Compositions And Methods Comprising Variant Microbial Proteases

Abstract

The present invention provides methods for protein engineering. Specifically, the invention provides methods utilizing site evaluation libraries to design libraries that optimize two or more properties of a protein. The present invention also provides variant subtilisins suitable for various uses.

1 . An isolated subtilisin variant of a  Bacillus  subtilisin, wherein said subtilisin variant is a mature form having proteolytic activity and comprising a substitution at one or more positions selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 33, 35, 36, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 64, 65, 66, 67, 71, 72, 73, 76, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106, 107, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 122, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 139, 141, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 203, 204, 206, 207, 209, 210, 212, 213, 214, 215, 216, 217, 218, 219, 220, 222, 223, 224, 225, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 267, 268, 269, 270, 271, 272, 273 and 274, wherein said positions correspond to the amino acid sequence of  B. amyloliquefaciens  subtilisin BPN′ set forth as SEQ ID NO:2. 
     
     
         2 . The isolated subtilisin variant of  claim 1 , wherein said one or more positions is selected from: 1, 2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 22, 23, 24, 25, 26, 28, 29, 30, 31, 33, 35, 36, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 64, 65, 66, 67, 71, 72, 73, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106, 107, 109, 110, 111, 112, 113, 114, 115, 116, 118, 119, 122, 124, 125, 126, 127, 128, 129, 131, 133, 134, 135, 136, 137, 139, 141, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 203, 204, 206, 207, 212, 213, 214, 215, 216, 217, 218, 219, 220, 222, 223, 224, 225, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 267, 268, 269, 270, 271, 272, 273 and 274, wherein said positions correspond to the amino acid sequence of  B. amyloliquefaciens  subtilisin BPN′ set forth as SEQ ID NO:2. 
     
     
         3 . The subtilisin variant of  claim 1 , wherein said one or more positions are selected from 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 21, 22, 23, 25, 26, 28, 29, 30, 31, 33, 35, 36, 37, 39, 40, 41, 42, 44, 46, 47, 48, 49, 50, 51, 53, 54, 55, 56, 57, 58, 59, 60, 64, 65, 66, 67, 71, 73, 77, 78, 79, 81, 82, 83, 84, 85, 86, 88, 89, 90, 91, 92, 93, 94, 95, 96, 102, 105, 106, 110, 111, 112, 113, 114, 115, 116, 122, 124, 125, 128, 129, 131, 133, 134, 135, 136, 137, 139, 141, 143, 145, 146, 147, 148, 149, 150, 151, 152, 153, 156, 157, 158, 159, 160, 162, 165, 166, 167, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 186, 187, 189, 190, 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 203, 204, 206, 207, 212, 214, 216, 217, 218, 219, 220, 222, 223, 224, 225, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 249, 250, 251, 253, 254, 255, 257, 258, 259, 260, 261, 262, 263, 264, 265, 267, 268, 269, 270, 271, 272, 273, and 274, wherein said positions correspond to the amino acid sequence of  B. amyloliquefaciens  subtilisin BPN′ set forth as SEQ ID NO:2. 
     
     
         4 . The subtilisin variant of  claim 1 , wherein said subtilisin variant further comprises a substitution at one or more positions selected from the group consisting of: 18, 52, 72, 117, 119, 127, 144, 185, 209 and 213, wherein said positions correspond to the amino acid sequence of  B. amyloliquefaciens  subtilisin BPN′ set forth as SEQ ID NO:2. 
     
