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(12) United States Patent (10) Patent No.: US 7,094,568 B2 Kozlowski Et Al

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(12) United States Patent (10) Patent No.: US 7,094,568 B2 Kozlowski Et Al US007094568B2 (12) United States Patent (10) Patent No.: US 7,094,568 B2 Kozlowski et al. (45) Date of Patent: Aug. 22, 2006 (54) METHOD FOR PRODUCING PROTEINS WO WO O1, 57198 A3 2, 2002 TAGGED AT THE N- OR C-TERMINUS (75) Inventors: Roland Kozlowski, Babraham (GB); OTHER PUBLICATIONS Michael B. McAndrew, Babraham (GB); Jonathan Michael Blackburn, Bordini, E., and Hamdan, M., “Investigation of Some Covalent and Cambridge (GB); Michelle Anne Noncovalent Complexes by Matrix-assisted Laser Desorption/Ion Mulder, Capetown (ZA); Mitali ization Time-of-flight and Electrospray Mass Spectrometry.” Rapid Commun. Mass Spectrom. 13:1143-1151, John Wiley & Sons, Ltd. Samaddar, Hyderabad (IN) (1999). Cai, J., et al., “Functional Expression of Multidrug Resistance (73) Assignee: Sense Proteomic Ltd., (GB) Protein 1 in Pichia pastoris.” Biochemistry 40:8307-8316, Ameri can Chemical Society (Jun. 2001). (*) Notice: Subject to any disclaimer, the term of this DeRisi, J., et al., “Use of a cDNA microarray to analyse gene patent is extended or adjusted under 35 expression patterns in human cancer,” Nat. Genet. 14:457-460, U.S.C. 154(b) by 477 days. Nature Publishing Co. (1996). Doellgast, G.J., et al., “Sensitive Enzyme-Linked Immunosorbent (21) Appl. No.: 10/114,334 Assay for Detection of Clostridium botulinum Neurotoxins A, B, and E Using Signal Amplification via Enzyme-Linked Coagulation (22) Filed: Apr. 3, 2002 Assay.” J. Clin. Microbiol. 3 1:2402-2409, American Society for Microbiology (1993). (65) Prior Publication Data Giuliani, C.D., et al., “Expression of an active recombinant lysine US 2003 FOOT3811 A1 Apr. 17, 2003 49 phospholipase A myotoxin as a fusion protein in bacteria.” Toxicon 39:1595-1600, Elsevier Science Ltd. (2001). Hara, H., et al., “Molecular cloning and functional expression Related U.S. Application Data analysis of a cDNA for human hepassocin, a liver-specific protein (63) Continuation-in-part of application No. PCT/GB01/ with hepatocyte mitogenic activity,” Biochim. Biophys. Acta 03693, filed on Aug. 17, 2001. 1520:45-53, Elsevier Science B.V. (2001). Rhodes, N., et al., “Expression and Purification of Active Recom (60) Provisional application No. 60/247,995, filed on Nov. binant ATM Protein from Transiently Transfected Mammalian 14, 2000. Cells.” Prot. Express. Purif 22:462-466, Academic Press, Inc. (Aug. 2001). (51) Int. Cl. Staudinger, J., et al., “Interactions among Vertebrate Helix-Loop CI2P 2/06 (2006.01) Helix Proteins in Yeast Using the Two-hybrid System.” J. Biol. CI2N IS/00 (2006.01) Chem. 268:4608-4611. The American Society for Biochemistry and C07K L/00 (2006.01) Molecular Biology, Inc. (1993). C7H 2L/02 (2006.01) Vojtek. A.B., et al., “Mammalian Ras Interacts Directly with the C7H 2L/00 (2006.01) Serine/Threonine Kinase Raf.” Cell 74:205-214, Cell Press (1993). (52) U.S. Cl. ................. 