A visible phagemid system for the estimation of Cre-mediated recombination efficiency

Kwon, Myung-Hee; Lee, Myung‐Shin; Hong, Seong Uk; Kim, Kyongmin; Shin, Ho‐Joon; Park, Sun; Lee, Chi-Hyung; Kim, Hyung-Il

doi:10.1016/s0022-1759(03)00261-8

Cited by 6 publications

(4 citation statements)

References 25 publications

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“…To examine intramolecular recombination, the construction of pFB2 was started by introducing the lacZ 0 gene into the 3 0 end of loxP wt of pRGB [9]. The lacZ 0 gene was amplified from pGEM-3Zf (+) using GalF-1 primer (5 0 -GAC TAG TAA TTC ACT GGC CGT CGT TTT AC) and GalF-2 primer (5 0 -GCA CGC GTT TAT CTC CAT TCG CCA TTC-3 0 ).…”

Section: Methodsmentioning

confidence: 99%

Analysis of spacer regions derived from intramolecular recombination between heterologous loxP sites

Jung¹,

Park²,

Lee³

et al. 2007

Biochemical and Biophysical Research Communications

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Section: Methodsmentioning

confidence: 99%

Analysis of spacer regions derived from intramolecular recombination between heterologous loxP sites

Jung¹,

Park²,

Lee³

et al. 2007

Biochemical and Biophysical Research Communications

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“…DNA encoding the 2C4 V H and V L was synthesized using specific primers and overlap PCR. Then AH_scFv15 and AH_scFv21 were synthesized via overlap PCR using (SG 4 ) 3 (15 aa) and LoxP511 (21 aa; used in the Cre-LoxP system for recombination; aa sequence: SGGSTITSYNVYYTKLSSSGT) 3,16 as the linker sequences, respectively. The PCR products were electrophoresed using 1.2% agarose gels and then subcloned into the double-digested pET-32a(+) vector to construct the expression plasmids AH_scFv15_P32 and AH_scFv21_P32.…”

Section: Methodsmentioning

confidence: 99%

“…Large phage libraries require repeated cycles of cloning, which is tedious and a waste of time. Therefore, we hypothesized that aspects of the Cre/LoxP recombinant system, 3 in which DNA fragments located between LoxP and LoxP511 sites from two different vectors recombine to generate exponential numbers of new gene sequences, might be used to construct large phage libraries.…”

Section: Introductionmentioning

confidence: 99%

A Single-Chain Antibody Using LoxP511 as the Linker Enables Large-Content Phage Library Construction via Cre/LoxP Recombination

Zhang

Wang

Lv³

et al. 2014

SLAS Discovery

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To obtain natural or "me-better" antibodies (e.g., affinity-maturated antibodies), phage display libraries are widely used. However, the likelihood of obtaining satisfactory antibodies depends on the library content. Here, we used computeraided design to model the use of the LoxP511 site as a linker between the heavy and light variable domains of an antibody for construction of a large single-chain fragment (scFv) antibody phage library by using the Cre/LoxP recombinant system. Then, we constructed two novel scFvs based on 2C4, namely, AH_scFv15 (15 amino acid [aa] linker; common [SG 4 ] 3 sequence) and AH_scFv21 (21-aa linker; LoxP511 sequence), to verify the use of the LoxP511 site as a linker. Our results indicate that LoxP511 could be used effectively for the construction of a large (e.g., 5 × 10 12 ) phage display library of scFv antibodies from which it was possible to isolate an antibody with the same epitope as 2C4 but with higher affinity.

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“…A higher initial antibody library size significantly increases the selectivity of high-affinity antigen-specific antibodies [54][55][56] . The human scFv library pRGA-scFv was provided by Prof. Myung-Hee Kwon (Department of Microbiology, Ajou University School of Medicine) 57 . The scFv gene fragments were inserted between the Sfi I and Not I restriction sites of a modified pCANTAB 5E phagemid vector.…”

Section: Subcloning For Yeast Surface Display the Splcv V1 Fragment mentioning

confidence: 99%

Development of novel detection system for sweet potato leaf curl virus using recombinant scFv

Cho

Kil

Cho

et al. 2020

Sci Rep

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Sweet potato leaf curl virus (SpLcV) causes yield losses in sweet potato cultivation. Diagnostic techniques such as serological detection have been developed because these plant viruses are difficult to treat. Serological assays have been used extensively with recombinant antibodies such as whole immunoglobulin or single-chain variable fragments (scfv). An scfv consists of variable heavy (V H) and variable light (V L) chains joined with a short, flexible peptide linker. An scFv can serve as a diagnostic application using various combinations of variable chains. Two SPLCV-specific scFv clones, F7 and G7, were screened by bio-panning process with a yeast cell which expressed coat protein (cp) of SpLcV. the scfv genes were subcloned and expressed in Escherichia coli. The binding affinity and characteristics of the expressed proteins were confirmed by enzyme-linked immunosorbent assay using SPLCV-infected plant leaves. Virus-specific scFv selection by a combination of yeast-surface display and scFv-phage display can be applied to detection of any virus.

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A visible phagemid system for the estimation of Cre-mediated recombination efficiency

Cited by 6 publications

References 25 publications

Analysis of spacer regions derived from intramolecular recombination between heterologous loxP sites

Analysis of spacer regions derived from intramolecular recombination between heterologous loxP sites

A Single-Chain Antibody Using LoxP511 as the Linker Enables Large-Content Phage Library Construction via Cre/LoxP Recombination

Development of novel detection system for sweet potato leaf curl virus using recombinant scFv

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