Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli - PubMed (original) (raw)

Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli

J S Huston et al. Proc Natl Acad Sci U S A. 1988 Aug.

Abstract

A biosynthetic antibody binding site, which incorporated the variable domains of anti-digoxin monoclonal antibody 26-10 in a single polypeptide chain (Mr = 26,354), was produced in Escherichia coli by protein engineering. This variable region fragment (Fv) analogue comprised the 26-10 heavy- and light-chain variable regions (VH and VL) connected by a 15-amino acid linker to form a single-chain Fv (sFv). The sFv was designed as a prolyl-VH-(linker)-VL sequence of 248 amino acids. A 744-base-pair DNA sequence corresponding to this sFv protein was derived by using an E. coli codon preference, and the sFv gene was assembled starting from synthetic oligonucleotides. The sFv polypeptide was expressed as a fusion protein in E. coli, using a leader derived from the trp LE sequence. The sFv protein was obtained by acid cleavage of the unique Asp-Pro peptide bond engineered at the junction of leader and sFv in the fusion protein [(leader)-Asp-Pro-VH-(linker)-VL]. After isolation and renaturation, folded sFv displayed specificity for digoxin and related cardiac glycosides similar to that of natural 26-10 Fab fragments. Binding between affinity-purified sFv and digoxin exhibited an association constant [Ka = (3.2 +/- 0.9) x 10(7) M-1] that was about a factor of 6 smaller than that found for 26-10 Fab fragments [Ka = (1.9 +/- 0.2) x 10(8) M-1] under the same buffer conditions, consisting of 0.01 M sodium acetate, pH 5.5/0.25 M urea.

PubMed Disclaimer

Similar articles

• Medical applications of single-chain antibodies.
  Huston JS, McCartney J, Tai MS, Mottola-Hartshorn C, Jin D, Warren F, Keck P, Oppermann H. Huston JS, et al. Int Rev Immunol. 1993;10(2-3):195-217. doi: 10.3109/08830189309061696. Int Rev Immunol. 1993. PMID: 8360586 Review.
• A bifunctional fusion protein containing Fc-binding fragment B of staphylococcal protein A amino terminal to antidigoxin single-chain Fv.
  Tai MS, Mudgett-Hunter M, Levinson D, Wu GM, Haber E, Oppermann H, Huston JS. Tai MS, et al. Biochemistry. 1990 Sep 4;29(35):8024-30. doi: 10.1021/bi00487a005. Biochemistry. 1990. PMID: 2261460
• Rapid and reliable cloning of antibody variable regions and generation of recombinant single chain antibody fragments.
  Gilliland LK, Norris NA, Marquardt H, Tsu TT, Hayden MS, Neubauer MG, Yelton DE, Mittler RS, Ledbetter JA. Gilliland LK, et al. Tissue Antigens. 1996 Jan;47(1):1-20. doi: 10.1111/j.1399-0039.1996.tb02509.x. Tissue Antigens. 1996. PMID: 8929708
• Engineering disulfide-linked single-chain Fv dimers [(sFv')2] with improved solution and targeting properties: anti-digoxin 26-10 (sFv')2 and anti-c-erbB-2 741F8 (sFv')2 made by protein folding and bonded through C-terminal cysteinyl peptides.
  McCartney JE, Tai MS, Hudziak RM, Adams GP, Weiner LM, Jin D, Stafford WF 3rd, Liu S, Bookman MA, Laminet AA, et al. McCartney JE, et al. Protein Eng. 1995 Mar;8(3):301-14. doi: 10.1093/protein/8.3.301. Protein Eng. 1995. PMID: 7479692
• Biosynthetic antibody binding sites: development of a single-chain Fv model based on antidinitrophenol IgA myeloma MOPC 315.
  McCartney JE, Lederman L, Drier EA, Cabral-Denison NA, Wu GM, Batorsky RS, Huston JS, Oppermann H. McCartney JE, et al. J Protein Chem. 1991 Dec;10(6):669-83. doi: 10.1007/BF01025718. J Protein Chem. 1991. PMID: 1815591 Review.

Cited by

• In vivo thrombin activity in the diatom Phaeodactylum tricornutum: biotechnological insights.
  Messaabi A, Merindol N, Bohnenblust L, Fantino E, Meddeb-Mouelhi F, Desgagné-Penix I. Messaabi A, et al. Appl Microbiol Biotechnol. 2024 Oct 8;108(1):481. doi: 10.1007/s00253-024-13322-z. Appl Microbiol Biotechnol. 2024. PMID: 39377797 Free PMC article.
• Therapeutic antibodies in oncology: an immunopharmacological overview.
  Toledo-Stuardo K, Ribeiro CH, González-Herrera F, Matthies DJ, Le Roy MS, Dietz-Vargas C, Latorre Y, Campos I, Guerra Y, Tello S, Vásquez-Sáez V, Novoa P, Fehring N, González M, Rodríguez-Siza J, Vásquez G, Méndez P, Altamirano C, Molina MC. Toledo-Stuardo K, et al. Cancer Immunol Immunother. 2024 Oct 3;73(12):242. doi: 10.1007/s00262-024-03814-2. Cancer Immunol Immunother. 2024. PMID: 39358613 Free PMC article. Review.
• Single-hit genome editing optimized for maturation in B cells redirects their specificity toward tumor antigens.
  Ueda N, Cahen M, Leonard J, Deleurme L, Dreano S, Sirac C, Galy A, Moreaux J, Danger Y, Cogné M. Ueda N, et al. Sci Rep. 2024 Sep 28;14(1):22432. doi: 10.1038/s41598-024-74005-3. Sci Rep. 2024. PMID: 39342013 Free PMC article.
• CAR T-cell therapy in cancer: Integrating nursing perspectives for enhanced patient care.
  Gupta A, Dagar G, Rehmani MU, Prasad CP, Saini D, Singh M, Shankar A. Gupta A, et al. Asia Pac J Oncol Nurs. 2024 Aug 23;11(10):100579. doi: 10.1016/j.apjon.2024.100579. eCollection 2024 Oct. Asia Pac J Oncol Nurs. 2024. PMID: 39315365 Free PMC article. Review.
• Evaluation of Anti-CAR Linker mAbs for CAR T Monitoring after BiTEs/bsAbs and CAR T-Cell Pretreatment.
  Grahnert A, Seiffert S, Wenk K, Schmiedel D, Boldt A, Vucinic V, Merz M, Platzbecker U, Klemann C, Koehl U, Friedrich M. Grahnert A, et al. Biomedicines. 2024 Jul 24;12(8):1641. doi: 10.3390/biomedicines12081641. Biomedicines. 2024. PMID: 39200107 Free PMC article.

References

1. 1. Methods Enzymol. 1972;26:3-27 - PubMed
2. 1. J Immunol. 1974 Apr;112(4):1373-82 - PubMed
3. 1. Biochemistry. 1976 Jun 15;15(12):2706-10 - PubMed
4. 1. J Mol Biol. 1977 May 25;112(3):535-42 - PubMed
5. 1. J Biol Chem. 1978 Jan 25;253(2):585-97 - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources

• Full Text Sources

  • Atypon
  • Europe PubMed Central
  • PubMed Central

• Other Literature Sources

  • The Lens - Patent Citations