GENETICALLY MODIFIED MICRORGANISMS THAT CARRY OUT THE HETEROLOGOUS PRODUCTION OF MODIFIED VERSIONS OF THE SURFACTANT PROTEIN LV-RANASPUMIN-1(LV-RSN-1), THE MODIFIED VERSIONS OF SAID SURFACTANT PROTEIN, THE SYNTHETIC GENES ENCODING SAID SURFACTANT PROTEIN, THE EXPRESSION CASSETTES CONTAINING SAID SYNTHETIC GENES, AND THE EXPRESSION VECTORS CONTAINING SAID SYNTHETIC GENES

Abstract

The present invention refers to the heterologous production in microorganisms of modified versions of a predicted isoform of the surfactant protein Lv-ranaspumin-1 (Lv-Rsn-1), whose sequence was inferred from analyzes of the protein extract of the nest foam from the Northeastern Pepper Frog (Leptodactylus vastus). More specifically, it refers to two surfactant proteins that consist of modified versions of the predicted isoform of Lv-Rsn-1; to two synthetic genes each encoding one of these modified versions of the predicted isoform of Lv-Rsn-1; to two expression cassettes each containing one of the synthetic genes encoding one of the modified versions of the predicted isoform of Lv-Rsn-1; to two expression vectors each containing one of the synthetic genes encoding modified versions of the predicted isoform of Lv-Rsn-1; and to two transgenic microorganisms, a bacterium and a yeast, each transformed with one of these synthetic genes and heterologously producing one of the modified versions of the predicted isoform of Lv-Rsn-1. Lv-Rsn-1 has surfactancy, emulsification and dispersancy properties, among others, and its heterologous production allows it to be used in various applications and industrial products, without the need to extract it from the frog nest foam.

Claims

1. A POLYNUCLEOTIDE characterized in that it encodes the predicted sequence of an isoform of the surfactant protein Lv-ranaspumin-1 (Lv-Rsn-1) and consisting of SEQ ID NO:2.

2. A POLYNUCLEOTIDE characterized in that it encodes the predicted sequence of an isoform of the surfactant protein Lv-ranaspumin-1 (Lv-Rsn-1) and in that it has the codon frequency optimized for expression in bacteria, consisting of SEQ ID NO:3.

3. A POLYNUCLEOTIDE characterized in that it encodes the predicted sequence of an isoform of the surfactant protein Lv-ranaspumin-1 (Lv-Rsn-1) and in that it has the codon frequency optimized for expression in yeast, consisting of SEQ ID NO:7.

4. A POLYPEPTIDE characterized in that it is a modified version of an isoform of the surfactant protein Lv-ranaspumin-1 (Lv-Rsn-1) and consisting of SEQ ID NO:6.

5. A POLYPEPTIDE characterized in that it is a modified version of an isoform of the surfactant protein Lv-ranaspumin-1 (Lv-Rsn-1) and consisting of SEQ ID NO:10.

6. AN EXPRESSION CASSETTE, characterized in that it comprises a polynucleotide according to claim 2 operably linked to a promoter that directs expression in bacteria.

7. AN EXPRESSION CASSETTE characterized in that it comprises a polynucleotide according to claim 3 operably linked to a promoter that directs expression in fungi, preferably in yeast.

8. AN EXPRESSION VECTOR characterized in that it comprises an expression cassette according to claim 6.

9. AN EXPRESSION AND TRANSFORMATION VECTOR characterized in that it comprises an expression cassette according to claim 7.

10. A GENETICALLY MODIFIED MICRO-ORGANISM characterized in that it is a bacterium that produces a protein whose encoding sequence comprises the polynucleotide according to claim 6.

11. A GENETICALLY MODIFIED MICRO-ORGANISM characterized in that it is a yeast that produces a protein whose encoding sequence comprises the polynucleotide according to claim 7.

12. A PROCESS OF PRODUCTION OF GENETICALLY MODIFIED ORGANISM characterized in that it results in a bacterium according to claim 10 and comprises: a) transforming a bacterial strain with the expression cassette according to claim 6; b) selecting the transformed bacteria.

13. A GENETICALLY MODIFIED ORGANISMS PRODUCTION PROCESS characterized in that it results in a yeast according to claim 11 and comprises: a) transforming a yeast strain with the expression cassette according to claim 7; b) selecting the transformed yeasts.

14. A PRODUCT characterized in that it comprises a polypeptide according to claim 4.

15. A PRODUCT characterized in that it comprises a polypeptide according to claim 5.

16. AN ADVANCED OIL RECOVERY PROCESS AND IMPROVEMENT OF RESERVOIR FLUID DYNAMICS, using a biosurfactant protein obtained from a genetically modified organism, according to claims 1 to 15, characterized in that the genetically modified organism is capable of synthesizing the biosurfactant protein Lv-ranaspumin-1.

