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

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.

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Expression in Escherichia Coli of a Single - Chain Variable - Fragment ( scFv ) Against the Amino Acid Motif DELLA of Plant Transcription Factor Is Toxic to E. Coli

International Journal for Sciences and Technology ◽

10.12816/0010080 ◽

2013 ◽

Vol 8 (4) ◽

pp. 19-26

Author(s):

Taha Al-Samarrai

Keyword(s):

Escherichia Coli ◽

Transcription Factor ◽

Amino Acid ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Amino Acid Motif ◽

E Coli ◽

Expression In Escherichia Coli

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E. coli GyrA Tower Domain Interacts with QnrB1 Loop B and Plays an Important Role in QnrB1 Protection from Quinolone Inhibition

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00402-21 ◽

2021 ◽

Author(s):

Chunhui Chen ◽

Yin Wang ◽

Hidemasa Nakaminami ◽

Eu Suk Kim ◽

George A. Jacoby ◽

...

Keyword(s):

Mass Spectrometry ◽

Amino Acids ◽

Amino Acid ◽

Dna Gyrase ◽

Amino Acid Residues ◽

Site Specific ◽

E Coli ◽

Small Deletion ◽

Interaction Sites ◽

Repeat Proteins

The Qnr pentapeptide repeat proteins interact with DNA gyrase and protect it from quinolone inhibition. The two external loops, particularly the larger loop B, of Qnr proteins are essential for quinolone protection of DNA gyrase. The specific QnrB1 interaction sites on DNA gyrase are not known. In this study, we investigated the interaction between GyrA and QnrB1 using site-specific photo crosslinking of QnrB1 loop B combined with mass spectrometry. We found that amino acid residues 286-298 on the Tower domain of GyrA interact with QnrB1 and play a key role in QnrB1 protection of gyrase from quinolone inhibition. Alanine replacement of arginine at residue 293 and a small deletion of amino acids 286-289 of GyrA resulted in a decrease in the QnrB1-mediated increase in quinolone MICs and also abolished the QnrB1 protection of purified DNA gyrase from ciprofloxacin inhibition.

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Molecular analysis of human anti-factor VIII antibodies by V gene phage display identifies a new epitope in the acidic region following the A2 domain

Blood ◽

10.1182/blood.v96.2.540.014k20_540_545 ◽

2000 ◽

Vol 96 (2) ◽

pp. 540-545 ◽

Cited By ~ 3

Author(s):

Edward N. van den Brink ◽

Ellen A. M. Turenhout ◽

Christine M. C. Bank ◽

Karin Fijnvandraat ◽

Marjolein Peters ◽

...

Keyword(s):

Amino Acid ◽

Phage Display ◽

Factor Viii ◽

Gene Segment ◽

Amino Acid Residues ◽

Germline Gene ◽

Single Chain ◽

Factor Viii Activity ◽

A2 Domain ◽

Acidic Region

One of the major binding sites for factor VIII inhibitors is located within the A2 domain. In this study, phage display technology was used to isolate 2 human monoclonal antibodies, termed VK34 and VK41, directed toward the heavy chain of factor VIII. The VHdomain of a single-chain variable domain antibody fragment (scFv) VK34 is encoded by germline gene segment DP-10. Epitope-mapping studies revealed that scFv VK34 is directed against amino acid residues Arg484–Ile508 , a previously identified binding site for factor VIII inhibitors in the A2 domain. ScFv VK34 inhibited factor VIII activity with a titer of 280 BU/mg. The VH domain of VK41 was encoded by germline gene segment DP-47. A phage corresponding to VK41 competed with a monoclonal antibody for binding to amino acid residues Asp712–Ala736 in the acidic region adjacent to the A2 domain. Reactivity of VK41 with a factor VIII variant in which we replaced amino acid residues Asp712–Ala736for the corresponding region of heparin cofactor II was strongly reduced. In addition, substitution of Tyr718719723 for Phe abrogated binding of VK41 to factor VIII. ScFv VK41 did not inhibit factor VIII activity. This study not only defines the primary structure of human anti-factor VIII antibodies reactive with the A2 domain, it also describes an antibody with an epitope not previously identified in the antibody repertoire of hemophilia patients with an inhibitor.

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Probe design for mining and selection of genes coding endo 1- 4 xylanase from dna metagenome data

TAP CHI SINH HOC ◽

10.15625/0866-7160/v40n1.9200 ◽

2018 ◽

Vol 40 (1) ◽

pp. 39-50

Author(s):

