A covalent chemical genotype-phenotype linkage for in vitro protein evolution

Stein, Viktor, Sielaff, India, Johnsson, Kai and Hollfelder, Florian (2007) A covalent chemical genotype-phenotype linkage for in vitro protein evolution. ChemBioChem, 8 18: 2191-2194. doi:10.1002/cbic.200700459

Author Stein, Viktor
Sielaff, India
Johnsson, Kai
Hollfelder, Florian
Title A covalent chemical genotype-phenotype linkage for in vitro protein evolution
Journal name ChemBioChem   Check publisher's open access policy
ISSN 1439-4227
Publication date 2007-12-17
Sub-type Article (original research)
DOI 10.1002/cbic.200700459
Volume 8
Issue 18
Start page 2191
End page 2194
Total pages 3
Place of publication Weinheim, Germany
Publisher Wiley - VCH Verlag
Language eng
Subject 0304 Medicinal and Biomolecular Chemistry
0601 Biochemistry and Cell Biology
Formatted abstract
The importance of functional proteins as therapeutics and tools for biological research is increasing. Directed evolution has emerged as the dominant approach for making functional proteins, often surpassing “rational” methods of protein design. The link between genotype and phenotype is the key strategic consideration in directed evolution experiments. In cells, genotype and phenotype are naturally linked; cell-based selection methods are, however, limited by a narrow dynamic range, low transformation efficiency, which generally restricts library sizes to well below 109, and protein toxicity (e.g., ~50% of E. coli proteins are growth-inhibiting or toxic when overexpressed in E. coli). Phage display, arguably the most widely used technique to generate protein binders, marks the boundary between in vivo and in vitro systems. Selections are carried out under nonphysiological conditions, but cloning and protein expression still occur in vivo. In the standard set-up, phage display is additionally limited by cytoplasmic export of proteins to the periplasm. Several in vitro approaches have consequently been developed aiming to by-pass these shortcomings: ribosome display, mRNA display, STABLE, M.Hae.III, CIS/CAD and bead display. Of these, ribosome display is currently the most widely used in vitro display method, but the ternary complex consisting of mRNA, ribosome and protein is noncovalent and requires stabilisation by low temperature and low magnesium ion concentrations. Furthermore, the coding nucleic acid is RNA rather than the chemically more stable DNA.

We report here an alternative method in which proteins are covalently linked to their coding DNA template by a SNAP-tag (i.e., via O6-alkylguanine-DNA alkyltransferase, AGT): linear DNA templates coding for a fusion protein comprised of an AGT mutant and the protein of interest are prepared by PCR by using primers derivatised with BG (O6-benzylguanine), a substrate analogue of AGT (Figure 1). The resulting BG-modified DNA then serves as a template in a coupled in vitro transcription–translation (TS/TL) reaction in water-in-oil emulsion droplets.
© 2007 Wiley-VCH Verlag GmbH& Co.KGaA, Weinheim
Keyword Directed evolution
Genotype-phenotype linkage
In vitro compartmentalization
Q-Index Code C1

Document type: Journal Article
Sub-type: Article (original research)
Collections: Excellence in Research Australia (ERA) - Collection
Institute for Molecular Bioscience - Publications
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Citation counts: TR Web of Science Citation Count  Cited 21 times in Thomson Reuters Web of Science Article | Citations
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Created: Wed, 24 Mar 2010, 09:07:25 EST by Jon Swabey on behalf of Institute for Molecular Bioscience