scholarly journals High-throughput retrieval of physical DNA for NGS-identifiable clones in phage display library

2018 ◽  
Author(s):  
Jinsung Noh ◽  
Okju Kim ◽  
Yushin Jung ◽  
Haejun Han ◽  
Jung-Eun Kim ◽  
...  
Keyword(s):  
High Throughput ◽  
Antibody Fragment ◽  
Phage Display Library ◽  
Sequence Information ◽  
Single Chain ◽  
Depth Analysis ◽  
Scfv Library ◽  
Linkage Information ◽  
Antibody Discovery ◽  
Human Hepatocyte Growth Factor

AbstractIn antibody discovery, in-depth analysis of an antibody library and high-throughput retrieval of clones in the library are crucial to identifying and exploiting rare clones with different properties. However, existing methods have several technical limitations such as low process throughput from laborious cloning process and waste of the phenotypic screening capacity from unnecessary repetitive tests on the dominant clones. To overcome the limitations, we developed a new high-throughput platform for the identification and retrieval of clones in the library, TrueRepertoire™. TrueRepertoire™ provides highly accurate sequences of the clones with linkage information between heavy and light chains of the antibody fragment. Additionally, the physical DNA of clones can be retrieved in high throughput based on the sequence information. We validated the high accuracy of the sequences and demonstrated that there is no platform-specific bias. Moreover, the applicability of TrueRepertoire™ was demonstrated by a phage-displayed single-chain variable fragment (scFv) library targeting human hepatocyte growth factor (hHGF) protein.

scholarly journals A High-Throughput Single-Clone Phage Fluorescence Microwell Immunoassay and Laser-Driven Clonal Retrieval System

Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 517
Author(s):  
Seohee Chang ◽  
Soohyun Kim ◽  
Jerome Han ◽  
Suji Ha ◽  
Hyunho Lee ◽  
...  
Keyword(s):  
High Throughput ◽  
Retrieval System ◽  
Low Cost ◽  
Therapeutic Antibodies ◽  
Amino Acid Residues ◽  
Single Chain ◽  
Scfv Library ◽  
Single Clone ◽  
Library Complexity ◽  
Lower Complexity

Phage display is one of the most frequently used platform technologies utilized to screen and select therapeutic antibodies, and has contributed to the development of more than 10 therapeutic antibodies used in the clinic. Despite advantages like efficiency and low cost, it has intrinsic technical limitations, such as the asymmetrical amplification of the library after each round of biopanning, which is regarded as a reason for it yielding a very limited number of antigen binders. In this study, we developed a high-throughput single-clonal screening system comprised of fluorescence immunoassays and a laser-driven clonal DNA retrieval system using microchip technology. Using this system, from a single-chain variable fragment (scFv) library displayed on phages with a complexity of 5.21 × 105 harboring random mutations at five amino acid residues, more than 70,000 clones—corresponding to ~14% of the library complexity—were screened, resulting in 78 antigen-reactive scFv sequences with mutations restricted to the randomized residues. Our results demonstrate that this system can significantly reduce the number of biopanning rounds, or even eliminate the need for this process for libraries with lower complexity, providing an opportunity to obtain more diverse clones from the library.

scholarly journals Functional analysis of the CXXC motif using phage antibodies that cross-react with protein disulphide-isomerase family proteins

2004 ◽  
Vol 382 (1) ◽  
pp. 169-176 ◽  
Author(s):  
Taiji KIMURA ◽  
Ai NISHIDA ◽  
Nobutoshi OHARA ◽  
Daisuke YAMAGISHI ◽  
Tomohisa HORIBE ◽  
...  
Keyword(s):  
Active Sites ◽  
Polyclonal Antibodies ◽  
Antibody Fragment ◽  
Variable Region ◽  
Phage Display Library ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Protein Disulphide Isomerase ◽  
Cxxc Motif ◽  
Phage Antibodies

Polyclonal antibodies that had been raised against particular PDI (protein disulphide-isomerase) family proteins did not cross-react with other PDI family proteins. To evade immune tolerance to the important self-motif Cys-Xaa-Xaa-Cys, which is present in PDI family proteins, we used the phage display library [established by Griffiths, Williams, Hartley, Tomlinson, Waterhouse, Crosby, Kontermann, Jones, Low, Allison et al. (1994) EMBO J. 13, 3245–3260] to isolate successfully the phage antibodies that can cross-react with human and bovine PDIs, human P5, human PDI-related protein and yeast PDI. By measuring the binding of scFv (single-chain antibody fragment of variable region) to synthetic peptides and to mutants of PDI family proteins in a surface plasmon resonance apparatus, we identified clones that recognized sequences containing the CGHC motif or the CGHCK sequence. By using the isolated phage antibodies, we demonstrated for the first time that a lysine residue following the CXXC motif significantly increases the isomerase activities of PDI family proteins. Moreover, we demonstrated that the affinity of isolated scFvs for mutant PDI family proteins is proportional to the isomerase activities of their active sites.

