Research Paper | Published:

Single Chain Antibody (SCA) Encoding Genes: One-Step Construction and Expression in Eukaryotic Cells

Bio/Technologyvolume 9pages165169 (1991) | Download Citation

Subjects

Abstract

We report the expression, in eukaryotic cells, of a gene encoding a single chain antibody (SCA) and a rapid method for the construction of such genes. A SCA directed against the aromatic dye fluorescein was synthesized from a gene constructed by means of the simultaneous use of four PCR primers and templates of both light and heavy chain immunoglobulin cDNAs in the form of either plasmid clones or reverse transcribed hybridoma RNA. Two of the primers were partially complementary to one another and encoded the polypeptide linker which joins the immunoglobulin light and heavy chain variable domains of the SCA polypeptide. A functional, hapten-binding product was synthesized from the gene thus constructed in both E. coli and the fission yeast, Schizosaccharomyces pombe. Our results demonstrate that gene constructs encoding single chain antigen binding proteins can be synthesized very rapidly with only limited sequence information about the pertinent light and heavy chain immunoglobulin genes, and, that neither murine codon usage bias, Thermus aquaticus DNA polymerase infidelity, nor the eukaryotic cellular environment preclude the synthesis of functional single chain antigen binding proteins in non-lymphatic, non-murine eukaryotic cells.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1

    Bird, R.E., Hardman, K.D., Jacobson, J.W., Johnson S., Kaufman, B.M., Lee, S.-M., Lee, T., Pope, S.H., Riordan, G.S. and Whitlow, M. 1988. Single-chain antigen-binding proteins. Science 242: 423–426.

  2. 2

    Huston, J.S., Levinson, D., Mudgett-Hunter, M., Tai, M.-S., Novotny, J., Margolies, M.N., Ridge, R.J., Bruccoleri, R.E., Haber, E., Crea, R. and Oppermann, H. 1988. Protein engineering of antibody binding sites: Recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 85: 5879–5883.

  3. 3

    Chaudhary, V.K., Batra, J.K., Gallo, M.G., Willingham, M.C., FitzGerald, D.J. and Pastan, I. 1990. A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins. Proc. Natl. Acad. Sci. USA 87: 1066–1070.

  4. 4

    Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K. and Pease, L.R. 1989. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77 61–68.

  5. 5

    Kranz, D.M., Herron J.N. and Voss,E.W.J. 1982. Mechanisms of ligand binding by monoclonal anti-fluorescyl antibodies. J. Biol. Chem. 257: 6987–6995.

  6. 6

    Bedzyk, W.D., Johnson, L.S., Riordan, G.S. and Voss, E.W.J. 1989. Comparison of variable region primary structures within an anti-fluorescein idiotype family. J. Biol. Chem. 264: 1565–1569.

  7. 7

    Herron, J.N., He, X.-m., Mason, M.L., Voss, E.W.J. and Edmundson, A.B. 1989. Three-dimensional structure of fluorescein-Fab complex crystallized in 2-methyl-2,4-pentanediol. Proteins 5: 271–280.

  8. 8

    Springer, B.A. and Sligar, S.G. 1987. High-level expression of sperm whale myoglobin Escherichia coll. Proc. Natl. Acad. Sci. USA 84: 8961–8965.

  9. 9

    Hamlyn, P.H., Gait, M.J. and Milstein, C. 1981. Complete sequence of an immunoglobulin mRNA using specific priming and the dideoxy-nucleotide method of RNA sequencing. Nucl. Acids Res. 9: 4485–4494.

  10. 10

    Kaartinen, M., Griffiths, G.M., Hamlyn, P.H., Markham, A.F., Karjalainen, K., Pelkonen, J.L.T., Makela, O. and Milstein, C. 1983. Anti-oxazolone hybridomas and the structure of the oxazolone idiotype. J. Immunol. 130: 937–945.

  11. 11

    Janada, K.D., Schloeder, D., Benkovic, S.J. and Lerner, R.A. 1988. Induction of an antibody that catalyzes the hydrolysis of an amide bond. Science 241: 1188–1191.

  12. 12

    Leboeuf, R.D., Galin, F.S., Hollinger, S.K., Peiper, S.C. and Blalock, J.E. 1989. Cloning and sequencing of immunoglobulin variable-region genes using degenerate oligodeoxyribonucleotides and the polymerase chain reaction. Gene 82: 371–377.

  13. 13

    Larrick, J.W., Danielsson, L., Brenner, C.A., Wallace, E.F., Abrahamson, M., Fry, K.E. and Borrebaeck, C.A.K. 1989. Polymerase chain reaction using mixed primers: cloning of human monoclonal antibody variable region genes from single hybridoma cells. Bio/Technology 7: 934–938.

  14. 14

    Chiang, Y.L., Sheng-Dong, R., Brow, M.A. and Larrick, J.W. 1989. Directed cDNA cloning of the rearranged immunoglobulin variable region. BioTechniques 7: 360–366.

  15. 15

    Orlandi, R., Gussow, D.H., Jones, P.T. and Winter, G. 1989. Cloning immunoglobulin variable domains by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA. 86: 3833–3837.

  16. 16

    Kawasaki, E. . 1989. Amplification of RNA sequences via complementary DNA (cDNA). Amplifications 3: 4–6.

  17. 17

    Dumais, M.M. and Nochumson, S. 1987. Small DNA fragment separation and M13 cloning directly in remelted NuSieve GTG agarose gels. BioTechniques 5: 62–67.

  18. 18

    Alexander, D.C. 1987. An efficient vector-primer cDNA cloning system. Meth. Enzymol. 154: 41–64.

  19. 19

    Holmes, D.S. and Quigley, M. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114: 193–197.

  20. 20

    Booher, R. and Beach, D. 1988. Involvement of cdc13+ in mitotic control of Schiwsaccharomyces pombe: possible interaction of the gene product with microtubules. EMBO J. 7: 2321–2327.

  21. 21

    Brokers, M. 1987. Transformation of intact Schizosaccharomyces pombe cells with plasmid DNA. BioTechniques 5: 516–518.

  22. 22

    Mitcheson, M. 1970. Physiological and cytological methods for Schizosaccharomyces pombe. p. 131–164. In: Methods in Cell Physiology. Prescott, E. M. (Ed.). Academic Press, New York.

  23. 23

    Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.

  24. 24

    Towbin, H., Staehelin, T. and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76: 4350–4354.

  25. 25

    Kranz, D.M. and Voss, E.W.J. 1983. Idiotypic analysis of monoclonal antifluorescyl antibodies: localization and characterization of idiotypic determinants. Molec. Immunol. 20: 1301–1312.

  26. 26

    Kraft,J., Tardiff,J., Krauter, K.S. and Leinwand, L.A. 1988. Using mini-prep plasmid DNA for sequencing double stranded templates with Sequenase. BioTechniques 6: 544–546.

Download references

Author information

Author notes

  1. Thomas W. Jacobs: Corresponding author.

Affiliations

  1. Department of Plant Biology, University of Illinois, Urbana, IL, 61801

    • George T. Davis
    •  & Thomas W. Jacobs
  2. Department of Microbiology, University of Illinois, Urbana, IL, 61801

    • William D. Bedzyk
    •  & Edward W. Voss

Authors

  1. Search for George T. Davis in:

  2. Search for William D. Bedzyk in:

  3. Search for Edward W. Voss in:

  4. Search for Thomas W. Jacobs in:

About this article

Publication history

Received

Accepted

Issue Date

DOI

https://doi.org/10.1038/nbt0291-165

Further reading