17. AN OIL BIOREMEDIATION PROCESS, using a biosurfactant protein obtained from a genetically modified organism, according to claims 1 to 15, characterized in that the genetically modified organism is capable of synthesizing the biosurfactant protein Lv-ranaspumin-1.

18. A TANK CLEANING PROCESS IN THE OIL AND GAS INDUSTRY, using a biosurfactant protein obtained from a genetically modified organism, according to claims 1 to 15, characterized in that the genetically modified organism is capable of synthesizing the biosurfactant protein Lv-ranaspumin-1.

Description

[0001] <110> Petróleo Brasileiro S. A.—Petrobras

[0002] <120> GENETICALLY MODIFIED MICROORGANISMS THAT CARRY OUT THE HETEROLOGICAL PRODUCTION OF MODIFIED VERSIONS OF THE SURFACTANT PROTEIN LV-RANASPUMIN-1 (LV-RSN-1), THE MODIFIED VERSIONS OF THIS SURFACTANT PROTEIN, THE SYNTHETIC GENES THAT ENCODE THIS SURFACTANT PROTEIN, THE EXPRESSION CASSETTES CONTAINING THESE SYNTHETIC GENES, AND THE EXPRESSION VECTORS CONTAINING THESE SYNTHETIC GENES

[0003] <160>10

[0004] <210>1

[0005] <211>216

[0006] <212> PRT

[0007] <213> Leptodactylus vastus

[0008] <223> predict sequence for one of the isoforms of the Lv-ranaspumin-1

[0009] <400>1

[0010] Leu Leu Glu Gly Phe Leu Val Gly Gly Gly Val Pro Gly Pro Gly Thr 5 10 15

[0011] Ala Cys Leu Thr Lys Ala Leu Lys Asp Ser Gly Asp Leu Leu Val Glu 20 25 30

[0012] Leu Ala Val Ile Ile Cys Ala Tyr Gln Asn Gly Lys Asp Leu Gln Glu

[0013] 35 40 45

[0014] Gln Asp Phe Lys Glu Leu Lys Glu Leu Leu Glu Arg Thr Leu Glu Arg

[0015] 55 60

[0016] Ala Gly Cys Ala Leu Asp Asp Ile Val Ala Asp Leu Gly Leu Glu Glu

[0017] 70 75 80

[0018] Leu Leu Gly Ser Ile Gly Val Ser Thr Gly Asp Ile Ile Gln Gly Leu

[0019] 85 90 95

[0020] Tyr Lys Leu Leu Lys Glu Leu Lys Ile Asp Glu Thr Val Phe Asn Ala

[0021] 100 105 110

[0022] Val Cys Asp Val Thr Lys Lys Met Leu Asp Asn Lys Cys Leu Pro Lys

[0023] 115 120 125

[0024] Ile Leu Gln Gly Asp Leu Val Lys Phe Leu Asp Leu Lys Tyr Lys Val 130 135 140

[0025] Cys Ile Glu Gly Gly Asp Pro Glu Leu Ile Ile Lys Asp Leu Lys Ile

[0026] 145 150 155 160

[0027] Ile Leu Glu Arg Leu Pro Cys Val Leu Gly Gly Val Gly Leu Asp Asp

[0028] 165 170 175

[0029] Leu Phe Lys Asn Ile Phe Val Lys Asp Gly Ile Leu Ser Phe Glu Gly

[0030] 180 185 190

[0031] Ile Ala Lys Pro Leu Gly Asp Leu Leu Ile Leu Val Leu Cys Pro Asn

[0032] 195 200 205

[0033] Val Lys Asn Ile Asn Val Ser Ser

[0034] 210 215

[0035] <210>2

[0036] <211>648

[0037] <212> DNA

[0038] <213> Artificial Sequence

[0039] <220>

[0040] <221> CDS

[0041] <222> (1) . . . (648)

[0042] <223> encoding sequence of one of the isoforms of Lv-ranaspumin-1 after reverse translation of the predicted amino acid sequence

