Microbead display of proteins by cell-free expression of anchored DNA.
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 formats, for different proteomics applications is discussed.
Nord
Olof
O
Department of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, SE-106 91 Stockholm, Sweden.
Uhlén
Mathias
M
Nygren
Per-Ake
PA
D000327:Adsorption; D002474:Cell-Free System; D020099:Coated Materials, Biocompatible / Q000138:chemical synthesis / Q000737:chemistry; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods; D008863:Microspheres; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods*; D040081:Protein Array Analysis / Q000379:methods; D011506:Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods
D020099:Coated Materials, Biocompatible; D029968:Escherichia coli Proteins; D019151:Peptide Library; D011506:Proteins; D004247:DNA