NEWS AND VIEWS

Phage display extends its reach

Damon Huber & Jonathan Beckwith
Proteins that could not be displayed on the surface of phage are now amenable to this powerful selection technology.
In phage display, a protein of interest is incorporated into the coat of a filamentous bacteriophage of Escherichia coli by genetically fusing it with one of several phage coat proteins. However, because the phage coat is assembled in the periplasm, phage display has been limited to the subset of proteins that can be efficiently exported to the periplasm. In this issue, Steiner et al.1 show that this limitation can be overcome by targeting proteins for export through the signal recognition particle (SRP)-dependent pathway. With this modification, the authors significantly improved the display of designed ankyrin-repeat proteins (DARPins). Ever since the discovery of signal sequences24, researchers have been attempting to export foreign proteins across the cytoplasmic membrane of microorganisms into extracytoplasmic compartments or into the medium by generating fusions between signal sequences and the protein of interest. This work has had various goals. For example, some covalent modifications, such as disulfide bonds and glycosylations, are formed only in extracytoplasmic compartments. In addition, purifying proteins expressed in these compartments may be easier in the absence of nucleic acids and other large cytoplasmic macromolecules. However, many proteins, especially those that normally localize to the cytoplasm, have defied all attempts to efficiently translocate them. This is a particularly frustrating problem for researchers who use phage display technology, because a protein of interest must be translocated across the E. coli cytoplasmic membrane to the periplasm to be incorporated into the coat of filamentous bacteriophage. For some proteins, fusion to any signal sequence is sufficient to export it to the appropriate compartment. In other cases, some feature of the protein interferes with translocation. An enormous amount of research time has probably been wasted on hit-or-miss attempts to solve this problem by trying randomly chosen signal sequences. However, with the exception
Damon Huber and Jonathan Beckwith are in the Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, Massachusetts 02115, USA. e-mail: jbeckwith@hms.harvard.edu

2006 Nature Publishing Group http://www.nature.com/naturebiotechnology

Post-translational translocation Unfolded coat protein p3 Unfolded DARPin

Cotranslational translocation SRP SRP signal sequence

Sec signal sequence Translating ribosome

Cytoplasm

X
Translocon

Folded DARPin

Periplasm

Folding in periplasm Filamentous bacteriophage p3

Assembly into phage coat

Bob Crimi

Figure 1 Comparison of the commonly used Sec signal sequence and the SRP signal sequence for phage display of proteins fused to the phage minor coat protein p3. (a) Many proteins (such as DARPins) cannot be efficiently exported to the periplasm with the most commonly used E. coli signal sequences because they rapidly fold before they can be exported. These proteins are not incorporated efficiently into the bacteriophage coat. (b) Signal sequences that are recognized by the SRP mediate cotranslational translocation of proteins and promote efficient display on phage.

of a few reports describing a handful of signal sequences that appear to promote better periplasmic localization, most of the results are not published because they are negative. Thus no coherent picture of what makes some signal sequences better than others can be drawn from published studies. One factor that determines whether a protein can be efficiently exported is protein folding. Until recently, it was thought that all cleavable bacterial signal sequences promoted post-translational export; that is, translocation begins only after a significant portion of the protein has been synthesized. Because a protein must be substantially unfolded to pass through the membrane-embedded translocation machinery, folded proteins remain trapped in the cytoplasm. However, we recently reported that, contrary to what was believed, there are a number of cleavable signal sequences in E. coli that promote cotranslational translocation across the

cytoplasmic membrane5. In cotranslational translocation, substrate proteins are translocated as they are being synthesized. These signal sequences were identified during studies on the export of the small (12 kDa) cytoplasmic protein thioredoxin-1. Thioredoxin-1 cannot be exported when fused to most commonly used signal sequences because of its rapid and stable folding6. Using a genetic screen, we found a new class of signal sequences that promotes very efficient translocation of thioredoxin-1 (ref. 5). These signal sequences are specifically recognized by the SRP, which targets them for export in what is likely a strictly cotranslational manner7. Because cotranslational translocation prevents any significant portion of a protein from being exposed to the cytoplasm, there is no interference with translocation from folding of the substrate protein. Steiner et al. describe the first practical application of SRP-dependent signal sequences. The authors sought to use phage display

