Display of polypeptides on bacteriophage (phage display) is a selection technique that allows to extract polypeptides with desired properties from a large collection of variants (Russel, M., Lowman, H. B., and Clackson, T., Introduction to phage biology and phage display, in “Phage Display”, Clackson, T. and Lowman, H. B., eds., Oxford University Press, 2004, pp. 1-26). Phage display has been intensively investigated for the selection from combinatorial antibody or peptide libraries.
By far the most widely used bacteriophages used in phage display are filamentous phages. Filamentous phages constitute a large family of bacterial viruses that infect many Gram-negative bacteria. The best-known filamentous phages are those that infect Escherichia coli; these are f1/M13/fd and IKe. Phages f1, M13, and fd are those that have so far been used for filamentous phage display. Their genomes are more than 98% identical and their gene products are interchangeable.
A unique aspect of filamentous phage assembly, in contrast to the assembly of many other bacteriophages, is that it is a secretory process. Incorporation of coat polypeptides into the growing phage occurs in the cytoplasmic membrane, and nascent phages are extruded from the cell as they assemble (Russel et al., loc. cit.). The E. coli cell does not lyse in this process. The five viral coat proteins (pIII, pVI, pVII, pVIII and pIX) are inserted in the cytoplasmic membrane prior to their incorporation into phage particles (FIG. 1). For example, the major part of pIII is translocated across the membrane into the periplasm, while its C-terminal hydrophobic tail anchors the protein in the membrane.
One prerequisite for filamentous phage display is the translocation of the polypeptide of interest (POI) across the cytoplasmic membrane. This is normally achieved by genetically fusing the POI to a phage coat protein and translocation of the corresponding fusion polypeptide. Alternatively, the POI and phage coat protein are translocated independently.
In this situation the POI is stably linked to the phage particle in the periplasm by, for example, formation of a disulfide bond (Cys-Display) or formation of a leucine-zipper (pJuFo system) with a corresponding phage coat protein. In conventional filamentous phage display using fusions to pIII, the Sec pathway is used for translocation of the fusion polypeptide comprising the POI. In this pathway, the polypeptide is first synthesized at the ribosome and then posttranslationally translocated, in its unfolded state, by the Sec translocon (FIG. 2, (3)-(4)-(5)). That is, the translocation across the cytoplasmic membrane begins only after a substantial amount of the polypeptide chain has been synthesized. However, the contribution of the mechanism of translocation for the success of phage display has not been fully elucidated, and the possibility to use a cotranslational translocation pathway was not explored in the prior art.
Intracellular and extracellular proteins of a wide range of sizes and structures have been functionally displayed on filamentous phage (Russel et al., loc. cit.). Nevertheless, some polypeptides are recalcitrant to display due to individual properties, mostly because of unknown reasons. This makes the success of the display of a certain protein unpredictable. Thus, it has usually been recommended to first test the efficiency of display on filamentous phage for each protein to be used. In addition, when a combinatorial library is created for phage display, not all clones will display with similar efficiency; this is especially true for libraries generated from cDNAs. The display problems of polypeptides may be a result of their interference with the phage production, their periplasmic aggregation, their proteolysis, their toxicity to E. coli or their incompatibility with the used translocation pathway. Especially, the step preceding translocation is an important factor influencing the incorporation of the fusion polypeptides into the phage particles. If the polypeptides fold prematurely, they can be refractory to translocation or even exhibit cytoplasmic toxicity. Thus, it is important whether the protein is translocated posttranslationally (potentially allowing premature folding) or cotranslationally (not permitting cytoplasmic folding). Current filamentous phage display methods use posttranslational pathways for translocation of the fusion polypeptide across the cytoplasmic membrane (Russel et al., loc. cit.; Paschke, M. and Höhne, W., Gene 350, 79-88, 2005). Thus, polypeptides incompatible with these pathways will be refractory to display, making phage display selections very inefficient or even impossible. For example, the posttranslational Sec pathway, which is almost exclusively used in phage display, is inherently incapable of translocating proteins that cannot remain in an unfolded state in the cytoplasm, since the Sec translocon itself can only transport unfolded polypeptides (Huber, D., Boyd, D., Xia, Y., Olma, M. H., Gerstein, M., and Beckwith, J., J. Bacteriol. 187, 2983-2991, 2005; Paschke et al., loc. cit.).
Thus, the technical problem underlying the present invention is to identify novel translocation approaches for the efficient display of those polypeptides on filamentous phages that are displayed inefficiently by using posttranslational translocation. The solution to this technical problem is achieved by providing the embodiments characterized in the claims.
USPTO Backgrounds
USPTO Backgrounds