     
         5 . The subtilisin variant of  claim 1 , wherein said substitution comprises one or more of: A001C, A001D, A001E, A001F, A001G, A001H, A001I, A001K, A001L, A001M, A001N, A001Q, A001R, A001S, A001V, A001W, Q002A, Q002D, Q002E, Q002G, Q002S, S003A, S003D, S003F, S003G, S003I, S003K, S003L, S003M, S003N, S003P, S003Q, S003R, S003T, S003V, S003W, S003Y, P005C, P005E, P005G, P005N, P005Q, P005T, Y006A, Y006C, Y006E, Y006F, Y006G, Y006K, Y006M, Y006N, Y006P, Y006Q, Y006R, Y006T, Y006V, Y006W, S009A, S009C, S009E, S009F, S009G, S009H, S009K, S009L, S009M, S009P, S009Q, S009R, S009T, S009V, S009W, Q010A, Q010C, Q010E, Q010F, Q010G, Q010H, Q010I, Q010K, Q010L, Q010M, Q010R, Q010S, Q010T, Q010V, Q010W, I011C, I011L, I011T, I011V, I011Y, K012A, K012C, K012D, K012E, K012F, K012G, K012H, K012I, K012N, K012Q, K012S, K012T, K012V, K012W, K012Y, A013C, A013F, A013G, A013H, A013L, A013R, A013S, A013T, A013V, P014A, P014C, P014D, P014E, P014F, P014G, P014I, P014K, P014L, P014M, P014N, P014Q, P014R, P014S, P014T, P014V, P014W, P014Y, A015C, A015D, A015E, A015F, A015G, A015H, A015I, A015K, A015L, A015M, A015P, A015Q, A015R, A015T, A015V, A015Y, L016A, L016C, L016I, L016M, L016Q, L016S, L016T, H017F, H017I, H017L, H017M, H017V, H017W, H017Y, S018A, S018E, S018F, S018G, S018H, S018I, S018K, S018L, S018M, S018N, S018P, S018Q, S018R, S018T, S018V, S018W, S018Y, Q019A, Q019C, Q019D, Q019E, Q019G, Q019I, Q019K, Q019M, Q019R, Q019T, Q019V, G020A, G020C, G020D, G020E, G020F, G020H, G020K, G020M, G020N, G020P, G020Q, G020R, G020S, G020W, Y021A, Y021C, Y021D, Y021E, Y021F, Y021G, Y021H, Y021K, Y021M, Y021R, Y021S, Y021T, Y021V, Y021W, T022A, T022C, T022E, T022F, T022G, T022H, T022I, T022K, T022L, T022M, T022N, T022Q, I022S, T022V, I022W, T022Y, S024C, S024F, S024G, S024H, S024I, S024K, S024L, S024M, S024N, S024Q, S024R, S024T, S024W, S024Y, N025C, N025E, N025F, N025G, N025H, N025I, N025K, N025L, N025M, N025P, N025R, N025S, N025T, N025V, N025W, K027A, K027D, K027E, K027F, K027G, K027H, K027L, K027M, K027P, K027R, K027S, K027W, K027Y, D032A, D032C, D032E, D032G, D032I, D032K, D032L, D032M, D032N, D032R, D032S, D032T, D032V, D032Y, S033A, S033F, S033G, S033H, S033P, S033T, S033W, I035A, I035C, I035D, I035R, I035S, I035T, I035V, D036A, D036C, D036E, D036Q, D036S, D036W, S037A, S037C, S037E, S037F, S037G, S037H, S037K, S037L, S037M, S037P, S037Q, S037R, S037T, S037W, H039C, H039Q, H039T, H039V, P040A, P040C, P040E, P040F, P040G, P040G, P040H, P040I, P040K, P040L, P040M, P040N, P040Q, P040R, P040S, P040T, P040V, P040W, D041E, L042I, L042M, L042V, K043A, K043C, K043D, K043E, K043F, K043G, K043I, K043L, K043M, K043N, K043R, K043S, K043T, K043V, K043W, K043Y, V044A, V044C, V044I, V044L, V044M, V044P, V044S, V044T, A045C, A045E, A045F, A045H, A045I, A045K, A045L, A045M, A045N, A045P, A045Q, A045S, A045T, A045V, A045Y, G046A, G046C, G046E, G046H, G046K, G046M, G046N, G046Q, G046T, G046W, G046Y, A048C, A048D, A048E, A048F, A048H, A048I, A048K, A048L, A048M, A048Q, A048R, A048S, A048T, A048V, A048W, A048Y, M050A, M050C, M050F, M050H, M050I, M050K, M050L, M050Q, M050R, M050S, M050T, M050V, M050W, M050Y, V051A, V051C, V051D, V051E, V051H, V051I, V051L, V051P, V051Q, P052C, P052D, P052E, P052F, P052H, P052I, P052K, P052L, P052Q, P052R, P052S, P052T, P052V, P052W, P052Y, S053E, S053F, S053G, S053H, S053I, S053K, S053L, S053M, S053N, S053Q, S053R, S053T, S053V, S053W, E054A, E054N, E054Q, E054S, T055A, T055C, T055D, T055F, T055G, T055H, T055I, T055K, T055M, T055P, T055Q, T055R, T055S, T055V, T055W, T055Y, N056D, N056M, N056P, N056Q, N056S, N056T, N056V, P057N, P057Q, P057T, P057V, P057W, F058C, F058E, F058G, F058H, F058L, F058M, F058N, F058Q, F058S, F058V, F058Y, Q059A, Q059C, Q059D, Q059E, Q059F, Q059G, Q059H, Q059K, Q059L, Q059M, Q059N, Q059P, Q059R, Q059S, Q059T, Q059V, Q059W, Q059Y, D060E, D060G, H064C, H064D, H064F, H064I, H064L, H064M, H064Q, H064R, H064S, H064T, H064V, H064Y, T066S, T066W, T066Y, H067A, H067C, H067F, H067I, H067L, H067M, H067N, H067P, H067R, H067S, H067T, T071I, T071Y, N077S, S078A, S078C, S078D, S078F, S078G, S078I, S078K, S078L, S078M, S078N, S078P, S078Q, S078R, S078T, S078V, S078W, S078Y, I079A, I079C, I079E, I079F, I079G, I079H, I079K, I079L, I079N, I079Q, I079R, I079S, I079T, I079V, I079W, I079Y, V081F, V081I, V081L, V081M, V081T, G083F, G083P, Q084A, Q084C, Q084H, Q084I, Q084N, Q084T, A085C, A085F, A085G, A085R, A085S, P086A, P086D, P086E, P086G, P086M, P086N, P086Q, P086R, P086S, P086T, P086W, P086Y, S089C, S089D, S089E, S089F, S089G, S089H, S089K, S089L, S089V, S089W, S089Y, L090A, L090D, L090E, L090G, L090H, L090M, L090Q, L090T, L090V, Y091A, Y091C, Y091D, Y091E, Y091F, Y091H, Y091I, Y091L, Y091M, Y091Q, Y091S, Y091T, Y091V, L096F, L096H, L096I, L096K, L096M, L096T, L096V, G097A, G097C, G097D, G097E, G097H, G097K, G097L, G097M, G097P, G097Q, G097R, G097S, G097T, G097V, A098C, A098D, A098F, A098G, A098H, A098I, A098L, A098P, A098Q, A098R, A098S, A098T, A098V, A098Y, D099C, D099E, D099I, D099K, D099L, D099N, D099P, D099Q, D099R, D099S, D099T, S101A, S101C, S101E, S101F, S101G, S101I, S101K, S101L, S101M, S101N, S101P, S101Q, S101R, S101T, S101V, S101Y, G102A, G102C, G102E, G102F, G102I, G102N, G102S, G102V, G102Y, Q103A, Q103C, Q103E, Q103F, Q103G, Q103I, Q103K, Q103L, Q103M, Q103N, Q103R, Q103S, Q103T, Q103V, Q103W, S105A, S105C, S105D, S105E, S105F, S105G, S105I, S105K, S105L, S105M, S105N, S105P, S105R, S105V, S105W, W106A, W106C, W106E, W106F, W106G, W106H, W106I, W106L, W106M, W106N, W106R, W106S, W106T, W106V, W106Y, N109A, N109C, N109E, N109F, N109G, N109H, N109L, N109M, N109P, N109Q, N109R, N109S, N109T, N109V, N109W, N109Y, I111C, I111F, I111L, I111M, I111N, I111P, I111T, I111V, I111W, E112A, E112C, E112D, E112F, E112G, E112I, E112L, E112M, E112N, E112Q, E112R, E112S, E112T, E112V, E112W, E112Y, W113D, W113E, W113F, W113N, W113P, W113Q, W113S, W113V, W113Y, I115E, I115H, I115N, I115R, I115T, I115V, I115W, I115Y, A116C, A116D, A116E, A116F, A116G, A116H, A116I, A116K, A116L, A116M, A116N, A116P, A116Q, A116R, A116S, A116T, A116V, A116W, A116Y, N118A, N118C, N118D, N118E, N118F, N118G, N118H, N118I, N118K, N118L, N118M, N118Q, N118R, N118S, N118T, N118V, N118W, N118Y, I122A, I220, I122H, I122K, I122L, I122M, I122N, I122P, M124C, M124D, M124I, M124L, M124S, M124T, M124V, M124W, S125A, S125C, S125E, S125F, S125I, S125K, S125M, S125N, S125P, S125Q, S125R, S125T, S125W, L126A, L126C, L126I, L126K, L126N, L126R, L126S, L126T, L126V, L126W, L126Y, G127A, G127C, G127E, G127F, G127I, G127K, G127M, G127N, G127P, G127Q, G127S, G127T, G127V, G127W, G127Y, G128A, G128D, G128F, G128H, G128L, G128N, G128P, G128S, G128T, G128V, G128W, G128Y, P129A, P129C, P129D, P129E, P129F, P129G, P129I, P129K, P129L, P129M, P129N, P129Q, P129S, P129T, P129V, P129Y, G131A, G131C, G131E, G131F, G131K, G131L, G131M, G131N, G131P, G131Q, G131R, G131S, G131T, G131V, G131W, G131Y, A133C, A133D, A133E, A133F, A133G, A133I, A133K, A133L, A133M, A133P, A133R, A133S, A133T, A133V, A133W, A133Y, A134D, A134E, A134F, A134G, A134I, A134K, A134L, A134M, A134P, A134Q, A134R, A134S, A134I, A134