Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Dec 15;25(24):5132–5134. doi: 10.1093/nar/25.24.5132

Antibody-ribosome-mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites.

M He 1, M J Taussig 1
PMCID: PMC147134  PMID: 9396828

Abstract

We describe a rapid, eukaryotic, in vitro method for selection and evolution of antibody combining sites using antibody-ribosome-mRNA (ARM) complexes as selection particles. ARMs carrying single-chain (VH/K) binding fragments specific for progesterone were selected using antigen-coupled magnetic beads; selection simultaneously captured the genetic information as mRNA, making it possible to generate and amplify cDNA by single-step RT-PCR on the ribosome-bound mRNA for further manipulation. Using mutant libraries, antigen-binding ARMs were enriched by a factor of 10(4)-10(5)-fold in a single cycle, with further enrichment in repeated cycles. While demonstrated here for antibodies, the method has the potential to be applied equally for selection of receptors or peptides from libraries.

Full Text

The Full Text of this article is available as a PDF (67.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arevalo J. H., Stura E. A., Taussig M. J., Wilson I. A. Three-dimensional structure of an anti-steroid Fab' and progesterone-Fab' complex. J Mol Biol. 1993 May 5;231(1):103–118. doi: 10.1006/jmbi.1993.1260. [DOI] [PubMed] [Google Scholar]
  2. Fedorov A. N., Baldwin T. O. Contribution of cotranslational folding to the rate of formation of native protein structure. Proc Natl Acad Sci U S A. 1995 Feb 14;92(4):1227–1231. doi: 10.1073/pnas.92.4.1227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Georgiou G., Poetschke H. L., Stathopoulos C., Francisco J. A. Practical applications of engineering gram-negative bacterial cell surfaces. Trends Biotechnol. 1993 Jan;11(1):6–10. doi: 10.1016/0167-7799(93)90068-K. [DOI] [PubMed] [Google Scholar]
  4. Hanes J., Plückthun A. In vitro selection and evolution of functional proteins by using ribosome display. Proc Natl Acad Sci U S A. 1997 May 13;94(10):4937–4942. doi: 10.1073/pnas.94.10.4937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. He M., Hamon M., Liu H., Kang A., Taussig M. J. Functional expression of a single-chain anti-progesterone antibody fragment in the cytoplasm of a mutant Escherichia coli. Nucleic Acids Res. 1995 Oct 11;23(19):4009–4010. doi: 10.1093/nar/23.19.4009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. He M., Kang A. S., Hamon M., Humphreys A. S., Gani M., Taussig M. J. Characterization of a progesterone-binding, three-domain antibody fragment (VH/K) expressed in Escherichia coli. Immunology. 1995 Apr;84(4):662–668. [PMC free article] [PubMed] [Google Scholar]
  7. High S., Görlich D., Wiedmann M., Rapoport T. A., Dobberstein B. The identification of proteins in the proximity of signal-anchor sequences during their targeting to and insertion into the membrane of the ER. J Cell Biol. 1991 Apr;113(1):35–44. doi: 10.1083/jcb.113.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kasahara N., Dozy A. M., Kan Y. W. Tissue-specific targeting of retroviral vectors through ligand-receptor interactions. Science. 1994 Nov 25;266(5189):1373–1376. doi: 10.1126/science.7973726. [DOI] [PubMed] [Google Scholar]
  9. Mattheakis L. C., Bhatt R. R., Dower W. J. An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):9022–9026. doi: 10.1073/pnas.91.19.9022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Nicholls P. J., Johnson V. G., Andrew S. M., Hoogenboom H. R., Raus J. C., Youle R. J. Characterization of single-chain antibody (sFv)-toxin fusion proteins produced in vitro in rabbit reticulocyte lysate. J Biol Chem. 1993 Mar 5;268(7):5302–5308. [PubMed] [Google Scholar]
  11. Sarkar G., Sommer S. S. The "megaprimer" method of site-directed mutagenesis. Biotechniques. 1990 Apr;8(4):404–407. [PubMed] [Google Scholar]
  12. Smith G. P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science. 1985 Jun 14;228(4705):1315–1317. doi: 10.1126/science.4001944. [DOI] [PubMed] [Google Scholar]
  13. Winter G., Griffiths A. D., Hawkins R. E., Hoogenboom H. R. Making antibodies by phage display technology. Annu Rev Immunol. 1994;12:433–455. doi: 10.1146/annurev.iy.12.040194.002245. [DOI] [PubMed] [Google Scholar]
  14. Wrighton N. C., Farrell F. X., Chang R., Kashyap A. K., Barbone F. P., Mulcahy L. S., Johnson D. L., Barrett R. W., Jolliffe L. K., Dower W. J. Small peptides as potent mimetics of the protein hormone erythropoietin. Science. 1996 Jul 26;273(5274):458–464. doi: 10.1126/science.273.5274.458. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES