Selection of an anti-IGF-1 Fab from a Fab phage library created by mutagenesis of multiple CDR loops

Three single chain variable fragment (scFv) antibodies against 2,4-dichlophenoxyacetic acid (2,4-D) herbicide were produced by the Griffin1.library. The selection of the scFv from the phage library was carried out by 2,4-D-protein coated tubes with different levels of hapten substitution in the conjugate. The scFv phage clones were isolated within the five round library panning and the antibodies were expressed in Escherichia coli HB2151. The recombinant products were purified by metal affinity chromatography yielding 200 g of pure scFv per 1 liter of bacterial culture. The antibody fragments provided steep curves in conventional indirect ELISA having the IC<sub>50</sub> values from 10.2 to 14.5 ng/ml established for 2,4-D standard. Interestingly enough, the recombinant ScFv E1 antibody exhibited 68% cross-reactivity with 2,4-dichlorphenol (2,4-D = 100%), and 38.0% with methylchlorophenoxyacetic acid (MCPA) whereas reaction with other phenoxyacetic compounds was low. Similar characteristics were obtained for other two recombinant products. Low stability for the isolated scFv antibodies was found in storage buffer even in the presence of stabilizers and protease inhibitors. Factors influencing stability of the recombinant antibodies are discussed.

Download Full-text

Selection of ganglioside GM1-binding peptides by using a phage library

FEBS Letters ◽

10.1016/s0014-5793(99)00962-x ◽

1999 ◽

Vol 456 (2) ◽

pp. 253-256 ◽

Cited By ~ 45

Author(s):

Teruhiko Matsubara ◽

Dai Ishikawa ◽

Takao Taki ◽

Yoshio Okahata ◽

Toshinori Sato

Keyword(s):

Phage Library ◽

Ganglioside Gm1 ◽

Binding Peptides ◽

Selection Of

Download Full-text

Selection of Muscle-Binding Peptides from Context-Specific Peptide-Presenting Phage Libraries for Adenoviral Vector Targeting

Journal of Virology ◽

10.1128/jvi.79.21.13667-13672.2005 ◽

2005 ◽

Vol 79 (21) ◽

pp. 13667-13672 ◽

Cited By ~ 20

Author(s):

Debadyuti Ghosh ◽

Michael A. Barry

Keyword(s):

Adenoviral Vector ◽

Viral Vectors ◽

C2c12 Cells ◽

Phage Library ◽

Structural Context ◽

Fiber Protein ◽

Genetically Engineer ◽

Context Specific ◽

Specific Peptide ◽

Selection Of

ABSTRACT Production of cell-targeting vectors in part involves the addition of new targeting ligands to the vector to mediate binding to the cells of interest. For viral vectors, the ideal approach is to genetically engineer new ligands into the capsid proteins of the virus to generate a single agent to mediate therapy. Although this is ideal, this insertion of an exogenous ligand from one structural context into the differing structural context of a capsid protein can ablate the function of the ligand or disrupt viral assembly and function. To address this context problem for adenoviral vectors, we have engineered a “context-specific” peptide-presenting phage library. We have displayed a 12-amino-acid (12-mer) random peptide library between the H and I sheets of the fiber protein of adenovirus type 5 on the pIII protein of fd bacteriophage. This library was used for peptide selection against C2C12 mouse skeletal muscle cells. Five rounds of selection combined with four rounds of clearing on nontarget cells selected one primary peptide designated 12.51, which bound target C2C12 cells approximately 100-fold better than the positive control RGD peptide. Translation of 12.51 back into the fiber protein produced a ligand-modified adenoviral vector that mediated 14-fold-better transduction of target C2C12 cells. These data suggest context-specific peptide-presenting libraries may allow selection of compatible peptide ligands for functional translation into viral vectors for retargeting.