[0043] <400>2

[0044] ctg ctg gaa ggc ttt ctg gtg ggc ggc ggc gtg ccg ggc ccg ggc acc 48

[0045] Leu Leu Glu Gly Phe Leu Val Gly Gly Gly Val Pro Gly Pro Gly Thr

[0046] 5 10 15

[0047] gcg tgc ctg acc aaa gcg ctg aaa gat agc ggc gat ctg ctg gtg gaa

[0048] 96

[0049] Ala Cys Leu Thr Lys Ala Leu Lys Asp Ser Gly Asp Leu Leu Val Glu

[0050] 20 25 30

[0051] ctg gcg gtg att att tgc gcg tat cag aac ggc aaa gat ctg cag gaa

[0052] 144

[0053] Leu Ala Val Ile Ile Cys Ala Tyr Gln Asn Gly Lys Asp Leu Gln Glu

[0054] 35 40 45

[0055] cag gat ttt aaa gaa ctg aaa gaa ctg ctg gaa cgc acc ctg gaa cgc

[0056] 192

[0057] Gln Asp Phe Lys Glu Leu Lys Glu Leu Leu Glu Arg Thr Leu Glu Arg

[0058] 50 55 60

[0059] gcg ggc tgc gcg ctg gat gat att gtg gcg gat ctg ggc ctg gaa gaa

[0060] 240

[0061] Ala Gly Cys Ala Leu Asp Asp Ile Val Ala Asp Leu Gly Leu Glu Glu

[0062] 65 70 75 80

[0063] ctg ctg ggc agc att ggc gtg agc acc ggc gat att att cag ggc ctg 288

[0064] Leu Leu Gly Ser Ile Gly Val Ser Thr Gly Asp Ile Ile Gln Gly Leu

[0065] 85 90 95

[0066] tat aaa ctg ctg aaa gaa ctg aaa att gat gaa acc gtg ttt aac gcg

[0067] 336

[0068] Tyr Lys Leu Leu Lys Glu Leu Lys Ile Asp Glu Thr Val Phe Asn Ala

[0069] 100 105 110

[0070] gtg tgc gat gtg acc aaa aaa atg ctg gat aac aaa tgc ctg ccg aaa

[0071] 384

[0072] Val Cys Asp Val Thr Lys Lys Met Leu Asp Asn Lys Cys Leu Pro Lys

[0073] 115 120 125

[0074] att ctg cag ggc gat ctg gtg aaa ttt ctg gat ctg aaa tat aaa gtg

[0075] 432

[0076] Ile Leu Gln Gly Asp Leu Val Lys Phe Leu Asp Leu Lys Tyr Lys Val

[0077] 130 135 140

[0078] tgc att gaa ggc ggc gat ccg gaa ctg att att aaa gat ctg aaa att

[0079] 480

[0080] Cys Ile Glu Gly Gly Asp Pro Glu Leu Ile Ile Lys Asp Leu Lys Ile

[0081] 145 150 155 160

[0082] att ctg gaa cgc ctg ccg tgc gtg ctg ggc ggc gtg ggc ctg gat gat 528

[0083] Ile Leu Glu Arg Leu Pro Cys Val Leu Gly Gly Val Gly Leu Asp Asp

[0084] 165 170 175

[0085] ctg ttt aaa aac att ttt gtg aaa gat ggc att ctg agc ttt gaa ggc

[0086] 576

[0087] Leu Phe Lys Asn Ile Phe Val Lys Asp Gly Ile Leu Ser Phe Glu Gly

[0088] 180 185 190

[0089] att gcg aaa ccg ctg ggc gat ctg ctg att ctg gtg ctg tgc ccg aac 624

[0090] Ile Ala Lys Pro Leu Gly Asp Leu Leu Ile Leu Val Leu Cys Pro Asn

[0091] 195 200 205

[0092] gtg aaa aac att aac gtg agc agc

[0093] 648

[0094] Val Lys Asn Ile Asn Val Ser Ser

[0095] 210 215

[0096] <210>3

[0097] <211>651

[0098] <212> DNA

[0099] <213> Artificial Sequence

[0100] <220>

[0101] <221> CDS

[0102] <222> (1) . . . (651)

[0103] <223> codon frequency optimization of SEQ ID NO:2 for expression in bacteria and addition of the ATG start codon

