A fusion protein is constructed in which the the domain of interest is fused to a C-terminal tether, such that this domain can fold while

Improved library quality by preselection

The success of selection experiments depends to a large extent on the quality of the library. Although the theoretical size of a library is virtually unlimited, the transformation efficiency for yeast (10⁷–10⁸ cells/μg DNA) and for Escherichia coli (10⁹–10¹⁰ cells/μg DNA) limits the achievable library size. By contrast, in vitro display technologies can handle libraries with up to 10¹⁴ members, depending only on the scale of the in vitro translation used. A common way to generate libraries

Directed evolution of proteins

Natural evolution has efficiently adapted proteins to their tasks under given environmental conditions. Nevertheless, the technological or medical application of proteins often places different demands on them; thus, their performance needs to be optimized. Using the Darwinian principle, evolution of polypeptides can now be conducted in the test tube: a pool of molecules (library) is subjected to alternating rounds of selection and randomization. If the randomization is carried out on the whole

Maturation of protein affinity

Protein affinity maturation with molecular evolution technologies is an important step in producing selective and high-affinity binding proteins for applications in biotechnology and medicine [23]. Both ribosome display and mRNA display have allowed selection for binding proteins to a wide variety of targets, such as small compounds 22••., 28••., peptides [20], whole proteins 21•., 23., 24., 34••. or even a specific DNA structure [35••].

Recent work now also demonstrates that in vitro display

Maturation of protein stability

A common requirement for most biotechnological and medical applications of proteins is that they possess an intrinsic high stability against denaturation. Stability engineering is still a difficult task 37., 38., 39., 40.. Evolutionary methods to perform stability engineering have shown promise, especially methods that employ phage display (reviewed in [41]). In a model system using antibody scFv fragments, Jermutus et al. [22••] have shown that ribosome display may be a valuable tool for in

Selection for enzymatic activity

It has been stated several times 6., 41., 45., 46. that a combination of directed evolution and the use of display technologies provides a powerful strategy to evolve improved biocatalysts. Although it is known that enzymes can be functionally displayed on the ribosome [47], ribosome display had so far not been used to select for enzymatic activity. In this technique the genetic information (i.e. the mRNA) is not covalently attached to the protein. Thus, the mRNA can be simply eluted, even in

Display of cDNA products

Phage display and two-hybrid systems are well-established methods to screen or select cDNA libraries for binders 1., 2., 48., 49.. Recently, two groups investigated the potential of in vitro display techniques for the display of cDNA products. Bieberich et al. [50] reported the specific isolation of the cDNA of sialyltransferase II by functional binding of the encoded enzyme to its substrate, ganglioside GD3, in a single-tube coupled ribosome display system. It remains unclear, however, if

Conclusions

In vitro display technologies, namely ribosome and mRNA display, prove to be valuable tools for many applications other than merely selecting polypeptide binders. They have great potential for directed evolution of protein stability and affinity, the generation of high-quality libraries by in vitro preselection, the selection of enzymatic activities, and the display of cDNA and random-peptide libraries. In addition, these technologies have several features that should make them amenable to

Acknowledgements

We thank Markus Kurz and Philip W Hammond for sharing unpublished results and Stephen F Marino and Christiane Schaffitzel for critically reading the manuscript and helpful suggestions.

References and recommended reading

Papers of particular interest, published within the annual period of review,have been highlighted as:

•of special interest
••of outstanding interest

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ReviewIn vitro display technologies: novel developments and applications

Abstract

Introduction

Section snippets

In vitro display technologies

Improved library quality by preselection

Directed evolution of proteins

Maturation of protein affinity

Maturation of protein stability

Selection for enzymatic activity

Display of cDNA products

Conclusions

Acknowledgements

References and recommended reading

Curr Opin Biotechnol

FEBS Lett

Curr Opin Biotechnol

Methods Enzymol

Methods Enzymol

Curr Opin Chem Biol

J Immunol Methods

Curr Opin Biotechnol

FEBS Lett

J Immunol Methods

J Immunol Methods

J Mol Biol

J Mol Biol

J Mol Biol

Curr Opin Chem Biol

J Biol Chem

J Mol Biol

J Mol Biol

Biochim Biophys Acta

Curr Opin Struct Biol

Trends Biotechnol

FEMS Microbiol Lett

Curr Opin Biotechnol

Curr Opin Biotechnol

J Biol Chem

Review
In vitro display technologies: novel developments and applications