V, A134W, L135D, L135E, L135M, L135R, L135W, L135Y, K136E, K136H, K136L, K136M, K136N, K136R, K136S, K136T, K136V, K136W, K136Y, A137C, A137E, A137F, A137H, A137K, A137L, A137M, A137N, A137P, A137Q, A137R, A137S, A137T, A137V, A137W, A137Y, V139C, V139I, V139L, V139N, V139S, V139T, K141A, K141C, K141D, K141E, K141F, K141G, K141H, K141I, K141L, K141M, K141N, K141Q, K141R, K141S, K141V, K141W, K141Y, V143A, V143C, V143D, V143E, V143F, V143G, V143K, V143L, V143M, V143N, V143Q, V143R, V143S, V143T, V143W, A144C, A144D, A144E, A144G, A144I, A144K, A144L, A144M, A144R, A144S, A144T, A144V, A144W, S145A, S145C, S145D, S145E, S145F, S145G, S145H, S145I, S145L, S145M, S145Q, S145R, S145T, S145V, S145W, S145Y, G146A, G146C, G146D, G146E, G146M, G146Q, G146R, G146S, G146T, G146Y, A153S, G154L, G154P, G154T, E156A, E156C, E156F, E156K, E156L, E156N, E156Q, E156R, E156S, E156T, E156V, E156W, E156Y, T158A, T158D, T158E, I158G, T158H, I158I, T158K, T158L, T158M, T158N, T158Q, T158S, T158V, T158Y, S159A, S159C, S159D, S159E, S159G, S159H, S159I, S159K, S159M, S159Q, S159R, S159T, G160D, G160E, G160K, G160N, G160P, G160Q, G160S, G160T, S162A, S162C, S162E, S162H, S162K, S162L, S162M, S162N, S162Q, S162R, S162T, S162V, S163G, S163P, Y167A, Y167F, Y167H, Y167I, P168D, P168G, P168I, P168M, P168S, P168Y, G169A, G169E, G169F, G169H, G169I, G169M, G169N, G169R, G169T, G169V, G169Y, K170A, K170C, K170F, K170G, K170H, K170R, K170V, K170W, K170Y, Y171G, Y171K, Y171P, P172A, P172C, P172E, P172K, P172L, P172M, P172N, P172Q, P172R, P172S, P172T, P172V, P172Y, S173A, S173C, S173E, S173I, S173T, S173V, V174C, V174F, V174H, V174R, V174T, I175G, I175L, I175M, I175R, I175T, I175V, A176C, A176E, A176F, A176K, A176M, A176S, A176Y, A179G, V180A, V180C, V180L, V180N, V180S, V180T, D181C, D181E, D181G, D181H, D181M, D181N, D181S, D181T, D181W, S182A, S182C, S182D, S182F, S182G, S182H, S182I, S182K, S182M, S182P, S182Q, S182R, S182V, S182Y, S183A, S183C, S183E, S183F, S183G, S183H, S183I, S183K, S183L, S183M, S183N, S183Q, S183R, S183T, S183V, S183W, S183Y, N184C, N184D, N184E, N184G, N184H, N184K, N184L, N184M, N184Q, N184S, N184T, N184V, N184W, Q185A, Q185C, Q185E, Q185F, Q185G, Q185H, Q185I, Q185K, Q185L, Q185M, Q185N, Q185S, Q185T, Q185V, Q185W, R186I, R186L, R186W, A187C, A187D, A187E, A187F, A187G, A187P, A187S, A187W, A187Y, S188A, S188C, S188D, S188E, S188F, S188G, S188H, S188I, S188K, S188L, S188M, S188P, S188Q, S188T, S188V, S188W, S188Y, S190F, S190H, S190I, S190K, S191A, S191G, S191N, S191P, V192A, V192S, V192T, V192Y, G193F, G193H, G193I, G193N, G193P, G193R, G193T, P194C, P194E, P194H, P194I, P194K, P194L, P194M, P194Q, P194T, P194V, P194W, L196A, M199P, M199S, A200C, A200G, A200K, A200Y, G202D, G202E, G202F, G202L, G202P, G202V, G202Y, Q206A, Q206C, Q206D, Q206E, Q206F, Q206G, Q206H, Q206I, Q206L, Q206M, Q206N, Q206P, Q206R, Q206S, Q206T, Q206V, Q206W, Q206Y, S207D, S207E, S207K, S207Q, S207T, S207V, P210A, P210C, P210S, P210T, N212A, N212C, N212E, N212F, N212G, N212H, N212K, N212L, N212M, N212P, N212Q, N212R, N212S, N212V, K213A, K213C, K213D, K213E, K213F, K213H, K213I, K213L, K213M, K213N, K213Q, K213R, K213S, K213T, K213V, K213Y, Y214W, G215A, G215C, G215D, G215E, G215I, G215M, G215N, G215Q, G215S, G215T, G215V, A216C, A216D, A216E, A216G, A216K, A216L, A216M, A216N, A216P, A216Q, A216R, A216S, A216V, A216W, Y217A, Y217C, Y217D, Y217E, Y217F, Y217G, Y217H, Y217I, Y217K, Y217L, Y217M, Y217N, Y217Q, Y217R, Y217S, Y217T, Y217V, Y217W, N218A, N218C, N218E, N218F, N218G, N218H, N218K, N218M, N218R, N218S, N218T, N218W, N218Y, G219A, G219C, G219D, G219H, G219I, G219M, G219P, G219Q, G219R, G219S, G219T, G219V, G219W, T220D, T220E, T220F, T220G, T220K, T220M, T220S, T220Y, A223E, A223F, A223L, A223M, A223R, A223S, A223V, A223W, A223Y, S224A, S224C, S224F, S224G, S224H, S224M, S224N, S224Q, S224R, S224T, P225A, P225C, P225F, P225G, P225H, P225I, P225K, P225L, P225M, P225R, P225S, P225I, P225V, P225Y, A228P, A228R, A228S, A228T, A228W, G229A, G229H, G229I, G229S, A230C, A230E, A230G, A230Q, A230R, A230S, A230T, A230V, A231C, A231I, A231P, A231R, I234A, I234C, I234L, I234M, I234N, I234P, I234Q, I234S, I234T, I234V, L235A, L235C, L235G, L235I, L235K, L235M, L235N, L235Q, L235R, L235S, L235T, L235V, L235W, L235Y, S236A, S236C, S236D, S236E, S236G, S236H, S236N, S236Q, S236T, S236V, S236Y, K237A, K237E, K237F, K237G, K237H, K237I, K237L, K237M, K237N, K237Q, K237R, K237S, K237T, K237V, K237W, K237Y, H238C, H238D, H238E, H238F, H238M, H238R, H238S, P239C, P239D, P239E, P239F, P239H, P239L, P239M, P239N, P239Q, P239R, P239S, P239T, P239V, P239W, P239Y, N240A, N240C, N240D, N240F, N240G, N240K, N240L, N240Q, N240R, N240S, N240T, N240V, N240W, N240Y, W241A, W241F, W241G, W241H, W241I, W241K, W241M, W241Q, W241T, W241V, T242A, T242C, T242E, T242F, T242G, T242K, T242M, T242N, T242P, T242R, T242S, T242W, T242Y, N243C, N243E, N243F, N243G, N243I, N243Q, N243S, N243T, N243V, N243W, N243Y, T244A, T244D, T244F, T244G, T244H, T244K, T244L, T244M, T244N, T244P, T244Q, T244R, T244S, T244V, T244W, T244Y, Q245A, Q245C, Q245D, Q245E, Q245H, Q245I, Q245K, Q245L, Q245M, Q245R, Q245T, Q245V, Q245Y, R247W, S248A, S248E, S248F, S248G, S248H, S248I, S248L, S248M, S248N, S248Q, S248R, S248T, S248V, S248W, S248Y, S249C, S249D, S249H, S249I, S249K, S249L, S249M, S249N, S249Q, S249R, S249T, S249V, S249W, S249Y, L250D, L250F, L250H, L250I, L250M, L250T, L250V, E251A, E251T, E251W, N252A, N252C, N252E, N252G, N252H, N252I, N252L, N252M, N252Q, N252R, N252S, N252T, N252V, N252Y, T253A, T253C, T253E, T253G, T253H, T253K, T253M, T253S, T253V, T254A, T254C, T254L, T254M, T254R, T254S, T254V, T255A, T255C, T255D, T255E, T255F, T255G, T255H, T255I, T255K, T255L, T255M, T255R, T255S, T255V, K256A, K256C, K256D, K256E, K256F, K256H, K256I, K256L, K256M, K256N, K256P, K256Q, K256R, K256S, K256T, K256V, K256W, K256Y, L257A, L257C, L257F, L257G, L257H, L257I, L257K, L257M, L257N, L257R, L257S, L257T, L257V, L257W, L257Y, G258Q, D259A, D259E, D259F, D259G, D259L, D259N, D259P, D259Q, D259R, D259S, D259T, D259V, D259W, D259Y, S260A, S260C, S260D, S260E, S260F, S260G, S260H, S260L, S260M, S260N, S260P, S260R, S260V, S260W, S260Y, F261H, F261W, Y262A, Y262C, Y262E, Y262F, Y262H, Y262L, Y262M, Y262N, Y263F, Y263M, Y263T, G264F, G264I, G264L, K265A, K265C, K265E, K265G, K265H, K265L, K265M, K265N, K265Q, K265R, K265S, K265W, K265Y, G266C, G266F, G266L, G266M, G266P, G266R, G266V, G266W, G266Y, L267A, L267C, L267E, L267G, L267H, L267I, L267M, L267N, L267Q, L267S, L267T, L267V, I268A, I268C, I268K, I268L, I268M, I268P, I268R, I268V, N269D, N269E, N269K, N269L, N269P, N269Q, N269S, Q271A, Q271C, Q271D, Q271E, Q271F, Q271G, Q271H, Q271I, Q271K, Q271L, Q271M, Q271N, Q271P, Q271R, Q271S, Q271T, Q271V, Q271W, Q271Y, A272E, A272F, A272G, A272H, A272K, A272L, A272M, A272N, A272Q, A272R, A272S, A272T, A272V, A272W, A272Y, A274C, A274D, A274F, A274G, A274H, A274I, A274K, A274L, A274Q, A274S, A274T, and A274V, wherein said positions correspond to the amino acid sequence of  B. amyloliquefaciens  subtilisin BPN′ set forth as SEQ ID NO:2. 
     
     
         6 . The subtilisin variant of  claim 1 , wherein said substitution comprises a combination of substitutions selected from: Y021H-Y217E, Y021H-Y217Q, S101E-Y217L, Y021H-Y217L, K213I-Y217Q, A045I-Y217Q, M119F-Y217Q, A045V-Y217L, Y021H-Y217L, K213I-Y217E, A092G-A114G, Y021W-S101E, V26Q-K213I, and Y021H-S101N, wherein said positions correspond to the amino acid sequence of  B. amyloliquefaciens  subtilisin BPN′ set forth as SEQ ID NO:2. 
     
     
         7 . The subtilisin variant of  claim 1 , wherein said substitution comprises a combination selected from: S101 N-K213I, S101N-M119H, K213L, M119N-K213N, K213N, K213I-Y217E, M119N-K213I, K213N-Y217Q, M119H-K213N, S101P-K213N, M119H-Y217Q, K213I-Y217Q, M119F-K213L, Y217Q, M119F-Y217Q, A048E-K213L, A048H-K213L, K213N, V026N-K213L, V026N-K213L, V026Y-K213N, A048D-K213N, A048H-K213N, A048H-K213L, K213L, K213N, V147D-K213L, K213I, A048E-K213L, A048D-K213N, K213I, A048H-K213N, V147D-K213N, Y021H-Y217L, Y021H-Y217Q, A045V-Y217L, S101E-Y217L, Y021H-Y217E, A045I-Y217Q, Y021H-Y217L, Y021H-Y217E, wherein said positions correspond to the amino acid sequence of  B. amyloliquefaciens  subtilisin BPN′ set forth as SEQ ID NO:2. 
     
     
         8 . The subtilisin variant of  claim 1 , wherein said substitution comprises a combination selected from: S101N-M119H, Y217L, M119N-K213N, S101E-Y217L, A045I-Y217Q, wherein said positions correspond to the amino acid sequence of  B. amyloliquefaciens  subtilisin BPN′ set forth as SEQ ID NO:2. 
     
     
         9 . An isolated nucleic acid encoding the subtilisin variant set forth in  claim 1 . 
     
     
         10 . An expression vector comprising the nucleic acid of  claim 9 . 
     
     
         11 . A host cell comprising the expression vector set forth  claim 10 . 
     
     
         12 . A cleaning composition comprising the subtilisin variant of  claim 1 . 
     
     
         13 . The cleaning composition of  claim 12 , wherein said cleaning composition is a laundry detergent. 
     
     
         14 . The cleaning composition of  claim 13 , wherein said laundry detergent is a cold water detergent, a low pH detergent, or a compact detergent. 
     
     
         15 . A method for producing a subtilisin variant of a  Bacillus  subtilisin, comprising the steps:
 a) transforming a host cell with an expression vector comprising a nucleic acid encoding the subtilisin variant of  claim 1 ; and   b) cultivating the transformed host cell under conditions suitable for the production of the subtilisin variant, to produce a subtilisin variant.   
     
     
         16 . The method of  claim 15 , further comprising the step of harvesting the produced subtilisin variant. 
     
     
         17 . The method of  claim 15 , wherein said host cell is a  Bacillus  species. 
     
     
         18 . The method of  claim 17 , wherein said  Bacillus  species is  B. subtilis.    
     
     
         19 . A method of cleaning, comprising the step of contacting a surface and/or an article comprising a fabric with a cleaning composition comprising an isolated subtilisin variant of  claim 1 . 
     
     
         20 . A method for protease engineering comprising:
 a) providing a plurality of site evaluation libraries (SELs) each comprising a plurality of protease variants having distinct substitutions at an identical amino acid position of said protease;   b) testing said protease variants of said SELs and a standard protease in a test of a property of interest;   c) determining a performance index (PI) for each of said protease variants for said test;   d) identifying two or more of said amino acid positions as non-restrictive positions, wherein at least one of said plurality of protease variants in each of two of said SELs has a PI greater than 0.5; and   f) providing a multiple mutation library comprising a plurality of multiply-substituted protease variants each comprising substitutions in said two or more non-restrictive positions.   
     
     
         21 . The method of  claim 20 , wherein said test comprises two or more different assays selected from the group consisting of stain removal assays (microswatch), LAS stability, EDTA stability, detergent stability assays, and specific activity assays. 
     
     
         22 . The method of  claim 20 , wherein said protease is a bacterial subtilisin. 
     
     
         23 . A method for producing a multiply substituted subtilisin variant of a  Bacillus  subtilisin, comprising:
 a) testing a plurality of singly-substituted subtilisin variants in a first test of a first property and a second test of a second property, wherein the property of a parent subtilisin is given a value of 1.0 in each test, a favorable first or second property has a value greater than 1.0, and an unduly unfavorable first or second property has a value less than about 0.80 or in some preferred embodiments, less than about 0.60;   b) identifying a substitution in at least one of the singly-substituted subtilisin variants that is associated with a favorable first property and which is not associated with an unduly unfavorable second property;   c) identifying a substitution in at least one of the singly-substituted subtilisin variants that is associated with a favorable second property and which is not associated with an unduly unfavorable first property;   d) introducing the substitution from the previous steps into a subtilisin to yield a multiply-substituted subtilisin variant.   
     
     
         24 . The method of  claim 23 , further comprising testing the multiply-substituted subtilisin variant in the first test and the second test, wherein an improved subtilisin variant achieves a value of greater than 1.0 in both of said first and second tests, or a value of greater than 1.0 in the first test and a value of 0.80 to 1.0 in the second test. 
     
     
         25 . The method of  claim 24 , further comprising producing the improved subtilisin variant(s). 
     
     
         26 . The method of  claim 25 , wherein said first and second properties are negatively correlated. 
     
     
         27 . The method of  claim 26 , wherein a favorable first or second property has a value greater than about 1.2. 
     
     
         28 . The method of  claim 24 , wherein an unduly unfavorable first or second property has a value less than about 0.40. 
     
     
         29 . The method of  claim 24 , wherein the first property is stability, and the second property is wash performance. 
     
     
         30 . The method of  claim 29 , wherein said stability comprises stability in detergent and said wash performance comprises blood milk ink (BMI) wash performance in detergent. 
     
     
         31 . The method of  claim 24 , wherein wash performance is tested in a powder or liquid detergent composition having a pH of between 5 and 12.0. 
     
     
         32 . The method of  claim 24 , wherein the parent bacterial subtilisin is a wild type mature form of a  B. amyloliquefaciens  subtilisin BPN′ having an amino acid sequence set forth as SEQ ID NO:2. 
     
     
         33 . The method of  claim 24 , wherein the positions are in a parent subtilisin having a solvent accessible surface (SAS) of greater than about 50%. 
     
     
         34 . The method of  claim 33 , wherein said one or more positions in a parent subtilisin are positions having a solvent accessible surface (SAS) of greater than about 65%.