[0104] <400>3

[0105] atg ctg ctg gaa ggt ttt ctg gtt ggg ggc ggt gtt ccg ggt cca ggc

[0106] 48

[0107] Met Leu Leu Glu Gly Phe Leu Val Gly Gly Gly Val Pro Gly Pro Gly

[0108] 5 10 15

[0109] acg gcc tgc ttg acg aag get ctg aaa gat agc ggt gac ctg ctg gtg

[0110] 96

[0111] Thr Ala Cys Leu Thr Lys Ala Leu Lys Asp Ser Gly Asp Leu Leu Val

[0112] 20 25 30

[0113] gag tta gcg gtt att att tgt gca tac cag aat ggc aaa gac ctt cag

[0114] 144

[0115] Glu Leu Ala Val Ile Ile Cys Ala Tyr Gln Asn Gly Lys Asp Leu Gln

[0116] 35 40 45

[0117] gag cag gac ttc aaa gaa ctg aag gaa ttg ctg gaa cgt aca ttg gaa

[0118] 192

[0119] Glu Gln Asp Phe Lys Glu Leu Lys Glu Leu Leu Glu Arg Thr Leu Glu

[0120] 55 60

[0121] cgt gcc ggt tgt gcc ctc gat gat att gtg gcc gat tta ggt ctg gaa

[0122] 240

[0123] Arg Ala Gly Cys Ala Leu Asp Asp Ile Val Ala Asp Leu Gly Leu Glu

[0124] 70 75 80

[0125] gaa ctg ctg ggc tcc atc ggc gtt agt acc ggc gat att atc cag ggt

[0126] 288

[0127] Glu Leu Leu Gly Ser Ile Gly Val Ser Thr Gly Asp Ile Ile Gln Gly

[0128] 85 90 95

[0129] ctg tat aaa ctg ttg aag gag tta aaa atc gac gag acc gtc ttt aat

[0130] 336

[0131] Leu Tyr Lys Leu Leu Lys Glu Leu Lys Ile Asp Glu Thr Val Phe Asn

[0132] 100 105 110

[0133] gcg gtc tgc gat gtg acc aaa aaa atg ctg gat aac aag tgc tta ccg

[0134] 384

[0135] Ala Val Cys Asp Val Thr Lys Lys Met Leu Asp Asn Lys Cys Leu Pro

[0136] 115 120 125

[0137] aaa att ctg caa gga gat ctg gta aag ttc ctt gat ctg aag tat aaa

[0138] 432

[0139] Lys Ile Leu Gln Gly Asp Leu Val Lys Phe Leu Asp Leu Lys Tyr Lys

[0140] 130 135 140

[0141] gtt tgt att gaa ggt ggc gat cca gaa ctg att att aag gat ctg aaa

[0142] 480

[0143] Val Cys Ile Glu Gly Gly Asp Pro Glu Leu Ile Ile Lys Asp Leu Lys

[0144] 145 150 155 160

[0145] atc atc ctg gaa cgg ctt ccg tgt gtg ttg ggt gga gtc ggt ttg gat

[0146] 528

[0147] Ile Ile Leu Glu Arg Leu Pro Cys Val Leu Gly Gly Val Gly Leu Asp

[0148] 165 170 175

[0149] gat ctc ttt aag aac att ttt gtt aag gat ggg att ctg tcc ttc gaa

[0150] 576

[0151] Asp Leu Phe Lys Asn Ile Phe Val Lys Asp Gly Ile Leu Ser Phe Glu

[0152] 180 185 190

[0153] ggt att gcg aaa cct ctt ggt gac ctt ctc atc ctt gtc tta tgc ccg

[0154] 624

[0155] Gly Ile Ala Lys Pro Leu Gly Asp Leu Leu Ile Leu Val Leu Cys Pro

[0156] 195 200 205

[0157] aac gtc aag aat atc aat gta tcc tct

[0158] 651

[0159] Asn Val Lys Asn Ile Asn Val Ser Ser

[0160] 210 215

[0161] <210>4

[0162] <211>697

[0163] <212> DNA

[0164] <213> Artificial Sequence

[0165] <220> CDS

[0166] <223> the SEQ ID NO:3 including the restriction site for Ndel,

[0167] the encoding sequence of the polyhistidine tag, the restriction site for EcoRI, the sequence encoding the cleavage site for TEV, the restriction site for Ndel, and the restriction site for Xhol