435/69.1; 435/91.1; 435/320.1; Walker, E.A., et al., “Functional Expression, Characterization, and Purification of the Catalytic Domain of Human 11-B- 530/350,536/23.1; 536/25.3: 536/25.32 Hydroxysteroid Dehydrogenase Type 1.” J. Biol. Chem. 276:21343 (58) Field of Classification Search ............... 435/69.1, 21350. The American Society for Biochemistry and Molecular 435/91.1, 320.1; 530/350, 536/23.1, 25.3, Biology, Inc. (2001). 536/25.32 Zwicker, N., et al., “Strep-tag.R II for One-Step Affinity Purification See application file for complete search history. of Active bHLHZip Domain of Human c-Myc,” BioTechniques (56) References Cited 27:368-370 and 372-375, Eaton Publishing Co. (1999). International Search Report for International Patent Application No. U.S. PATENT DOCUMENTS PCT/GB01/03693, mailed Nov. 7, 2002. 6,087,103 A 7/2000 Burmer * cited by examiner 6,239,209 B1 5/2001 Yang et al. Primary Examiner Bennett Celsa 2002/0162622 A1 * 1 1/2002 Gut ............................ 156,233 Assistant Examiner Mark L. Shibuya (74) Attorney, Agent, or Firm Ivor R. Elrifi, Esq.; Mintz Levin Cohn Ferris Glovsky and Popeo PC FOREIGN PATENT DOCUMENTS (57) ABSTRACT WO WO 99/11777 A1 3, 1999 WO WO 99.3921.0 A1 8, 1999 The present invention relates to novel methods of producing WO WO 99.51773 A1 10, 1999 proteins in which one or more domains are full length and WO WOOOO4382 A1 1, 2000 correctly folded and which are each tagged at either the N WO WOOOO9654 A2 2, 2000 or C-terminus with one or more marker moieties and arrays WO WOOOO9654 A3 6, 2000 containing Such proteins, as well as the use of Such proteins WO WO 01/04265 A2 1, 2001 in arrays for rapid screening. WO WO 01/04265 A3 4/2001 WO WO O1, 57198 A2 8, 2001 22 Claims, 6 Drawing Sheets U.S. Patent Aug. 22, 2006 Sheet 1 of 6 US 7,094,568 B2 Hid GFPuy 200 Scal (S) / o o Ndel -lactamase N S B o ge o (ASn)5(His)6 tag C . s go Hpal pMM106H a. (3549bp) 3 N S d s O191a 1 9. BSOMp 'a s non-coding Ooo sequence (672bp) / Oog O Ov NCol P trC Amber Hpa I Stop GTAACAACAACAACAACCACCACCACCACCACCACTAGGGCTCTATGAGT CAATTC, TCTTGTTGGGTGGTGGTGGTGGTGGTGATCCCGAGATACTCA N N N N N H H H H H H (E) G. S M S > ASN-HIS TAG GFP FIG. U.S. Patent Aug. 22, 2006 Sheet 2 of 6 US 7,094,568 B2 Aat NCO 'STforward' TGA ha Glutathione Stransferase gene (in pGSTN) OS-dNTP:dNTP y Nde STreverse 1:3 PCR amplify gene and digest product with Aat II Y 3S TGCAGWCM W\-MMAM-'': ... A W.M. R. PCR amplifieds GST gene Aat exonuclease IIT Y 35 TGCAGCWNM WNM W/03MSM (, M 5 5 CWNM WM//WNM 3 3 TGCAG WWM M//\M- AC'? M 5' nested set of deletions from 3'-end of GST gene; 5 C M. W^M///NM-3' -- 3 IGCAG WYY^^ MACE M 5' all deletions terminate at 5 CM wan/WNM M33. an O-S-dNTP base 3 TGCAG WNM M/M AC WM 5 FIG. 2 U.S. Patent Aug. 22, 2006 Sheet 3 of 6 US 7,094,568 B2 5 CW CCATGGWNM ATGM 3 3 TGCAGWNM GGTACCWYYY TACM WM- (- M. 5 nested Set of 5 CM, CCATGG M. WNMM 3" 3'-recessed deletio 3 TGCAGM GGTACCWm M/YYYYY ACT Y^^^^^ 