Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selection
Phage display technologies are powerful tools for selecting binding ligands against purified molecular targets, live cells, and organ vasculature. However, the selection of natural ligands using phage display has been limited because of significant problems associated with the display of complex cDN...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2004-06-01
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| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/04366RR03 |
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| _version_ | 1850151881551118336 |
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| author | Peggy Ho Faix Michael A. Burg Michelle Gonzales Edward P. Ravey Andrew Baird David Larocca |
| author_facet | Peggy Ho Faix Michael A. Burg Michelle Gonzales Edward P. Ravey Andrew Baird David Larocca |
| author_sort | Peggy Ho Faix |
| collection | DOAJ |
| description | Phage display technologies are powerful tools for selecting binding ligands against purified molecular targets, live cells, and organ vasculature. However, the selection of natural ligands using phage display has been limited because of significant problems associated with the display of complex cDNA repertoires. Here we describe the use of cDNA fragmentation and open reading frame (ORF) selection to display a human placental cDNA library on the pIII coat protein of filamentous phage. The library was enriched for ORFs by selecting cDNA-β-lactamase fusion proteins on ampicillin, resulting in a cDNA population having 97% ORFs. The ORF-selected cDNAs were fused to pIII in the phagemid vector, pUCMG4CT-198, and the library was rescued with a pIII-deleted helper phage for multivalent display. The resulting phagemid particle library consisted of 87% ORFs, compared to only 6% ORFs when prepared without ORF selection. Western blot analysis indicated cDNA-pIII fusion protein expression in eight out of nine ORF clones tested, and seven of the ORF encoded peptides were displayed multivalently. The high level of cDNA expression obtained by ORF selection suggests that ORF-enriched phage cDNA libraries prepared by these methods will be useful as functional genomics tools for identifying natural ligands from various source tissues. |
| format | Article |
| id | doaj-art-429f19afa5fb4bcfba85ab356418dac3 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2004-06-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-429f19afa5fb4bcfba85ab356418dac32025-08-20T02:26:07ZengTaylor & Francis GroupBioTechniques0736-62051940-98182004-06-013661018102910.2144/04366RR03Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selectionPeggy Ho Faix0Michael A. Burg1Michelle Gonzales2Edward P. Ravey3Andrew Baird4David Larocca51Selective Genetics, Inc., San Diego, CA, USA1Selective Genetics, Inc., San Diego, CA, USA1Selective Genetics, Inc., San Diego, CA, USA1Selective Genetics, Inc., San Diego, CA, USA2King's College London, London, UK1Selective Genetics, Inc., San Diego, CA, USAPhage display technologies are powerful tools for selecting binding ligands against purified molecular targets, live cells, and organ vasculature. However, the selection of natural ligands using phage display has been limited because of significant problems associated with the display of complex cDNA repertoires. Here we describe the use of cDNA fragmentation and open reading frame (ORF) selection to display a human placental cDNA library on the pIII coat protein of filamentous phage. The library was enriched for ORFs by selecting cDNA-β-lactamase fusion proteins on ampicillin, resulting in a cDNA population having 97% ORFs. The ORF-selected cDNAs were fused to pIII in the phagemid vector, pUCMG4CT-198, and the library was rescued with a pIII-deleted helper phage for multivalent display. The resulting phagemid particle library consisted of 87% ORFs, compared to only 6% ORFs when prepared without ORF selection. Western blot analysis indicated cDNA-pIII fusion protein expression in eight out of nine ORF clones tested, and seven of the ORF encoded peptides were displayed multivalently. The high level of cDNA expression obtained by ORF selection suggests that ORF-enriched phage cDNA libraries prepared by these methods will be useful as functional genomics tools for identifying natural ligands from various source tissues.https://www.future-science.com/doi/10.2144/04366RR03 |
| spellingShingle | Peggy Ho Faix Michael A. Burg Michelle Gonzales Edward P. Ravey Andrew Baird David Larocca Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selection BioTechniques |
| title | Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selection |
| title_full | Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selection |
| title_fullStr | Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selection |
| title_full_unstemmed | Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selection |
| title_short | Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selection |
| title_sort | phage display of cdna libraries enrichment of cdna expression using open reading frame selection |
| url | https://www.future-science.com/doi/10.2144/04366RR03 |
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