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(12) Patent Application Publication (10) Pub. No.: US 2005/0181366 A1 Ostermeier (43) Pub US 2005O181366A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0181366 A1 Ostermeier (43) Pub. Date: Aug. 18, 2005 (54) MOLECULAR SWITCHES AND METHODS Publication Classification FOR MAKING AND USING THE SAME (51) Int. Cl." ........................... C12O 1/68; G01N 33/53; (75) Inventor: Mark Ostermeier, Baltimore, MD (US) C12P 21/04; C12N 15/74 (52) U.S. Cl. .............................. 435/6; 435/7.1; 435/69.7; Correspondence Address: 435/252.3; 530/350; 435/471 EDWARDS & ANGELL, LLP P.O. BOX 55874 BOSTON, MA 02205 (US) (73) Assignee: Johns Hopkins University, Baltimore, MD (US) (57) ABSTRACT The invention provides molecular Switches which couple (21) Appl. No.: 10/507,466 external signals to functionality and to methods of making and using the Same. The Switches according to the invention (22) PCT Fed: Mar. 10, 2003 can be used, for example, to regulate gene transcription, target drug delivery to Specific cells, transport drugs intra (86) PCT No.: PCT/US03/07380 cellularly, control drug release, provide conditionally active Related U.S. Application Data proteins, perform metabolic engineering, and modulate cell Signaling pathways. Libraries comprising the Switches and (60) Provisional application No. 60/362.588, filed on Mar. expression vectors and host cells for expressing the Switches 11, 2002. are also provided. Patent Application Publication Aug. 18, 2005 Sheet 1 of 10 US 2005/0181366A1 protein whose activity we wish to modulate / a) domain insertion b) conditional heterodinertzation Enzyme-Binding Protein Hybrid. an O Patent Application Publication Aug. 18, 2005 Sheet 2 of 10 US 2005/0181366 A1 FGURE A. Ea -C)Est -ac -actatase a.a. - Ear- 24.25-26-27.28.2s &Saetías5. i:siggestigii-sistianisatisatist:aest s eagaascetgets------- Satsatagica gaagasatctggttca -s." e?t digest wif Eart solate desired fragment 2.25.2s. 27.28.2s & 2S 2. S v sessearrera es coacg tastgattaagatagtctgagcatatggttc-5 fin overhangs with Kenow 3.s -SESSSSSSSS353 -- stctgageatagtggttcesteaéSate Sat-3 al FGURE B. create one randon frat double stranded break by nuclease digestion ---, -o- dephosphorylate pDMC8-Male E s blunt bis(24-286 - OOO desired constructs OOO Patent Application Publication Aug. 18, 2005 Sheet 3 of 10 US 2005/0181366 A1 FGu Re 20, s Xibal x Xbal Xibal ligate DNase Xibal al- 120Obp saw 22 Xaba - a -st malE2 polMC8 2xMa EcoRV e a na ligate pDIMC8-2xMalE Xba ibrary OOOO!.Xaba randomly kocated incremental truncation blunt bla(24-286) map --> Patent Application Publication Aug. 18, 2005 Sheet 4 of 10 US 2005/0181366 A1 Guy RE. 2d Sertion libra 2% maltose soup Arpicillin Ampicillin SCREEN 1 growth? growth? Yes No Yes No area *::: SCREEN 2 growth? growth? Yes No Yes No Patent Application Publication Aug. 18, 2005 Sheet 5 of 10 US 2005/0181366 A1 AG-3A - X) -> est 8-gacggcs. te crgottscraggagaciega s T G.33 s b)Signal Transduction ra ii)s i? FG3 Apoptosis. Differentiation, etc. c) Targeted Drug Delivery ?i 2. surface protein unique or O g is diseasedpresentathigher cellor pathogen level on FG, 3D healthy cell diseased cellor pathogen drug d) Drug Transport gi small molecule, protein or other signal (e.g. pH G. g .) --> drugchange inside ce. protein with domain that is taken up by cells (e.g. transferrin) FC.3E e) Conditionally-active toxic proteins inactive form of toxin active form of toxin healthy cell diseased cellor pathogen E. e.g., FG.3F pathogenE. in diseased cellor f) Metabolic Engineering O s 1. X O FG, 3G NVA/ ea is f Patent Application Publication Aug. 18, 2005 Sheet 6 of 10 US 2005/0181366 A1 (uret n drug and iron elease Patent Application Publication Aug. 18, 2005 Sheet 7 of 10 US 2005/0181366 A1 FG V FESSA - C Protein Wewish to control Patent Application Publication Aug. 18, 2005 Sheet 8 of 10 US 2005/0181366 A1 Patent Application Publication Aug. 18, 2005 Sheet 9 of 10 US 2005/0181366 A1 Gu Re A 2006 GSGGno linker linker 12S OOOO5 GSGGGSGG linker S 3. OOOO4 9.R OOOO3 s Sa Ooooe ) L O oooon Kanamycin (ug/ml) war . Frequency of active heterodimers of Neo whose assembly is assisted by antiparallel leucine zippers. First Amino Acid of C-terminal Fragnent Patent Application Publication Aug. 18, 2005 Sheet 10 of 10 US 2005/0181366 A1 $LIGTOSE) malijioi B-cyclodextrin mallose Sugar (5 nM US 2005/0181366 A1 Aug. 18, 2005 MOLECULAR SWITCHES AND METHODS FOR previously shown to be one tolerant of bisection (Pelletier, MAKING AND USING THE SAME et al., 1998, Proc. Natl. Acad. Sci. USA 95: 12141-12146). The two fragments of DHFR divided at 107 were found to FIELD OF THE INVENTION be unable to reassemble to form an active enzyme unless the fragments were fused to domains that dimerized (e.g., Such 0001. The invention relates to fusion molecules which as leucine zippers). Yeast expressing the FKBP12-DHFR or function as molecular Switches and to methods for making ERC-DHFR fusion proteins had an approximate two-fold and