Do Thi Huyen ◽

Nguyen Minh Giang ◽

Nguyen Thu Nguyet ◽

Truong Nam Hai

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Probe Design ◽

Amino Acid Residues ◽

High Similarity ◽

Metagenomic Dna ◽

Free Living ◽

Conserved Residues ◽

Living Bacteria ◽

Selection Of

According to the CAZY classification, endo 1- 4 xylanase belongs to GH 5, 8, 10, 11, 30, 51, 98. However only 03 sequences of GH8, 27 sequences of GH10, 18 sequence of GH11, only one sequence of each GH30 and GH51 from CAZy and NCBI database were thouroughly experimentally studied for biological activity and characteristics of the enzyme. Through the collected sequences, two probes for endo 1- 4 xylanase of GH10 and GH11 were designed, based on the sequence homology. The GH10 probe was 338 amino acids lenghth contained all the conserved amino acid residues (16 conserved residues in all sequences, 13 residues similar in almost sequences, 14 residues conserved in many sequences) with the lowest maxscore of 189, coverage of 88% and identity of 39%. The GH11 probe was 204 amino acids contained all the conserved amino acid residues (54 conserved residues were identity in all sequences, 25 residues similar in almost sequences, 24 residues conserved in many sequences) with the lowest maxscore of 165, coverage of 84% and identity of 50%. Using the two probes, we mined only one sequence (GL0018509) for endo 1- 4 xylanase from metagenomic DNA data of free-living bacteria in Coptotermes termite gut. Prediction of three-dimention structure of GL0018509 sequence by Phyre2 and Swiss Prot showed that this sequence was high similarity (95% by Phyre2 and 93,4% by Swiss Prot) with endo 1- 4 xylanase with the 100% confidence.

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Construction and directional evolution of anti-enrofloxacin ScFv antibody for the immunoassay of fluoroquinolones

10.21203/rs.3.rs-310107/v2 ◽

2021 ◽

Author(s):

Fangyu Wang ◽

Ning Li ◽

Yunshang Zhang ◽

Xuxefeng Sun ◽

Yali Zhao ◽

...

Keyword(s):

Amino Acid ◽

Scfv Antibody ◽

Phage Library ◽

Enzyme Linked Immunosorbent Assay ◽

Amino Acid Residues ◽

Single Chain ◽

Single Chain Antibody ◽

Relative Standard ◽

Directional Evolution ◽

Scfv Library

Abstract A recombinant anti-enrofloxacin single-chain antibody (scFv) was produced for the detection of enrofloxacin. An immunized mouse phage display scFv library with a capacity of 2.35×109 CFU/mL was constructed and used for anti-enrofloxacin scFv screening. After four rounds of bio-panning, 10 positives were isolated and identified successfully. The highest positive scFv was expressed in E. coli BL21. Then, its recognition mechanisms were studied using the molecular docking method. The result showed the amino acid residues Leu121 were the key residues for the binding of ScFv to ENR. Based on the results of virtual mutation, the ScFv antibody was evolved by directional mutagenesis of contact amino acid residue Leu121 to Asn. After the expression and purification, an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) based on the parental and mutant ScFv were established for enrofloxacin respectively. The IC50 value of the assay established with the ScFv mutant was 1.63 ng/mL, while the parental ScFv was 21.08 ng/mL, this result showed highly increased affinity with up to 12.9-folds improved sensitivity. The mean recovery for ENR ranged from 71.80% to 117.35% with 10.46% relative standard deviation between the intra-assay and the inter-assay. The results indicate that we have obtained a highly sensitive anti-ENR scFv by the phage library construction and directional evolution, and the scFv-based IC-ELISA is suitable for the detection of ENR residue in animal derived edible tissues and milk.

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Molecular analysis of human anti-factor VIII antibodies by V gene phage display identifies a new epitope in the acidic region following the A2 domain

Blood ◽

10.1182/blood.v96.2.540 ◽

2000 ◽

Vol 96 (2) ◽

pp. 540-545 ◽

Cited By ~ 20

Author(s):

Edward N. van den Brink ◽

Ellen A. M. Turenhout ◽

Christine M. C. Bank ◽

Karin Fijnvandraat ◽

Marjolein Peters ◽

...

Keyword(s):

Amino Acid ◽

Phage Display ◽

Factor Viii ◽

Gene Segment ◽

Amino Acid Residues ◽

Germline Gene ◽

Single Chain ◽

Factor Viii Activity ◽

A2 Domain ◽

Acidic Region

Abstract One of the major binding sites for factor VIII inhibitors is located within the A2 domain. In this study, phage display technology was used to isolate 2 human monoclonal antibodies, termed VK34 and VK41, directed toward the heavy chain of factor VIII. The VHdomain of a single-chain variable domain antibody fragment (scFv) VK34 is encoded by germline gene segment DP-10. Epitope-mapping studies revealed that scFv VK34 is directed against amino acid residues Arg484–Ile508 , a previously identified binding site for factor VIII inhibitors in the A2 domain. ScFv VK34 inhibited factor VIII activity with a titer of 280 BU/mg. The VH domain of VK41 was encoded by germline gene segment DP-47. A phage corresponding to VK41 competed with a monoclonal antibody for binding to amino acid residues Asp712–Ala736 in the acidic region adjacent to the A2 domain. Reactivity of VK41 with a factor VIII variant in which we replaced amino acid residues Asp712–Ala736for the corresponding region of heparin cofactor II was strongly reduced. In addition, substitution of Tyr718719723 for Phe abrogated binding of VK41 to factor VIII. ScFv VK41 did not inhibit factor VIII activity. This study not only defines the primary structure of human anti-factor VIII antibodies reactive with the A2 domain, it also describes an antibody with an epitope not previously identified in the antibody repertoire of hemophilia patients with an inhibitor.

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