Microbead display by in vitro compartmentalisation: selection for binding using flow cytometry

Sepp, Armin; Tawfik, Dan S.; Griffiths, Andrew D.

doi:10.1016/s0014-5793(02)03740-7

Cited by 102 publications

(78 citation statements)

References 29 publications

(35 reference statements)

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“…Beads were sorted and individual beads were used as templates for conventional PCR by using the same primers used for BEAMing. Because each bead contains thousands of bound template molecules, single beads were expected to generate robust PCR products (23), and this was confirmed experimentally. These PCR products then were subjected to sequenc- The data in A and B were generated by including 10 and 1 g of human genomic DNA, respectively, in the microemulsions, querying the MID42 locus.…”

Section: Characteristics Of Microemulsionsmentioning

confidence: 79%

“…Beads generated from random fragments of whole genomes (26) rather than from individual genes as described above could be used to identify gene segments that bind to specific DNA-binding proteins (27). If the beads made by BEAMing were used in compartmentalized in vitro transcription-translation reactions, variant proteins would be bound to beads containing the corresponding variant DNA sequences (23), which could allow facile flow-cytometric evaluation of rare mutations by using antibodies that distinguish between wild-type and mutant gene products (28).…”

Section: Discussionmentioning

confidence: 99%

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Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations

Dressman

Yan

Traverso

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

735

479

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Many areas of biomedical research depend on the analysis of uncommon variations in individual genes or transcripts. Here we describe a method that can quantify such variation at a scale and ease heretofore unattainable. Each DNA molecule in a collection of such molecules is converted into a single magnetic particle to which thousands of copies of DNA identical in sequence to the original are bound. This population of beads then corresponds to a one-to-one representation of the starting DNA molecules. Variation within the original population of DNA molecules can then be simply assessed by counting fluorescently labeled particles via flow cytometry. This approach is called BEAMing on the basis of four of its principal components (beads, emulsion, amplification, and magnetics). Millions of individual DNA molecules can be assessed in this fashion with standard laboratory equipment. Moreover, specific variants can be isolated by flow sorting and used for further experimentation. BEAMing can be used for the identification and quantification of rare mutations as well as to study variations in gene sequences or transcripts in specific populations or tissues.

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Section: Characteristics Of Microemulsionsmentioning

confidence: 79%

Section: Discussionmentioning

confidence: 99%

Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations

Dressman

Yan

Traverso

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

735

479

View full text Add to dashboard Cite

show abstract

“…Although phage display has been available for more than two decades (23,32,43), it is still the in vitro selection method of choice for the majority of laboratories working in the field of combinatorial protein engineering. Nevertheless, today there are a number of more or less established competing technologies, including, among others, ribosome display (13,21,57), other cell-free selection systems (3,18,28,29,41), protein complementation assays (16), and various formats of cell display (4, 7-9, 14, 33, 56), all with their respective advantages and disadvantages. We have previously described a system for display of proteins and peptides on the cell surface of the gram-positive bacterium Staphylococcus carnosus (17, 36-39, 45, 51-55).…”

mentioning

confidence: 99%

Evaluation of Staphylococcal Cell Surface Display and Flow Cytometry for Postselectional Characterization of Affinity Proteins in Combinatorial Protein Engineering Applications

Löfblom

Sandberg

Wernérus

et al. 2007

Appl Environ Microbiol

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For efficient generation of high-affinity protein-based binding molecules, fast and reliable downstream characterization platforms are needed. In this work, we have explored the use of staphylococcal cell surface display together with flow cytometry for affinity characterization of candidate affibody molecules directly on the cell surface. A model system comprising three closely related affibody molecules with different affinities for immunoglobulin G and an albumin binding domain with affinity for human serum albumin was used to investigate advantages and differences compared to biosensor technology in a side-by-side manner. Equilibrium dissociation constant (K D ) determinations as well as dissociation rate analysis were performed using both methods, and the results show that the on-cell determinations give both K D and dissociation rate values in a very fast and reproducible manner and that the relative affinities are very similar to the biosensor results. Interestingly, the results also show that there are differences between the absolute affinities determined with the two different technologies, and possible explanations for this are discussed. This work demonstrates the advantages of cell surface display for directed evolution of affinity proteins in terms of fast postselectional, on-cell characterization of candidate clones without the need for subcloning and subsequent protein expression and purification but also demonstrates that it is important to be aware that absolute affinities determined using different methods often vary substantially and that such comparisons therefore could be difficult.

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“…Existing DNA-based in vitro selection systems are based on emulsion encapsulation of DNA and are limited to libraries of 10 9 to 10 10 per ml (20,21). Here we describe an alternate DNA-based approach that does not require any compartmentalization of the library-encoding nucleic acid.…”

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confidence: 99%

CIS display: In vitro selection of peptides from libraries of protein–DNA complexes

Odegrip¹,

Coomber²,

Eldridge³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

131

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We describe the development of an in vitro library selection system (CIS display) that exploits the ability of a DNA replication initiator protein (RepA) to bind exclusively to the template DNA from which it has been expressed, a property called cis-activity. A diverse peptide library is created by ligation of DNA fragments of random sequence to a DNA fragment that encodes RepA. After in vitro transcription and translation, a pool of protein-DNA complexes is formed where each protein is stably associated with the DNA that encodes it. These complexes are amenable to the affinity selection of ligands to targets of interest. Here we show that RepA is a highly faithful cis-acting DNA-binding protein and demonstrate that libraries encoding >10 12 random 18-mer peptides can be constructed and used to isolate peptides that bind specifically to disparate targets. The use of DNA to encode the displayed peptides offers advantages over in vitro peptide display systems that use mRNA.

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Microbead display by in vitro compartmentalisation: selection for binding using flow cytometry

Cited by 102 publications

References 29 publications

Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations

Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations

Evaluation of Staphylococcal Cell Surface Display and Flow Cytometry for Postselectional Characterization of Affinity Proteins in Combinatorial Protein Engineering Applications

CIS display: In vitro selection of peptides from libraries of protein–DNA complexes

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