[0168] <400>4

[0169] g aat tct gaa aac ttg tat ttc cag ggc agc cat atg atg ctg ctg

[0170] 46

[0171] Asn Ser Glu Asn Leu Tyr Phe Gln Gly Ser His Met Met Leu Leu

[0172] 5 10 15

[0173] gaa ggt ttt ctg gtt ggg ggc ggt gtt ccg ggt cca ggc acg gcc tgc

[0174] 94

[0175] Glu Gly Phe Leu Val Gly Gly Gly Val Pro Gly Pro Gly Thr Ala Cys

[0176] 20 25 30

[0177] ttg acg aag get ctg aaa gat agc ggt gac ctg ctg gtg gag tta gcg

[0178] 142

[0179] Leu Thr Lys Ala Leu Lys Asp Ser Gly Asp Leu Leu Val Glu Leu Ala

[0180] 35 40 45

[0181] gtt att att tgt gca tac cag aat ggc aaa gac ctt cag gag cag gac

[0182] 190

[0183] Val Ile Ile Cys Ala Tyr Gln Asn Gly Lys Asp Leu Gln Glu Gln Asp

[0184] 50 55 60

[0185] ttc aaa gaa ctg aag gaa ttg ctg gaa cgt aca ttg gaa cgt gcc ggt

[0186] 238

[0187] Phe Lys Glu Leu Lys Glu Leu Leu Glu Arg Thr Leu Glu Arg Ala Gly

[0188] 70 75

[0189] tgt gcc ctc gat gat att gtg gcc gat tta ggt ctg gaa gaa ctg ctg

[0190] 286

[0191] Cys Ala Leu Asp Asp Ile Val Ala Asp Leu Gly Leu Glu Glu Leu Leu

[0192] 85 90 95

[0193] ggc tcc atc ggc gtt agt acc ggc gat att atc cag ggt ctg tat aaa

[0194] 334

[0195] Gly Ser Ile Gly Val Ser Thr Gly Asp Ile Ile Gln Gly Leu Tyr Lys

[0196] 100 105 110

[0197] ctg ttg aag gag tta aaa atc gac gag acc gtc ttt aat gcg gtc tgc

[0198] 382

[0199] Leu Leu Lys Glu Leu Lys Ile Asp Glu Thr Val Phe Asn Ala Val Cys

[0200] 115 120 125

[0201] gat gtg acc aaa aaa atg ctg gat aac aag tgc tta ccg aaa att ctg

[0202] 430

[0203] Asp Val Thr Lys Lys Met Leu Asp Asn Lys Cys Leu Pro Lys Ile Leu

[0204] 130 135 140

[0205] caa gga gat ctg gta aag ttc ctt gat ctg aag tat aaa gtt tgt att

[0206] 478

[0207] Gln Gly Asp Leu Val Lys Phe Leu Asp Leu Lys Tyr Lys Val Cys Ile

[0208] 145 150 155

[0209] gaa ggt ggc gat cca gaa ctg att att aag gat ctg aaa atc atc ctg

[0210] 526

[0211] Glu Gly Gly Asp Pro Glu Leu Ile Ile Lys Asp Leu Lys Ile Ile Leu

[0212] 160 165 170 175

[0213] gaa cgg ctt ccg tgt gtg ttg ggt gga gtc ggt ttg gat gat ctc ttt

[0214] 574

[0215] Glu Arg Leu Pro Cys Val Leu Gly Gly Val Gly Leu Asp Asp Leu Phe

[0216] 180 185 190

[0217] aag aac att ttt gtt aag gat ggg att ctg tcc ttc gaa ggt att gcg

[0218] 622

[0219] Lys Asn Ile Phe Val Lys Asp Gly Ile Leu Ser Phe Glu Gly Ile Ala

[0220] 195 200 205

[0221] aaa cct ctt ggt gac ctt ctc atc ctt gtc tta tgc ccg aac gtc aag

[0222] 670

[0223] Lys Pro Leu Gly Asp Leu Leu Ile Leu Val Leu Cys Pro Asn Val Lys

[0224] 210 215 220

[0225] aat atc aat gta tcc tct taactcgag

[0226] 697

[0227] Asn Ile Asn Val Ser Ser

[0228] 225

[0229] <210>5

[0230] <211>6395

[0231] <212> DNA

[0232] <213> Artificial Sequence

[0233] <220>

[0234] <223> pPBUFCBac-LvRsnl expression vector resulting from the insertion of SEQ ID NO:4 into SEQ ID NO:5