Download Full-text

Selection of a Single Chain Variable Fragment Antibody (scFv) against Subtilisin BRC and its Interaction with Subtilisin BRC

Current Biotechnology ◽

10.2174/2211550108666190417113342 ◽

2019 ◽

Vol 8 (1) ◽

pp. 24-31

Author(s):

Chol-Jin Kim ◽

Sunll Choe ◽

Kum-Chol Ri ◽

Chol-Ho Kim ◽

Hyon-Gwang Li ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Phage Display ◽

Affinity Purification ◽

Phage Library ◽

Amino Acid Residues ◽

Single Chain Variable Fragment ◽

Single Chain ◽

E Coli ◽

Selection Of

Background: The focus of this study was the selection of a single chain variable fragment antibody (scFv) against subtilisin BRC, a fibrinolytic enzyme using phage display, and to characterize the interaction between the antibody and subtilisin BRC. Methods: The subtilisin BRC-specific phage clones were selected using Griffin.1 scFv phage library and sequenced. The gene of subtilisin BRC-specific scFv (scFv-BRC) from selected phage clone was expressed in E. coli and scFv-BRC was characterized. Molecular modeling of the three-dimensional (3D) structures of scFv-BRC was performed using MODELLER 9.19 modeling software and assessed by PROCHEK. Molecular docking of subtilisin BRC with scFv-BRC was carried out using PATCHDOCK. Results: The size of scFv-BRC gene is 635bp and it consists of 54bp of heavy chain region (VH), 336bp of light chain region (VL), 45bp of a linker. scFv-BRC was actively expressed by E. coli expression vector pET28a-scFv in E. coli BL21 (DE3), and the amount of expressed scFv-BRC was about 50 mg/L. Its molecular weight is ~26kDa. The CDR domain of scFv-BRC consists of 6 amino acids in CDR L1, 3 amino acids in CDR L2 and 9 amino acids in CDR L3. Docking results of subtilisin BRC and scFv-BRC showed global energy of - 56.29 kJ/mol. Furthermore, the results showed that amino acid residues in subtilisin BRC for binding with scFv-BRC are Tyr6, Ser182, Ser204, and Gln206. Conclusion: scFv against subtilisin BRC selected using phage display showed relatively strong binding energy with subtilisin BRC. The amino acid residues in subtilisin BRC for binding with scFv-BRC are not relevant to that in subtilisin BRC for binding with its substrates. These results suggested that scFv-BRC can be used as a ligand for detection and affinity purification of subtilisin BRC.

Download Full-text

Evolutional selection of a combinatorial phage library displaying randomly-rearranged various single domains of immunoglobulin (Ig)-binding proteins (IBPs) with four kinds of Ig molecules

BMC Microbiology ◽

10.1186/1471-2180-8-137 ◽

2008 ◽

Vol 8 (1) ◽

pp. 137 ◽

Cited By ~ 6

Author(s):

Hua Yang ◽

Jie Cao ◽

Lian-Qing Li ◽

Xia Zhou ◽

Qiu-Li Chen ◽

...

Keyword(s):

Binding Proteins ◽

Phage Library ◽

Selection Of

Download Full-text

Novel Strategy for Selection of Monoclonal Antibodies Against Highly Conserved Antigens: Phage Library Panning Against Ephrin-B2 Displayed on Yeast

PLoS ONE ◽

10.1371/journal.pone.0030680 ◽

2012 ◽

Vol 7 (1) ◽

pp. e30680 ◽

Cited By ~ 2

Author(s):

Xiaoling Gu ◽

Yogindra Vedvyas ◽

Xiaoyue Chen ◽

Tanwi Kaushik ◽

Chang-Il Hwang ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Phage Library ◽

Novel Strategy ◽

Selection Of

Download Full-text

Practical Tips for Construction of Custom Peptide Libraries and Affinity Selection by Using Commercially Available Phage Display Cloning Systems

Journal of Nucleic Acids ◽

10.1155/2012/295719 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 20

Author(s):

Keisuke Fukunaga ◽

Masumi Taki

Keyword(s):

Phage Display ◽

Basic Amino Acid ◽

Phage Library ◽

Affinity Selection ◽

Phage Display Technology ◽

Display Technology ◽

Phage Libraries ◽

Type 3 ◽

T7 Phage ◽

Selection Of

Phage display technology is undoubtedly a powerful tool for affinity selection of target-specific peptide. Commercially available premade phage libraries allow us to take screening in the easiest way. On the other hand, construction of a custom phage library seems to be inaccessible, because several practical tips are absent in instructions. This paper focuses on what should be born in mind for beginners using commercially available cloning kits (Ph.D. with type 3 vector and T7Select systems for M13 and T7 phage, respectively). In the M13 system, Pro or a basic amino acid (especially, Arg) should be avoided at the N-terminus of peptide fused to gp3. In both systems, peptides containing odd number(s) of Cys should be designed with caution. Also, DNA sequencing of a constructed library before biopanning is highly recommended for finding unexpected bias.

Download Full-text