Change search
ReferencesLink to record
Permanent link

Direct link
Microbead display of proteins by cell-free expression of anchored DNA
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0001-8993-048X
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0003-4214-6991
2003 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 106, no 1, 1-13 p.Article in journal (Refereed) Published
Abstract [en]

Gene expression technologies where nucleic acid sequences remain physically linked to their corresponding gene products are important tools for selection and identification of rare variants in large protein libraries. Here, we describe a gene expression system, which combines the potential of bead-based suspension array technology (SAT) with gene expression and clonal identification. Using streptavidin-coated polystyrene micrometer-sized beads as solid supports for anchored PCR products, we have investigated conditions for cell-free expression and bioaffinity technology to provide clonal co-anchoring of corresponding gene products. Experiments showed that coupled transcription and translation of PCR product expression cassettes resulted in display of affinity-anchored proteins whose binding characteristics could be analyzed via direct and selective interaction with a fluorescently labeled target protein. Interestingly, experiments performed with differently biotinylated PCR products showed that the efficiency of display was dependent on the directionality of the expression cassette relative to the bead surface. In spiked systems, using small immunoglobulin binding proteins as models, we demonstrate efficient flow cytometric sorting of beads corresponding to the target interacting clones, verified by post-sorting analysis and clonal identification at DNA level. The use of this technology, including alternative for-mats, for different proteomics applications is discussed.

Place, publisher, year, edition, pages
2003. Vol. 106, no 1, 1-13 p.
Keyword [en]
cell-free expression, microbeads, selection, sorting, flow cytometry, immobilization, affinity, enrichment, library, in-vitro selection, surface display, phage-display, evolution, technology, affinity, library, immobilization, purification, domain
URN: urn:nbn:se:kth:diva-23006DOI: 10.1016/j.jbiotec.2003.09.002ISI: 000187026900001OAI: diva2:341704
QC 20100525Available from: 2010-08-10 Created: 2010-08-10Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full text

Search in DiVA

By author/editor
Uhlén, MathiasNygren, Per-Åke
By organisation
In the same journal
Journal of Biotechnology

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 20 hits
ReferencesLink to record
Permanent link

Direct link