[0235] <400>5

[0236] gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat 60

[0237] tcccctctag aaataatttt gtttaaactt taagaaggag atatacatat g cat cat 117

[0238] His His

[0239] cat cat cat cac gtg aat tct gaa aac ttg tat ttc cag ggc agc cat 165

[0240] His His His His Val Asn Ser Glu Asn Leu Tyr Phe Gln Gly Ser His

[0241] 5 10 15

[0242] atg atg ctg ctg gaa ggt ttt ctg gtt ggg ggc ggt gtt ccg ggt cca 213

[0243] Met Met Leu Leu Glu Gly Phe Leu Val Gly Gly Gly Val Pro Gly Pro

[0244] 25 30

[0245] ggc acg gcc tgc ttg acg aag gct ctg aaa gat agc ggt gac ctg ctg 261

[0246] Gly Thr Ala Cys Leu Thr Lys Ala Leu Lys Asp Ser Gly Asp Leu Leu

[0247] 40 45 50

[0248] gtg gag tta gcg gtt att att tgt gca tac cag aat ggc aaa gac ctt 309

[0249] Val Glu Leu Ala Val Ile Ile Cys Ala Tyr Gln Asn Gly Lys Asp Leu

[0250] 55 60 65

[0251] cag gag cag gac ttc aaa gaa ctg aag gaa ttg ctg gaa cgt aca ttg 357

[0252] Gln Glu Gln Asp Phe Lys Glu Leu Lys Glu Leu Leu Glu Arg Thr Leu

[0253] 70 75 80

[0254] gaa cgt gcc ggt tgt gcc ctc gat gat att gtg gcc gat tta ggt ctg 405

[0255] Glu Arg Ala Gly Cys Ala Leu Asp Asp Ile Val Ala Asp Leu Gly Leu 85 90 95

[0256] gaa gaa ctg ctg ggc tcc atc ggc gtt agt acc ggc gat att atc cag 453

[0257] Glu Glu Leu Leu Gly Ser Ile Gly Val Ser Thr Gly Asp Ile Ile Gln

[0258] 100 105 110

[0259] ggt ctg tat aaa ctg ttg aag gag tta aaa atc gac gag acc gtc ttt 501

[0260] Gly Leu Tyr Lys Leu Leu Lys Glu Leu Lys Ile Asp Glu Thr Val Phe

[0261] 115 120 125 130

[0262] aat gcg gtc tgc gat gtg acc aaa aaa atg ctg gat aac aag tgc tta 549

[0263] Asn Ala Val Cys Asp Val Thr Lys Lys Met Leu Asp Asn Lys Cys Leu

[0264] 135 140 145

[0265] ccg aaa att ctg caa gga gat ctg gta aag ttc ctt gat ctg aag tat 597

[0266] Pro Lys Ile Leu Gln Gly Asp Leu Val Lys Phe Leu Asp Leu Lys Tyr

[0267] 150 155 160

[0268] aaa gtt tgt att gaa ggt ggc gat cca gaa ctg att att aag gat ctg 645

[0269] Lys Val Cys Ile Glu Gly Gly Asp Pro Glu Leu Ile Ile Lys Asp Leu

[0270] 165 170 175

[0271] aaa atc atc ctg gaa cgg ctt ccg tgt gtg ttg ggt gga gtc ggt ttg 693

[0272] Lys Ile Ile Leu Glu Arg Leu Pro Cys Val Leu Gly Gly Val Gly Leu

[0273] 180 185 190

[0274] gat gat ctc ttt aag aac att ttt gtt aag gat ggg att ctg tcc ttc 741

[0275] Asp Asp Leu Phe Lys Asn Ile Phe Val Lys Asp Gly Ile Leu Ser Phe

[0276] 195 200 205 210

[0277] gaa ggt att gcg aaa cct ctt ggt gac ctt ctc atc ctt gtc tta tgc 789

[0278] Glu Gly Ile Ala Lys Pro Leu Gly Asp Leu Leu Ile Leu Val Leu Cys

[0279] 215 220 225

[0280] ccg aac gtc aag aat atc aat gta tcc tct taactcgaga tcgatgatat 819

[0281] Pro Asn Val Lys Asn Ile Asn Val Ser Ser

[0282] 230 235

[0283] tcgagcctag gtataatcgg atccggctgc taacaaagcc cgaaaggaag ctgagttggc 879

[0284] tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag 939

[0285] gggttttttg ctgaaaggag gaactatatc cggatatccc gcaagaggcc cggcagtacc 999

[0286] ggcataacca agcctatgcc tacagcatcc agggtgacgg tgccgaggat gacgatgagc 1059

[0287] gcattgttag atttcataca cggtgcctga ctgcgttagc aatttaactg tgataaacta 1119

[0288] ccgcattaaa gctagcttat cgatgataag ctgtcaaaca tgagaattaa ttcttgaaga 1179

[0289] cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct 1239

[0290] tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 1299

[0291] taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 1359

[0292] tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 1419

[0293] gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 1479

[0294] gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 1539

[0295] cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 1599

[0296] tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac 1659

[0297] tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 1719

[0298] atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 1779

[0299] ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 1839

[0300] gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 1899

[0301] gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc 1959

[0302] gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 2019

[0303] gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 2079

[0304] gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 2139

[0305] cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 2199

[0306] atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 2259

[0307] tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 2319

[0308] ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 2379

[0309] gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 2439

[0310] tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 2499

[0311] ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 2559

[0312] ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 2619

[0313] gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 2679

[0314] ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 2739

[0315] tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 2799

[0316] ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 2859

[0317] agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 2919

[0318] agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 2979

[0319] gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 3039

[0320] tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 3099

[0321] accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 3159

[0322] gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt 3219

[0323] atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc 3279

[0324] agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac 3339

[0325] acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt 3399

[0326] gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag 3459

[0327] gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc 3519

[0328] atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg 3579

[0329] ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt 3639

[0330] tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac 3699

[0331] tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat 3759

[0332] gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt 3819

[0333] aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca 3879

[0334] gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt 3939

[0335] tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg 3999

[0336] tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag 4059

[0337] gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatgc gccgcgtgcg 4119