using the same. increase in growth rate in the presence of their respective ligands (FK106 and estrogen) when DHFR activity limited BACKGROUND OF THE INVENTION growth. The fusion proteins were either fortuitously tem 0002 Gene fusion technology, the fusion of two or more perature sensitive (ERC-DHFR) or designed to be so by genes into a Single gene, has been widely used as a tool in mutation (FKBP12-DHFR) in order that subtle changes in protein engineering, localization and purification. There are growth could be detected upon addition of the ligand. two conceptually different methods of making fusions. The 0007 Generally, methods for generating fusion mol Simplest method, end-to-end fusions, has been used almost ecules have not provided a Systematic way to functionally exclusively. The Second method, insertional fusion, com couple protein domains. prises the insertion of one gene into the middle of another gene. Insertions can result in a continuous domain being SUMMARY OF THE INVENTION Split into a discontinuous domain. 0008. The invention provides molecular switches which 0003. One of the first reports of successful insertion of couple external Signals, including, but not limited to, the one protein into another was a study by Ehrmann, et al., presence, absence or level of molecules, ligands, metabo Proc. Natl. Acad. Sci. USA 87: 7574-8, who described the lites, ions, and the like, the presence, absence, or level of insertion of alkaline phosphatase (AP) into the E. coli outer chemical, optical or electrical conditions, to functionality. membrane protein MalF, as a tool for Studying membrane Preferably, the Switches are fusion molecules comprising an topology. High levels of alkaline phosphatase activity were insertion Sequence and an acceptor Sequence for receiving obtained in the fusions despite the fact that alkaline phos the insertion Sequence, wherein the State of the insertion phatase requires dimerization for activity. Since then, AP has Sequence is coupled to the State of the acceptor Sequence. been Successfully inserted into a number of integral mem For example, the activity of the insertion sequence can be brane proteins (see, e.g., Bibi and Beja, 1994, J. Biol. Chem. coupled to the activity/state of the acceptor Sequence. 269: 19910-5; Cosgriff and Pittard, 1997, J. Bacteriol. 179: 3317-23; Lacatena, et al., 1994, Proc. Natl. Acad. Sci. USA 0009. The “state' of a molecule can comprise its ability 91: 10521-5; Pi and Pittard, 1996, J. Bacteriol. 178: 2650-5; or latent ability to emit or absorb light, its ability or latent Pigeon and Silver, 1994, Mol. Microbiol. 14: 871-81). ability to change conformation, its ability or latent ability to bind to a ligand, to catalyze a Substrate, transfer electrons, 0004) Other proteins, including green fluorescent protein and the like. Preferably, molecular Switches according to the GFP (Biondi, et al., 1998, Nucleic Acids Res. 26: 4946-4952; invention are multistable, i.e., able to Switch between at least Kratz, et al., 1999, Proc. Natl. Acad. Sci. USA 96: Siegel and two States. In one aspect, the fusion molecule is bistable, i.e., Isacoff, 1997, Neuron 19: 73541; Siegel and Isacoff, 2000, a state is either “ON” or “OFF", for example, able to emit Methods Enzymol. 327:249-59), TEM1 B-lactamase (Betton, light or not, able to bind or not, able to catalyze or not, able et al., 1997, Nat. Biotechnology 15: 1276-1279; Collinet, et to transfer electrons or not, and So forth. In another aspect, al., 2000, J. Biol. Chem. 275: 17428-33; Ehrmann, et al., the fusion molecule is able to Switch between more than two 1990, Proc. Natl. Acad. Sci. USA 87: 7574-8), thioredoxin States. For example, in response to a particular threshold (Lu, et al., 1995, Biotechnology (NY) 13:366-72); dihy State exhibited by an insertion Sequence or acceptor drofolate reductase (Collinet, et al., 2000, J. Biol. Chem. Sequence, the respective other Sequence of the fusion may 275: 17428-33); FKBP12 (Tucker and Fields, Nat. Biotech exhibit a range of States (e.g., a range of binding activity, a nol. 19: 1042-6); estrogen receptor-O. (Tucker and Fields, range of enzyme catalysis, etc.). Thus, rather than Switching 2000, Supra), and B-xylanase (Ay, et al., 1998, Proc. Natl. from “ON” or “OFF", the fusion molecule can exhibit a Acad. Sci. USA 95: 6613-6618); have been successfully graded response to a Stimulus. More generally, a molecular inserted into other proteins. Such fusions at least partially Switch is one which generates a measurable change in State retain the function of the inserted protein. in response to a signal. 0005) Doi, et al., 1999, FEBS Letters 453: 305-307, 0010. In one aspect, a molecular switch can comprise a describe a fusion which comprises an insertion of the plurality of fusion molecules responsive to a Signal, which f-lactamase inhibiting protein (BLIP) polypeptide into a mediate a function in response to a change in State of at least surface loop of the GFP protein.
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