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technology to select for DARPins with novel binding specificities. However, like thioredoxin, DARPins cannot be efficiently exported to the periplasm using the signal sequences encoded in commonly available cloning vectors, such as the PhoA signal sequence, a model signal sequence that has been extensively studied, and the PelB signal sequence, found in many pET vectors (Fig. 1a). To get around this problem, the authors tested several of the SRP-dependent signal sequences (Fig. 1b). In every case, expression of the DARPins on the phage surface was dramatically improved. This report is not only a significant step forward for phage display technology, but also suggests that SRP-dependent signal sequences may be useful for efficient periplasmic expression of proteins for a wide variety of purposes. Premature cytoplasmic folding may have been the major obstacle in many failed attempts to export proteins to the periplasm. Indeed, that E. coli has evolved so many mechanisms to prevent exported proteins from folding in the cytoplasm suggests that protein folding is an issue for translocation of native substrates. For example, some translocated proteins, such as alkaline phosphatase, require disulfide bonds, which can be formed only in the periplasm, to fold stably8. Other proteins, such as maltosebinding protein, are maintained in an unfolded state in the cytoplasm by chaperones (such as SecB)9. Indeed, SRP targeting itself may prevent cytoplasmic folding of native proteins. Nevertheless, the use of SRP-dependent signal sequences may not suffice to achieve protein export in every case, as protein folding is only one of several factors that can interfere with protein translocation. Other possible factorswhich pertain to both post-translational and cotranslational translocation include positive charges in the N-terminal region of the protein attached to the signal sequence10, signal sequence processing defects and the length of the protein11. Systematic study of these factors should be fruitful both for researchers wishing to understand the details of these processes and for those wishing to exploit these processes for biotechnological ends.
1. Steiner, D., Forrer, P., Stumpp, M.T. & Plckthun, A. Nat. Biotechnol. 24, 823831 (2006). 2. Blobel, G. & Dobberstein, B. J. Cell Biol. 67, 835851 (1975). 3. Takeishi, K., Yasumura, M., Pirtle, R. & Inouye, M. J. Biol. Chem. 251, 62596266 (1976). 4. Inouye, H. & Beckwith, J. Proc. Natl. Acad. Sci. USA 74, 14401444 (1977). 5. Huber, D. et al. J. Bacteriol. 187, 29832991 (2005). 6. Huber, D. et al. Proc. Natl. Acad. Sci. USA 102, 1887218877 (2005). 7. Schierle, C.F. et al. J. Bacteriol. 185, 57065713 (2003). 8. Sone, M., Kishigami, S., Yoshihisa, T. & Ito, K. J. Biol. Chem. 272, 61746178 (1997). 9. Kumamoto, C.A. & Gannon, P.M. J. Biol. Chem. 263, 1155411558 (1988). 10. Li, P., Beckwith, J. & Inouye, H. Proc. Natl. Acad. Sci. USA 85, 76857689 (1988). 11. Andersson, H. & von Heijne, G. Embo. J. 12, 683691 (1993).

Drugging the PI3 kinome

Paul Workman, Paul A Clarke, Sandrine Guillard & Florence I Raynaud
Inhibitors of the PI3 kinase family of enzymes show promise for treating brain tumors.
Protein kinases have proved their mettle as drug targets in oncology. The strategy of drugging the cancer kinome1 has led to trastuzumab (Herceptin), targeted to ERBB2/HER2; imatinib (Gleevec), targeted to BRC-ABL, KIT and PDFGR; and gefitinib/erlotinib (Iressa/ Tarceva), targeted to EGFR. Yet the spotlight on protein kinases has overshadowed another
Paul Workman, Paul A Clarke, Sandrine Guillard and Florence I Raynaud are at the Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, 15 Cotswold Road, Sutton, Surrey, UK. e-mail: paul.workman@icr.ac.uk

important potential kinase target, the phosphatidylinositol 3-kinase (PI3 kinase) family of lipid kinases, which has been implicated by a wealth of evidence in the etiology of cancer, inflammation, autoimmune conditions, thrombosis and viral infection2. This situation is about to change. Recent papers by Knight et al.3 in Cell and by Fan et al.4 in Cancer Cell describe the use of a library of small molecules to define patterns of inhibitory selectivity within the PI3 kinase family. As part of this tour de force of chemical biology, the papers demonstrate proof of concept for the exciting potential of compounds that simultaneously inhibit PI3 kinase p110 and mTOR for the treatment of malignant brain tumors (gliomas).

Although the same chemical probe approach revealed that p110 is critical for insulin signaling3 (also demonstrated in ref. 5), the dual p110/mTOR inhibitor showed clear evidence of a therapeutic window without undue toxicity in an animal model of human glioma4. Cancer treatment is the arena in which information from the human genome project is being translated most readily into personalized treatment1. Malignant progression is driven by molecular abnormalities, many of which result in the hijacking of signal transduction pathways2. This causes cancer cells to become highly dependent upon particular signaling pathways, a process known as oncogene addiction. Hence, pharmacologic inhibitors of oncogenic pathways show therapeutic selectivity towards cancer versus normal cells. The PI3 kinase pathway controls a range of cellular processes, including cell growth, survival, differentiation, chemotaxis and metabolism. Responding to receptor tyrosine kinases and Ras, PI3 lipid kinases activate many downstream signaling pathways by generating second messengers, particularly phosphatidylinositol3,4,5-trisphosphate (PIP3) (Fig. 1). The overall family of around 16 PI3 kinases6 includes the four class I lipid kinase isoforms p110, p110, p110 and p110, which generate PIP3. The class IV isoformsknown as PIKKSare protein kinases that monitor genomic integrity (through DNA-PK, ATM, ATR and hSmg-1) or integrate nutrient signaling to regulate cell growth (through mTOR). The importance of each of these PI3 kinase family members in health and disease continues to be defined. Of particular note, p110 is frequently overexpressed and mutated in many cancers, including gliomas and colon, breast, prostate and gynecological tumors, among others6. Moreover, additional players in the PI3 kinase signaling pathway are also commonly deregulated in malignancy. For example, loss of the PTEN phosphatase and overexpression and activation of the upstream receptor tyrosine kinases and the downstream serine/threonine kinase PKB/Akt have been associated with tumorigenesis7 (Fig. 1). Knight et al. and Fan et al. applied libraries of chemical probes to help determine the biological functions of particular PI3 kinase isoforms and to identify therapeutic opportunities for the cognate inhibitors. The paper by Knight et al. is especially innovative in two ways. First, it applies a diverse matrix of chemical structures to probe the function of PI3 kinases using a range of techniques. These include enzyme, cell and whole-animal assays, X-ray crystallography and molecular modeling, as well as chemical synthesis and chemoinformatics. Second, it applies these technologies to help understand the

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