US 20090098627A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0098.627 A1 Darzins et al. (43) Pub. Date: Apr. 16, 2009 (54) METHOD OF IMMOBILIZING A PROTEIN Related U.S. Application Data ORMOLECULEVA A MUTANT DEHALOGENASE THAT IS BOUND TO AN (60) Division of application No. 11/006,031, filed on Dec. MMOBILIZED DEHALOGENASE 6, 2004, now Pat. No. 7,429,472, which is a continua SUBSTRATE AND LINKED DIRECTLY OR tion-in-part of application No. 10/768,976, filed on INDIRECTLY TO THE PROTEIN OR Jan. 30, 2004, now Pat. No. 7,238,842. MOLECULE (60) Provisional application No. 60/444,094, filed on Jan. 31, 2003, provisional application No. 60/474,659, (75) Inventors: Aldis Darzins, Verona, WI (US); filed on May 30, 2003. Lance Encell, Fitchburg, WI (US); Tonny Johnson, Madison, WI Publication Classification (US); Dieter Klaubert, Arroyo (51) Int. Cl. Grande, CA (US); Georgyi V. Los, CI2N II/2 (2006.01) Madison, WI (US); Mark CI2N II/00 (2006.01) McDougall, Arroyo Grande, CA CI2N II/4 (2006.01) (US); Keith V. Wood, Mt. Horeb, WI (US); Monika G. Wood, Mt. (52) U.S. Cl. .......................... 435/176; 435/174; 435/179 Horeb, WI (US); Chad Zimprich, (57) ABSTRACT Stoughton, WI (US) A mutant hydrolase optionally fused to a protein of interest is Correspondence Address: provided. The mutant hydrolase is capable of forming a bond Schwegman, Lundberg, Woessner & Kluth, P.A. with a substrate for the corresponding nonmutant (wild-type) P.O. Box 2938 hydrolase which is more stable than the bond formed between Minneapolis, MN 55402 (US) the wild-type hydrolase and the substrate and has at least two amino acid substitutions relative to the wild-type hydrolase. (73) Assignee: Promega Corporation Substrates for hydrolases comprising one or more functional groups are also provided, as well as methods of using the (21) Appl. No.: 12/220,478 mutant hydrolase and the substrates of the invention. Also provided is a fusion protein capable of forming a stable bond (22) Filed: Jul. 24, 2008 with a substrate and cells which express the fusion protein. Cis TAGGED OR UNTAGGED PROTEINS HYDROLASE SUBSTRATE MODIFIED ano Cis (EC. SUCCINIMIDYL CLUTHTHONE, NTA ECT) | SUBSTRATE BINDING is Y BINDING OF SUBSTRATE-PROTEIN COMPLEX TOMMOBILIZED HYDROLASE f f HYDROLASE SOLID PHASE areer-r s PROTEIN MIXTURE $5. DETECTION Patent Application Publication Apr. 16, 2009 Sheet 1 of 28 US 2009/0098.627 A1 H H H C Nucleophilic displacement of halide CF. Asp106-8. 0. ( group by Asp106 carboxylate HS272 ) Asp106 1CN OV SN HS272 R e G N H Formation of covalent b ester interediate \ h -cs O Q Glu130 /Cso - Trp107 YN N - CF |AeroAsp106- ) Asp106 1Ca O '.... 'a Release of products His2 2- o \- His 2722NH from the active site 6) Activated water (His272) SN ar molecule hydrolyzes COValent intermediate O m O '... W /So Glu130 stabilizes positive Gf130 charge on His272 G130 FIG. IB Patent Application Publication Apr. 16, 2009 Sheet 2 of 28 US 2009/0098.627 A1 60 dhaa MSEIGTGFPFDPHYWEVLGERMHYWDVGPRDGTPWLFLHGNPTSSYLWRNIIPHVAPSHR Dhaa. H272 - - - - - - - - - - - - - - on a mm - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Dhaad106C a as as as a a - - - - - - - - - - - - - - - - - - - - - - - - - - PP so a saab as as as as a useum to a 120 Dhaa CIAPDLIGMGKSDKPDLDYFEDDHVRYLDAFI EALGLEEVVLVIHDWGSALGFHWAKRNP Dhaa. H272 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Dhala. 106C - - - - - - - - - - - - - - - - - - - - - - - - - -a - - - - - - - - - - - - - - - - - - - C- - - - - - - - - - - - 180 Dhaa ERVKGIACMEFIRPIP TWDEWPEFARET FOAFRTADVGRELTIDONAFIEGALPKCVVRP Dhaa. H272 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - asses - - - - - - - - - - - - - - - - - - - - - - - - - - - - Dhaa. D106C - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 240 Dhaa LTEVEMDHYREPFLKPVDREPLWRFPNELPIAGEPANIVALVEAYMNWLHOSPVPKLLEW Dhaal. 272 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ----------------------------- Dhaa. D106C - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 290 Dhaa GTPGVLIPPAEAARLAESLPNCKTVDIGPGLHYLQEDNPDLIGSEIARWLPAL Dhaal. 272 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Dhaa. D106C - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - FIG 2 Patent Application Publication Apr. 16, 2009 Sheet 3 of 28 US 2009/0098.627 A1 TAGGED OR UNTAGGED PROTEINS HYDROLASE SUBSTRATE MODIFIED arO Cis (E.G. SUCCINIMIDYL, GLUTHTHONE, NTAECT) SUBSTRATE BINDING is city BINDING OF SUBSTRATE-PROTEIN COMPLEX TO IMMOBILIZED HYDROLASE f f HYDROLASE O SOLID PHASE DETECTION Patent Application Publication Apr. 16, 2009 Sheet 4 of 28 US 2009/0098.627 A1 s'. HYDROLASE PROTEIN MIXTURE WASHING, ELUTION SUBSTRATE-ANTIBODY COMPLEX AND ANALYSIS IMMOBILIZED HYDROLASE IN AN EPPENDORFF TUBE FIG. 4 Patent Application Publication Apr. 16, 2009 Sheet 5 of 28 US 2009/0098.627 A1 FLUORESCENTLY SUBSTRATE WITH LABELED SUBSTRATE QUENCHER DYE PKA PKA / REGULATORYSUBUNIT N CATALYTICSUBUNIT HYDROLASE HYDROLASE FLUORESCENCE FIG 5A Patent Application Publication Apr. 16, 2009 Sheet 6 of 28 US 2009/0098.627 A1 FLUORESCENTLY s: LABELED SUBSTRATE fi N &** N. PKA REGULATORY SUBUNIT PKA HYDROLASE REGULATORY SUBUNIT BREI - RS res FLUORESCENCE/LUMINESCENCE FIG. 5B Patent Application Publication Apr. 16, 2009 Sheet 7 of 28 US 2009/0098.627 A1 NO CASPASE OR PROTEASE EXPRESSION IN CELS O 8. - REPRESSOR PROTEIN WITH CASPASE/PROTEASE STE P T R REPORTER (LUCIFERASE/EMDROLASE-NO EXPRESSION IN PRESENCE OF CASPASE OR PROTEASE EXPRESSION IN CELLS P T R REPORTER (UCFERASE/HYDROLASE)-- s: LUCIFERASE/FLUORESCENCE FIG, 6A Patent Application Publication Apr. 16, 2009 Sheet 8 of 28 US 2009/0098.627 A1 NO CASPASE OR PROTEASE EXPRESSION IN CELS CASPASE/PROTEASE NG PROMOT TET REPRESSOR - O'E' REPRESSOR REPRESS REPORTER (UCIFERASE/HYDROLASE) -- NO EXPRESSION X PROMOT TET REPRESSOR XTET REPRESSOR PROMOTREPRESSREPORTER (LUCIFERASE/EMDROLASE-LUCIFERASE/FLUORESCENCE FIG. 6B Patent Application Publication Apr. 16, 2009 Sheet 9 of 28 US 2009/0098.627 A1 NO CASPASE OR PROTEASE EXPRESSION IN CELLS CFO-NO EXPRESSION DEGRADATION CASPASE/PROTEASE REPORTER INSTABILITY RECOGNITION DOMAIN SITE IN PRESENCE OF CASPASE OR PROTEASE EXPRESSION IN CELLS OF O / N N DECRA6ATION CASPASE/PROTEASE REPORTER UCFERASE/FLUORESCENCE INSTABILITY RECOGNITION DOMAIN SITE FIG, 6C Patent Application Publication Apr. 16, 2009 Sheet 10 of 28 US 2009/0098627 A1 O 60 120 180 240 TIME (MIN) FIG 7 Patent Application Publication Apr. 16, 2009 Sheet 11 of 28 US 2009/0098.627 A1 FLUORESCEN u ~ C 8 O H FLUORESCEN u ~N~ C 12 O O FLUORESCEN l ~~~~ C 12 H O FLUORESCEN - ~...~~~~ C 14 BIOTINN-nu-0---nu-N-N-1 Cl 18 O H H FLUORESCEIN u --~~~~ C 2 O O ANTHRACENYL ~g --~~~~ C 16 FIG, 8A Patent Application Publication Apr. 16, 2009 Sheet 12 of 28 US 2009/0098.627 A1 'l-'---~.H Biotin-14-C H N --on--1a1a-Cl O H Biotin-X-14-C H H H N-1Non-o-o-o-n-ron-o--~C O O FIG. 8B Patent Application Publication Apr. 16, 2009 Sheet 13 of 28 US 2009/0098.627 A1 4OOOOOO / / 3OOOOOO 20OOOOO 1OOOOOO ol/47, 47, L - L. 1/ WT.17(-) WT.17(+) F.17(-) F.17(+) R.Luc-DhaA chimeras FIG. 9 Patent Application Publication Apr. 16, 2009 Sheet 14 of 28 US 2009/0098.627 A1 R 1 R2 R t 6 ) COO-me- ...O.R. HO 9 coo Ser70 Ser70 A FIG 10 Patent Application Publication Apr. 16, 2009 Sheet 15 of 28 US 2009/0098.627 A1 2 3 FIG 11B Patent Application Publication Apr. 16, 2009 Sheet 16 of 28 US 2009/0098627 A1 BO--WASH -BO+WASH +BO-WASH FIG 11C Patent Application Publication Apr. 16, 2009 Sheet 17 of 28 US 2009/0098.627 A1 Clone Codon 175 Codon 176 Codon 273 TTG (Leu) ATG (Met) TGT (Cys) FIG, 12A Patent Application Publication Apr. 16, 2009 Sheet 18 of 28 US 2009/0098.627 A1 Clone Codon 175 Codon 176 FIG, 12B Patent Application Publication Apr. 16, 2009 Sheet 19 of 28 US 2009/0098.627 A1 -- 35nM HALO TAG 10nM TMR-LIGAND -0-35nM STREPTAVIDIN 10nM TMR-LIGAND O 100 200 300 TIME (SEC) FIG, 13 Patent Application Publication Apr. 16, 2009 Sheet 20 of 28 US 2009/0098.627 A1 TEMPERATURE (C) FIG, 14A O 10 20 30 40 50 60 70 TEMPERATURE (C) FIG, 14B Patent Application Publication Apr. 16, 2009 Sheet 21 of 28 US 2009/0098.627 A1 Patent Application Publication Apr. 16, 2009 Sheet 22 of 28 US 2009/0098.627 A1 a-GST-HRPITMB (450 nm) GST Gs GST DhaA DhaA C C C Cl MN M MNs Streptavidin coated plate FIG, 16A O 100 200 300 400 500 600 Dha (pmol/100ul) FIG, 16B Patent Application Publication Apr. 16, 2009 Sheet 23 of 28 US 2009/0098.627 A1 as a raw at are a as aw axe as as at aw aw aw an axe are a w w w w w w w w w W. My re M ry we ew. We r w w re rw 1051 GCTAGCCAG CTGGCGCGGA TATCGCCACC ATGGGATCCG AAATCGGTAC CGATCGGTC GACCGCGCCT ATAGCGGTGG TACCCTAGGC TTTAGCCATG GlyPhe Pro PheaspproHis TyrValGlu Val Leugly Glu ArgMetHis aw My re. Mw raw w M M W M M w w w w Y1-1aw w w W. M. she Me Me Aw as M. M. M. M. Me Me W raw was a rvae as as a re rae as a r a War as a 1101 AGGCTTCCCC TTCGACCCCC ATTATGTGGA AGTCCTGGGC GAGCGTATGC TCCGAAGGGG AAGCTGGGGG TAATACACCT TCAGGACCCG CTCGCATACG HTyrValAsp ValGlyPro ArgAspGlyThr ProValleu Phelieu His M w. W. M M Aw w My M. 4W M W w w AW M W M W MW W aw MW Aw Mw a 4W 4W MW axe M MY AY A M A Me Y are MY aw MY A Me A M as Me a r a was re. 1151 ACTACGTCGA TGTTGGACCG CGGGATGGCA CGCCTGTGCT GTTCCTGCAC TGATGCAGCT ACAACCTGGC GCCCTACCGT GCGGACACGA CAAGGACGTG GlyAsnProThir