Functional preference of the constituent amino acid residues in a phage-library-based nonphosphorylated inhibitor of the Grb2-SH2 domain

2001 ◽  
Vol 57 (6) ◽  
pp. 447-454 ◽  
Author(s):  
F.-D. T. Lung ◽  
Y.-Q. Long ◽  
Peter P. Roller ◽  
C. R. King ◽  
J. Varady ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sh2 Domain ◽  
Phage Library ◽  
Amino Acid Residues ◽  
Constituent Amino Acid

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

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.

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

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.

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

2021 ◽  
Author(s):  
Guangxu Xing ◽  
Yunshang Zhang ◽  
Fangyu Wang ◽  
Liuding Wen ◽  
Gaiping Zhang
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.

Substrate Recognition by Vitamin K-Dependent Carboxylase

1987 ◽  
Vol 57 (01) ◽  
pp. 017-019 ◽  
Author(s):  
Magda M W Ulrich ◽  
Berry A M Soute ◽  
L Johan M van Haarlem ◽  
Cees Vermeer
Keyword(s):  
Amino Acid ◽  
Vitamin K ◽  
Substrate Recognition ◽  
Bovine Liver ◽  
Amino Acid Residues

SummaryDecarboxylated osteocalcins were prepared and purified from bovine, chicken, human and monkey bones and assayed for their ability to serve as a substrate for vitamin K-dependent carboxylase from bovine liver. Substantial differences were observed, especially between bovine and monkey d-osteocalcin. Since these substrates differ only in their amino acid residues 3 and 4, it seems that these residues play a role in the recognition of a substrate by hepatic carboxylase.

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