5' from 3'-end of GST 5 CM CCATGGM MMM- 3 gene, all deletions 3 TGCAGW GGTACCM YM/YYYYY ACT M 5' terminate at a 3 35 TGCAGWNMCM GGTACCYYYYCCATGG WNM WM3M/WNM ACT WWNM 5' O-S-dNTP base mung bean nuclease y 5 C M CCATGGWNM M/M 3 3 GWN GGTACC WNYNM. WNM W S ' nested Set of blunt-end 5 C. M. CCATGGM MM. 3 deletions from 3'-end of GST W GGTACCWYY wm-MM 5 gene, all deletions terminate 5 C M CCATGGWNM WYM 3' a 3'-O-S-dNTP base 3 GW GGTACCM Wm/M- S 5 CWNM CCATGGM M/MTGA M 3 3 GW GGTACCWM M/MACT WW 5 Digest with Nco I and clone fragments in to Nco I/Hpal sites of pMM106H to add tag specifically to nested set of 3'- deletions of GST. Visualise colonies which fluoresce green under uV light to select correctly folded, Y in-frame fusions to the His tag U.S. Patent Aug. 22, 2006 Sheet 4 of 6 US 7,094,568 B2 Grow individual colonies in liquid culture and induce expression. O N , ty"Yare bead Lyse cells and add Ni'- 3. -- o, N-H 2 NTA coated magnetic O So-o beads to crude lysate 2 H Nickel-nitrillotracetic acid (NTA) Wash beads to remove other proteins Measure enzymatic ()Masamaawmawwa. acfivitv nn heads 1-chloro-2,4-dinitrobenzene NO2 NO O O 2 ul-ul, H C -O y N -- NO NH4+ - N O n GlutathioneS NH 3 -- O transferase O H Glutathione Amax = 340mm FIG. 4 U.S. Patent Aug. 22, 2006 Sheet S of 6 US 7,094,568 B2 FIG. 5 T5 promoter / Dra III (126) TSma I (175) beta lactamase resistance gene GFP Pst I (920) BCCP Hind III (1180) plFM101-Clstuffer Lambda to transcription terminato 5743 bp ColE1 oigin of replication Nco I (1853) Y rrnBT1 transcription terminator lac repressor coding sequence U.S. Patent Aug. 22, 2006 Sheet 6 of 6 US 7,094,568 B2 F.G. 6 cDNA library in plasmid form (pTripi Ex2) W Linearise plasmids by digestion at Not restriction site downstream of cDNA inserts W Digest the 3’-ends of the linearised duplex plasmid DNA in 3’-5’ direction using exonuclease III under controlled conditions to generate a nested set of 3'-deletions w Remove single-stranded DNA overhangs with Mung Bean Nuclease to render the nested deletions blunt w Digest at Sfil restriction site upstream of cDNA inserts to release cDNA fragments w Size fractionate cDNA fragments W Ligate cDNA fragments into DraIEI SmaI sites of pIFM101-A/stuffer, -B/stuffer & -C/stuffer W Transform E. coli cells with library and plate onto solid media w Induce protein expression W Identify and pick clones based on folding marker activity (i.e. fluorescence) w Further characterise and store library of solubly expressed, folded, C-terminally tagged proteins US 7,094,568 B2 1. 2 METHOD FOR PRODUCING PROTEINS protein interactions. Functional proteome expression arrays, TAGGED AT THE N- OR C-TERMINUS or “proteome chips, will enable the specificity of protein protein interactions and the specificity of any drug-mediated CROSS-REFERENCE TO RELATED effect to be determined in an in vitro format. They will APPLICATIONS therefore have enormous potential because they will simply revolutionize this area of research.
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