Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

An in vivo library-versus-library selection of optimized protein–protein interactions


We describe a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into Escherichia coli. Interaction between the library polypeptides reconstituted enzymatic activity of mDHFR, allowing bacterial growth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We enriched the best-performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagation. This competitive growth allowed small differences among the pairs to be amplified, and different sequence positions were enriched at different rates. We applied these selection processes to a library-versus-library sample of 2.0 × 106 combinations and selected a novel leucine zipper pair that may be appropriate for use in further in vivo heterodimerization strategies.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

Figure 1: (A) DNA constructs code for fusions between library proteins (shown as α-helical leucine zippers) and either fragment of murine DHFR (mDHFR).
Figure 2: (A) Schematic representation of a leucine zipper pair visualized from the N-terminus illustrating e/g-interactions and the hydrophobic core formed by the a- and d-positions.
Figure 3: Efficiency of competition in a model selection.
Figure 4: Competition selection and chain shuffling.
Figure 5: Sequencing profile of pools from passages of the chain shuffling WinZip-B1-DHFR[2:I114A] + LibA-DHFR[1].


  1. Fields, S. & Song, O. A Novel genetic system to detect protein-protein interactions. Nature 340, 245– 246 (1989).

    Article  CAS  Google Scholar 

  2. Chien, C.T., Bartel, P.L., Sternglanz, R. & Fields, S. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl. Acad. Sci. USA 88, 9578–9582 ( 1991).

    Article  CAS  Google Scholar 

  3. Smith, G.P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315 –1317 (1985).

    Article  CAS  Google Scholar 

  4. Pelletier, J.N., Remy, I. & Michnick, S.W. Protein-fragment complementation assays: a general strategy for the in vivo detection of protein-protein interactions. J. Biomol. Techniques,

  5. Pelletier, J.N., Campbell-Valois, F.X. & Michnick, S.W. Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments. Proc. Natl. Acad. Sci. USA 95, 12141– 12146 (1998).

    Article  CAS  Google Scholar 

  6. Remy, I. & Michnick, S.W. Clonal selection and in vivo quantitation of protein interactions with protein fragment complementation assays. Proc. Natl. Acad. Sci. USA 96, 5394 –5399 (1999).

    Article  CAS  Google Scholar 

  7. Remy, I., Wilson, I.A. & Michnick S.W. Erythropoietin receptor activation by a ligand-induced conformation change. Science 283, 990– 993 (1999).

    Article  CAS  Google Scholar 

  8. Sydor, J.R., Engelhard, M., Wittinghofer, A., Goody, R.S. & Herrmann, C. Transient kinetic studies on the interaction of Ras and the Ras-binding domain of c-Raf-1 reveal rapid equilibration of the complex. Biochemistry 37, 14292– 14299. (1998).

    Article  CAS  Google Scholar 

  9. Chen, J., Zheng, X.F., Brown, E.J. & Schreiber, S.L. Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. Proc. Natl. Acad. Sci. USA 92, 4947–4951 (1995).

    Article  CAS  Google Scholar 

  10. O'Shea, E.K., Lumb, K.J. & Kim, P.S. Peptide 'Velcro': design of a heterodimeric coiled coil. Curr. Biol. 3, 658–667 (1993).

    Article  CAS  Google Scholar 

  11. Jelesarov, I. & Bosshard, H.R. Thermodynamic characterization of the coupled folding and association of heterodimeric coiled coils (leucine zippers). J. Mol. Biol. 263, 344– 358 (1996).

    Article  CAS  Google Scholar 

  12. Zhou, N.E., Kay, C.M. & Hodges, R.S. The role of interhelical ionic interactions in controlling protein folding and stability. De novo designed synthetic two-stranded alpha-helical coiled-coils. J. Mol. Biol. 237, 500– 512 (1994).

    Article  CAS  Google Scholar 

  13. Müller, K.M., Arndt, K.M., Strittmatter, W. & Plückthun, A. The first constant domain (CH1 and CL) of an antibody used as heterodimerization domain for bispecific miniantibodies. FEBS Lett. 422, 259–264 (1998).

    Article  Google Scholar 

  14. Virnekäs, B. et al. Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis. Nucleic Acids Res. 22, 5600–5607 ( 1994).

    Article  Google Scholar 

  15. Zeng, X., Herndon, A.M. & Hu, J.C. Buried asparagines determine the dimerization specificities of leucine zipper mutants. Proc. Natl. Acad. Sci. USA 94, 3673–3678 (1997).

