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Anti-estradiol-17beta single-chain Fv fragments: Generation, characterization, gene randomization, and optimized phage display.
Steroids. 2008 Dec 22; 73(14):1485-99.S

Abstract

A single-chain Fv fragment (scFv) against estradiol-17beta (E(2)) was generated to begin the construction of a library of various mutated anti-steroid antibodies with an improved affinity and/or specificity. A hybridoma clone secreting a specific anti-E(2) antibody (Ab#E4-4) was established by the cell fusion using splenocytes from a mouse immunized with an immunogenic E(2)-carrier conjugate. DNA fragments encoding the variable heavy and light domains (V(H) and V(L)) of the Ab#E4-4 were cloned and combined to give the scFv gene fragment encoding the sequence 5'-V(H)-(GGGGS)(3)-V(L)-3'. Compared to the Ab#E4-4 Fab fragment, soluble scFv (scFv#E4-4) protein showed a similar affinity to E(2) (K(a)=8.6x10(7)M(-1)) and a similar cross-reaction profile. To further study the fundamentals for creating a comprehensive library of mutated scFvs, the scFvV(H) and V(L) genes were amplified using error-prone PCR conditions and the frequency and pattern of incorporated mutations were investigated. For this, regular Taq polymerase was used in the presence of unequal concentrations of dNTPs. At 1.0mM MnCl(2), the error frequency reached to 8.5% and 11% for the V(H) and V(L) respectively, although a significant transition/transversion bias was observed. ScFv#E4-4 and the mutated polyclonal scFvs were then displayed on filamentous phage under various packaging conditions. Cultivation of the transformed bacteria was more suitable at 25 degrees C than at higher temperatures for the packaging of scFv-bearing phagemid particles. Based on these experimental conditions, an scFv-displaying phage library, each scFv member in which has mutated complementarity-determining region (CDR) H2, H3, L1, and L3, was constructed. A soluble scFv clone (scFv#m1-e7) with a mutated amino acid (I-->V) in CDR L1, isolated from this library, showed threefold higher affinity (K(a)=2.6 x 10(8)M(-1)) than that of scFv#4-4.

Authors+Show Affiliations

Kobe Pharmaceutical University, 4-19-1 Motoyama-Kitamachi, Higashinada-ku, Kobe 658-8558, Japan. no-kobay@kobepharma-u.ac.jpNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

18824188

Citation

Kobayashi, Norihiro, et al. "Anti-estradiol-17beta Single-chain Fv Fragments: Generation, Characterization, Gene Randomization, and Optimized Phage Display." Steroids, vol. 73, no. 14, 2008, pp. 1485-99.
Kobayashi N, Kato Y, Oyama H, et al. Anti-estradiol-17beta single-chain Fv fragments: Generation, characterization, gene randomization, and optimized phage display. Steroids. 2008;73(14):1485-99.
Kobayashi, N., Kato, Y., Oyama, H., Taga, S., Niwa, T., Sun, P., Ohtoyo, M., & Goto, J. (2008). Anti-estradiol-17beta single-chain Fv fragments: Generation, characterization, gene randomization, and optimized phage display. Steroids, 73(14), 1485-99. https://doi.org/10.1016/j.steroids.2008.08.009
Kobayashi N, et al. Anti-estradiol-17beta Single-chain Fv Fragments: Generation, Characterization, Gene Randomization, and Optimized Phage Display. Steroids. 2008 Dec 22;73(14):1485-99. PubMed PMID: 18824188.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Anti-estradiol-17beta single-chain Fv fragments: Generation, characterization, gene randomization, and optimized phage display. AU - Kobayashi,Norihiro, AU - Kato,Yoshinori, AU - Oyama,Hiroyuki, AU - Taga,Shiori, AU - Niwa,Toshifumi, AU - Sun,Pi, AU - Ohtoyo,Mamoru, AU - Goto,Junichi, Y1 - 2008/09/09/ PY - 2008/04/22/received PY - 2008/08/12/revised PY - 2008/08/13/accepted PY - 2008/10/1/pubmed PY - 2009/4/18/medline PY - 2008/10/1/entrez SP - 1485 EP - 99 JF - Steroids JO - Steroids VL - 73 IS - 14 N2 - A single-chain Fv fragment (scFv) against estradiol-17beta (E(2)) was generated to begin the construction of a library of various mutated anti-steroid antibodies with an improved affinity and/or specificity. A hybridoma clone secreting a specific anti-E(2) antibody (Ab#E4-4) was established by the cell fusion using splenocytes from a mouse immunized with an immunogenic E(2)-carrier conjugate. DNA fragments encoding the variable heavy and light domains (V(H) and V(L)) of the Ab#E4-4 were cloned and combined to give the scFv gene fragment encoding the sequence 5'-V(H)-(GGGGS)(3)-V(L)-3'. Compared to the Ab#E4-4 Fab fragment, soluble scFv (scFv#E4-4) protein showed a similar affinity to E(2) (K(a)=8.6x10(7)M(-1)) and a similar cross-reaction profile. To further study the fundamentals for creating a comprehensive library of mutated scFvs, the scFvV(H) and V(L) genes were amplified using error-prone PCR conditions and the frequency and pattern of incorporated mutations were investigated. For this, regular Taq polymerase was used in the presence of unequal concentrations of dNTPs. At 1.0mM MnCl(2), the error frequency reached to 8.5% and 11% for the V(H) and V(L) respectively, although a significant transition/transversion bias was observed. ScFv#E4-4 and the mutated polyclonal scFvs were then displayed on filamentous phage under various packaging conditions. Cultivation of the transformed bacteria was more suitable at 25 degrees C than at higher temperatures for the packaging of scFv-bearing phagemid particles. Based on these experimental conditions, an scFv-displaying phage library, each scFv member in which has mutated complementarity-determining region (CDR) H2, H3, L1, and L3, was constructed. A soluble scFv clone (scFv#m1-e7) with a mutated amino acid (I-->V) in CDR L1, isolated from this library, showed threefold higher affinity (K(a)=2.6 x 10(8)M(-1)) than that of scFv#4-4. SN - 0039-128X UR - https://www.unboundmedicine.com/medline/citation/18824188/Anti_estradiol_17beta_single_chain_Fv_fragments:_Generation_characterization_gene_randomization_and_optimized_phage_display_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0039-128X(08)00216-X DB - PRIME DP - Unbound Medicine ER -