scholarly journals High-Throughput Sequencing of Phage Display Libraries Reveals Parasitic Enrichment of Indel Mutants Caused by Amplification Bias

2021 ◽  
Vol 22 (11) ◽  
pp. 5513
Author(s):  
Sander Plessers ◽  
Vincent Van Deuren ◽  
Rob Lavigne ◽  
Johan Robben
Keyword(s):  
Amino Acid ◽  
Phage Display ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Target Selection ◽  
Acid Modification ◽  
Phage Display Technology ◽  
Amplification Bias ◽  
Large Deletions ◽  
Depth Analysis

The combination of phage display technology with high-throughput sequencing enables in-depth analysis of library diversity and selection-driven dynamics. We applied short-read sequencing of the mutagenized region on focused display libraries of two homologous nucleic acid modification eraser proteins—AlkB and FTO—biopanned against methylated DNA. This revealed enriched genotypes with small indels and concomitant doubtful amino acid motifs within the FTO library. Nanopore sequencing of the entire display vector showed additional enrichment of large deletions overlooked by region-specific sequencing, and further impacted the interpretation of the obtained amino acid motifs. We could attribute enrichment of these corrupted clones to amplification bias due to arduous FTO display slowing down host cell growth as well as phage production. This amplification bias appeared to be stronger than affinity-based target selection. Recommendations are provided for proper sequence analysis of phage display data, which can improve motive discovery in libraries of proteins that are difficult to display.

Cross-clade HIV-1 neutralization by an antibody fragment from a lupus phage display library

AIDS ◽  
2004 ◽  
Vol 18 (2) ◽  
pp. 329-331 ◽  
Author(s):  
Sangeeta Karle ◽  
Stephanie Planque ◽  
Yasuhiro Nishiyama ◽  
Hiroaki Taguchi ◽  
Yong-Xin Zhou ◽  
...  
Keyword(s):  
Phage Display ◽  
Antibody Fragment ◽  
Phage Display Library ◽  
Hiv 1

Injectable formulations for an intravitreal sustained-release application of a novel single-chain VEGF antibody fragment

2015 ◽  
Vol 95 ◽  
pp. 250-260 ◽  
Author(s):  
Lutz R. Asmus ◽  
John P.A. Grimshaw ◽  
Philipp Richle ◽  
Barbara Eicher ◽  
David M. Urech ◽  
...  
Keyword(s):  
Sustained Release ◽  
Antibody Fragment ◽  
Single Chain

scholarly journals Characterization of LL25X, a Newly Isolated Anti-SIV Monoclonal Antibody, to Determine Binding Specificity

Elements ◽  
2017 ◽  
Vol 13 (1) ◽  
Author(s):  
Zackary Tajin Park
Keyword(s):  
Phage Display ◽  
Mammalian Cells ◽  
Expression Vector ◽  
Restriction Enzymes ◽  
Phage Display Library ◽  
Single Chain ◽  
Phage Display Technology ◽  
Mammalian Expression ◽  
Mammalian Expression Vector ◽  
Single Chain Fv

A phage display library was previously constructed from an SIV-infected rhesus macaque. Several single chain Fv (scFv), including SU24, SU343 and LL25X, were selected using phage display technology. Sequences corresponding to SU24, SU343 and LL25X were optimized for expression in a mammalian system and commercially synthesized. SU24 and SU343 had previously been cloned into a mammalian expression vector. In this study, we aimed to characterize the specificity of SU24, SU343, and LL25X.. The codon-optimized version of the scFv LL25X gene sequence was cloned into a mammalian expression vector (pCEP4).  LL25X DNA was amplified by PCR, and the PCR product and mammalian expression vector were both digested with KpnI/SapI restriction enzymes. Digested fragments were purified, and the fragments were ligated using T4DNA ligase. E. coli cells were transformed with the ligation reaction. Single colonies were selected on LB agar plates containing the selective antibiotic (ampicillin). Positive colonies were identified after DNA mini-preparation and test-digestion with KpnI and SapI. Sanger sequencing confirmed cloning results and DNA sequence accuracy. Following transfection of mammalian cells (293T), LL25X-Fc cells, and purifying our protein, the binding of LL25X-Fc to the SIV gp140 envelope protein was confirmed via ELISA and Western Blotting.

Sign in / Sign up

Export Citation Format

Share Document