[0338] gctgctggag atggcggacg cgatggatat gttctgccaa gggttggttt gcgcattcac 4179

[0339] agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt tagcgaggtg 4239

[0340] ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg 4299

[0341] aggcagacaa ggtatagggc ggcgcctaca atccatgcca acccgttcca tgtgctcgcc 4359

[0342] gaggcggcat aaatcgccgt gacgatcagc ggtccaatga tcgaagttag gctggtaaga 4419

[0343] gccgcgagcg atccttgaag ctgtccctga tggtcgtcat ctacctgcct ggacagcatg 4479

[0344] gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat ggggaaggcc 4539

[0345] atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc cgccatgccg 4599

[0346] gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga 4659

[0347] gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag 4719

[0348] cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc 4779

[0349] atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag 4839

[0350] gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg 4899

[0351] agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 4959

[0352] tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 5019

[0353] cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg 5079

[0354] gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg 5139

[0355] tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac 5199

[0356] taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag 5259

[0357] cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg 5319

[0358] catggtttgt tgaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga 5379

[0359] atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa 5439

[0360] cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg 5499

[0361] cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag 5559

[0362] acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg 5619

[0363] tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc 5679

[0364] gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc 5739

[0365] agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga 5799

[0366] ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg 5859

[0367] ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa 5919

[0368] acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct 5979

[0369] gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg 6039

[0370] cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg 6099

[0371] ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag 6159

[0372] caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac 6219

[0373] ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg 6279

[0374] atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt 6339

[0375] gatgccggcc acgatgcgtc cggcgtagag gatcga 6375

[0376] <210>6

[0377] <211>236

[0378] <212> PRT

[0379] <213> Artificial Sequence

[0380] <220>

[0381] <223> amino acid sequence of the modified version of the Lv-Rsn-1 surfactant protein encoded by the nucleotide sequence SEQ ID NO:4, which comprises the SEQ ID NO:3

[0382] <400>6

[0383] His His His His His His Val Asn Ser Glu Asn Leu Tyr Phe Gln Gly

[0384] 5 10 15

[0385] Ser His Met Met Leu Leu Glu Gly Phe Leu Val Gly Gly Gly Val Pro

[0386] 20 25 30

[0387] Gly Pro Gly Thr Ala Cys Leu Thr Lys Ala Leu Lys Asp Ser Gly Asp

[0388] 35 40 45

[0389] Leu Leu Val Glu Leu Ala Val Ile Ile Cys Ala Tyr Gln Asn Gly Lys

[0390] 55 60

[0391] Asp Leu Gln Glu Gln Asp Phe Lys Glu Leu Lys Glu Leu Leu Glu Arg

[0392] 70 75 80

[0393] Thr Leu Glu Arg Ala Gly Cys Ala Leu Asp Asp Ile Val Ala Asp Leu

[0394] 85 90 95

[0395] Gly Leu Glu Glu Leu Leu Gly Ser Ile Gly Val Ser Thr Gly Asp Ile

[0396] 100 105 110

[0397] Ile Gln Gly Leu Tyr Lys Leu Leu Lys Glu Leu Lys Ile Asp Glu Thr

[0398] 115 120 125

[0399] Val Phe Asn Ala Val Cys Asp Val Thr Lys Lys Met Leu Asp Asn Lys

[0400] 130 135 140

[0401] Cys Leu Pro Lys Ile Leu Gln Gly Asp Leu Val Lys Phe Leu Asp Leu

[0402] 145 150 155 160

[0403] Lys Tyr Lys Val Cys Ile Glu Gly Gly Asp Pro Glu Leu Ile Ile Lys

[0404] 165 170 175

[0405] Asp Leu Lys Ile Ile Leu Glu Arg Leu Pro Cys Val Leu Gly Gly Val

[0406] 180 185 190

[0407] Gly Leu Asp Asp Leu Phe Lys Asn Ile Phe Val Lys Asp Gly Ile Leu

[0408] 195 200 205

[0409] Ser Phe Glu Gly Ile Ala Lys Pro Leu Gly Asp Leu Leu Ile Leu Val

[0410] 210 215 220

[0411] Leu Cys Pro Asn Val Lys Asn Ile Asn Val Ser Ser

[0412] 225 230 235

[0413] <210>7

[0414] <211>648

[0415] <212> DNA

[0416] <213> Artificial Sequence

[0417] <220>

[0418] <221> CDS

[0419] <222> (1) . . . (648)

[0420] <223> codon frequency optimization of the SEQ ID NO:2 for expression in yeasts