    Article  CAS  Google Scholar 

  16. Lumb, K.J. & Kim, P.S. A buried polar interaction imparts structural uniqueness in a designed heterodimeric coiled coil. Biochemistry 34, 8642–8648 (1995).

    Article  CAS  Google Scholar 

  17. O'Shea, E.K., Klemm, J.D., Kim, P.S. & Alber, T. X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science 254, 539–544 ( 1991).

    Article  CAS  Google Scholar 

  18. Zhou, N.E., Kay, C.M. & Hodges, R.S. The net energetic contribution of interhelical electrostatic attractions to coiled-coil stability. Protein Eng. 7, 1365–1372 (1994).

    Article  CAS  Google Scholar 

  19. Monera, O.D., Kay, C.M. & Hodges, R.S. Electrostatic interactions control the parallel and antiparallel orientation of alpha-helical chains in two-stranded alpha-helical coiled-coils. Biochemistry 33, 3862– 3871 (1994).

    Article  CAS  Google Scholar 

  20. John, M., Briand, J.P., Granger-Schnarr, M. & Schnarr, M. Two pairs of oppositely charged amino acids from Jun and Fos confer heterodimerization to GCN4 leucine zipper. J. Biol. Chem. 269, 16247–16253 (1994).

    CAS  PubMed  Google Scholar 

  21. Lumb, K.J. & Kim, P.S. Measurement of interhelical electrostatic interactions in the GCN4 leucine zipper. Science 268 , 436–439 (1995).

    Article  CAS  Google Scholar 

  22. Buchwalder, A., Szadkowski, H. & Kirschner, K. A fully active variant of dihydrofolate reductase with a circularly permuted sequence. Biochemistry 31, 1621–1630 (1992).

    Article  CAS  Google Scholar 

  23. Hu, J.C., O'Shea, E.K., Kim, P.S. & Sauer, R.T. Sequence requirements for coiled-coils: analysis with lambda repressor-GCN4 leucine zipper fusions. Science 250, 1400–1403 (1990).

    Article  CAS  Google Scholar 

  24. Zeng, X., Zhu, H., Lashuel, H.A. & Hu, J.C. Oligomerization properties of GCN4 leucine zipper e and g position mutants. Protein Sci. 6, 2218–2226 (1997).

    Article  CAS  Google Scholar 

  25. Spada, S. & Plückthun, A. Selectively infective phage (SIP) technology: a novel method for in vivo selection of interacting protein-ligand pairs. Nat. Med. 3, 694– 696 (1997).

    Article  CAS  Google Scholar 

  26. Rudert, F., Woltering, C., Frisch, C., Rottenberger, C. & Ilag, L.L. A phage-based system to select multiple protein-protein interactions simultaneously from combinatorial libraries. FEBS Lett. 440, 135–140 (1998).

    Article  CAS  Google Scholar 

  27. Bartel, P.L., Roecklein, J.A., SenGupta, D. & Fields, S. A protein linkage map of Escherichia coli bacteriophage T7. Nat. Genet. 12, 72–77 ( 1996).

    Article  CAS  Google Scholar 

  28. Fromont-Racine, M., Rain, J.C. & Legrain, P. Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens. Nat. Genet. 16, 277–282 (1997).

    Article  CAS  Google Scholar 

  29. SenGupta, D.J. et al. A three-hybrid system to detect RNA-protein interactions in vivo. Proc. Natl. Acad. Sci. USA 93, 8496 –8501 (1996).

    Article  CAS  Google Scholar 

Download references


J.N.P. was a recipient of a fellowship from les Fonds de la Recherche en Santé du Québec, and currently holds a fellowship from le Conseil de Recherche en Sciences Naturelles et en Génie du Canada. K.M.A. is a recipient of a doctoral grant from the Stipendienfonds der Basler Chemischen Industrie. This work was funded by The Burroughs-Wellcome Fund (S.W.M.) and by grant 0311628 from the Bundesministerium für Bildung und Forschung (AP). We thank Günter Wellnhofer (Morphosys AG, Munich, Germany) for the synthesis of the oligonucleotides with the mixed trinucleotide building blocks. We thank Michel Denault for his painstaking help in probabilistic analysis, and François-Xavier Campbell-Valois, Alexis Vallée-Belisle, and Patrick Forrer for critical reading of the manuscript.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Stephen W. Michnick.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Pelletier, J., Arndt, K., Plückthun, A. et al. An in vivo library-versus-library selection of optimized protein–protein interactions. Nat Biotechnol 17, 683–690 (1999).

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing