Expression screen by enzyme-linked immunofiltration assay designed for high-throughput purification of affinity-tagged proteins

Kéry, Vladimír; Savage, Justin R.; Widjaja, Kartika; Blake, B. Kelly; Conklin, David R.; Ho, Yew-Seng J.; Long, Xinghua; Rechenberg, Moritz von; Zarembinski, Thomas I.; Boniface, J. Jay

doi:10.1016/s0003-2697(03)00116-7

Cited by 5 publications

(4 citation statements)

References 3 publications

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“…The Shewanella strain with the construct containing mtrA was first used to determine whether expression of recombinant protein was inducible with L-arabinose using enzyme-linked immunofiltration assay (ELIFA) (10). Results of ELIFA showed that expression levels of MtrA increased from less than 0.01% to 0.9% of total soluble proteins 14 h after induction with 1 mM L-arabinose, demonstrating that expression of recombinant protein could be regulated in Shewanella cells.…”

mentioning

confidence: 99%

Overexpression of Multi-Heme C-type Cytochromes

et al. 2005

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confidence: 99%

Overexpression of Multi-Heme C-type Cytochromes

et al. 2005

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“…52 This should result in a better enrichment of the scarce high-affinity clones and allow for a more efficient sampling of the large variety in an unselected library. Afterwards, the reduced library diversity can be further depleted through subsequent RISE panning rounds as described in this article.…”

Section: Discussionmentioning

confidence: 99%

“…A more powerful RISE selection could be attained by using greater culture volumes in deep-well plates during the initial panning rounds, possibly in combination with immunofiltration. 52 This should result in a better enrichment of the scarce high-affinity clones and allow for a more efficient sampling of the large variety in an unselected library. Afterwards, the reduced library diversity can be further depleted through subsequent RISE panning rounds as described in this article.…”

Section: Discussionmentioning

confidence: 99%

Rescue and In Situ Selection and Evaluation (RISE): A Method for High-Throughput Panning of Phage Display Libraries

Vanhercke¹,

Ampè²,

Tirry³

et al. 2005

SLAS Discovery

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Phage display has proven to be an invaluable instrument in the search for proteins and peptides with optimized or novel functions. The amplification and selection of phage libraries typically involve several operations and handling large bacterial cultures during each round. Purification of the assembled phage particles after rescue adds to the labor and time demand. The authors therefore devised a method, termed rescue and in situ selection and evaluation (RISE), which combines all steps from rescue to binding in a single microwell. To test this concept, wells were precoated with different antibodies, which allowed newly formed phage particles to be captured directly in situ during overnight rescue. Following 6 washing steps, the retained phages could be easily detected in an enzyme-linked immunosorbent assay (ELISA), thus eliminating the need for purification or concentration of the viral particles. As a consequence, RISE enables a rapid characterization of phagedisplayed proteins. In addition, this method allowed for the selective enrichment of phages displaying a hemagglutinin (HA) epitope tag, spiked in a 10 4 -fold excess of wild-type background. Because the combination of phage rescue, selection, or evaluation in a single microwell is amenable to automation, RISE may boost the high-throughput screening of smaller sized phage display libraries. (Journal of Biomolecular Screening 2005:108-117)

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“…nih.gov/). Subsets of these collections have been transferred into various vectors that enable highly parallel functional screens (Walhout et al 2000a(Walhout et al , 2002Davy et al 2001;Walhout and Vidal 2001;Boulton et al 2002;Li et al 2004) and protein expression/ purification (Huang et al 2003;Kery et al 2003).…”

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confidence: 99%

Concerted Assembly and Cloning of Multiple DNA Segments Using In Vitro Site-Specific Recombination: Functional Analysis of Multi-Segment Expression Clones

Cheo¹,

Titus²,

Byrd³

et al. 2004

Genome Res.

126

103

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The ability to clone and manipulate DNA segments is central to molecular methods that enable expression, screening, and functional characterization of genes, proteins, and regulatory elements. We previously described the development of a novel technology that utilizes in vitro site-specific recombination to provide a robust and flexible platform for high-throughput cloning and transfer of DNA segments. By using an expanded repertoire of recombination sites with unique specificities, we have extended the technology to enable the high-efficiency in vitro assembly and concerted cloning of multiple DNA segments into a vector backbone in a predefined order, orientation, and reading frame. The efficiency and flexibility of this approach enables collections of functional elements to be generated and mixed in a combinatorial fashion for the parallel assembly of numerous multi-segment constructs. The assembled constructs can be further manipulated by directing exchange of defined segments with alternate DNA segments. In this report, we demonstrate feasibility of the technology and application to the generation of fusion proteins, the linkage of promoters to genes, and the assembly of multiple protein domains. The technology has broad implications for cell and protein engineering, the expression of multidomain proteins, and gene function analysis.[Supplemental material is available online at www.genome.org.]The cloning and manipulation of DNA segments, typically encoding functional elements such as promoters, genes, protein domains, or fusion tags, are central to methods of cell engineering, protein production, and gene-function analysis. The large number of available genome sequences now makes it possible to create and apply repositories of defined functional elements to conduct high-throughput, genome-wide analyses. The Gateway Cloning Technology (Hartley et al. 2000) uses in vitro sitespecific recombination to clone and subsequently transfer DNA segments between vector backbones. This approach has been used to generate several large clone collections (Entry Clones), in some cases comprising the entire or nearly entire coding capacity of model genomes as open reading frames (ORFs). These ORFeomes include Caenorhabditis elegans (Walhout et al. 2000b;Reboul et al. 2001Reboul et al. , 2003, Pseudomonas aeruginosa (LaBaer et al. 2004), and Saccharomyces cerevisiae (G. Marsischky, pers. comm.), Arabidopsis (Yamada et al. 2003; also see Atome project http:// genoplante-info.infobiogen.fr/Databases/CT_Nouveaux_Outils/ NO2001054/), human (clones available from several commercial sources), and an incipient collection of Drosophila ORFs (http:// www.fruitfly.org/EST/gateway.shtml). A collection of sequenced, full-length Arabidopsis cDNAs in the Gateway Vector pCMV-SPORT6 will shortly be made available through INRA-Genoscope (Castelli et al. 2004). Repositories of full-length clones, some of which are in the Gateway format, are available for Xenopus (http://xgc.nci.nih.gov/), zebrafish (http://zgc.nci.nih.gov/), as well as many hu...

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Expression screen by enzyme-linked immunofiltration assay designed for high-throughput purification of affinity-tagged proteins

Cited by 5 publications

References 3 publications

Overexpression of Multi-Heme C-type Cytochromes

Overexpression of Multi-Heme C-type Cytochromes

Rescue and In Situ Selection and Evaluation (RISE): A Method for High-Throughput Panning of Phage Display Libraries

Concerted Assembly and Cloning of Multiple DNA Segments Using In Vitro Site-Specific Recombination: Functional Analysis of Multi-Segment Expression Clones

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