[0421] <400>7

[0422] ttg ttg gaa gga ttt ttg gtc gga ggt ggt gtc cct ggt cct ggt aca

[0423] 48

[0424] Leu Leu Glu Gly Phe Leu Val Gly Gly Gly Val Pro Gly Pro Gly Thr

[0425] 5 10 15

[0426] gca tgt ttg act aag gca ttg aaa gac agt gga gac ttg ttg gtt gag

[0427] 96

[0428] Ala Cys Leu Thr Lys Ala Leu Lys Asp Ser Gly Asp Leu Leu Val Glu

[0429] 20 25 30

[0430] ttg gct gtt att att tgt gct tac caa aac ggt aaa gat ttg caa gag

[0431] 144

[0432] Leu Ala Val Ile Ile Cys Ala Tyr Gln Asn Gly Lys Asp Leu Gln Glu

[0433] 35 40 45

[0434] caa gat ttc aag gaa ttg aag gag ttg ttg gaa aga act ttg gaa aga

[0435] 192

[0436] Gln Asp Phe Lys Glu Leu Lys Glu Leu Leu Glu Arg Thr Leu Glu Arg

[0437] 55 60

[0438] gct ggt tgt gct ttg gat gat att gtt gct gat ttg ggt ttg gaa gag

[0439] 240

[0440] Ala Gly Cys Ala Leu Asp Asp Ile Val Ala Asp Leu Gly Leu Glu Glu

[0441] 70 75 80

[0442] ttg ttg ggt tct att ggt gtt tct act gga gat atc atc caa ggt ttg

[0443] 288

[0444] Leu Leu Gly Ser Ile Gly Val Ser Thr Gly Asp Ile Ile Gln Gly Leu

[0445] 85 90 95

[0446] tac aag ttg ttg aag gag ttg aag atc gat gaa act gtt ttt aac get

[0447] 336

[0448] Tyr Lys Leu Leu Lys Glu Leu Lys Ile Asp Glu Thr Val Phe Asn Ala

[0449] 100 105 110

[0450] gtt tgt gat gtt act aag aaa atg ttg gat aac aag tgt ttg cca aag

[0451] 384

[0452] Val Cys Asp Val Thr Lys Lys Met Leu Asp Asn Lys Cys Leu Pro Lys

[0453] 115 120 125

[0454] atc ttg caa gga gat ttg gtt aag ttc ttg gat ttg aag tac aag gtt

[0455] 432

[0456] Ile Leu Gln Gly Asp Leu Val Lys Phe Leu Asp Leu Lys Tyr Lys Val

[0457] 130 135 140

[0458] tgt atc gaa ggt gga gat cca gaa ttg att att aag gat ttg aag atc

[0459] 480

[0460] Cys Ile Glu Gly Gly Asp Pro Glu Leu Ile Ile Lys Asp Leu Lys Ile

[0461] 145 150 155 160

[0462] atc ttg gag aga ttg cct tgt gtt ttg ggt ggt gtt ggt ttg gat gat

[0463] 528

[0464] Ile Leu Glu Arg Leu Pro Cys Val Leu Gly Gly Val Gly Leu Asp Asp

[0465] 165 170 175

[0466] ttg ttt aaa aac atc ttc gtt aag gat ggt att ttg tct ttc gaa ggt

[0467] 576

[0468] Leu Phe Lys Asn Ile Phe Val Lys Asp Gly Ile Leu Ser Phe Glu Gly

[0469] 180 185 190

[0470] att get aag cct ttg gga gat ttg ttg att ttg gtt ttg tgt cct aat

[0471] 624

[0472] Ile Ala Lys Pro Leu Gly Asp Leu Leu Ile Leu Val Leu Cys Pro Asn

[0473] 195 200 205

[0474] gtc aag aat atc aat gtt tca tca

[0475] 648

[0476] Val Lys Asn Ile Asn Val Ser Ser

[0477] 210 215

[0478] <210>8

[0479] <211>685

[0480] <212> DNA

[0481] <213> Artificial Sequence

[0482] <220>

[0483] <221> CDS

[0484] <222> (1) . . . (648)

[0485] <223> the SEQ ID NO:8 after addition of the restriction site for

[0486] the Pstl endonuclease, of two nucleotides to place the encoding sequence in the same frame of translation as the secretion factor alpha, and of the restriction site for the endonuclease Notl

[0487] <400>8

[0488] ct gca gga ttg ttg gaa gga ttt ttg gtc gga ggt ggt gtc cct ggt

[0489] 47

[0490] Ala Gly Leu Leu Glu Gly Phe Leu Val Gly Gly Gly Val Pro Gly

[0491] 5 10 15

[0492] cct ggt aca gca tgt ttg act aag gca ttg aaa gac agt gga gac ttg

[0493] 95

[0494] Pro Gly Thr Ala Cys Leu Thr Lys Ala Leu Lys Asp Ser Gly Asp Leu

[0495] 20 25 30

[0496] ttg gtt gag ttg get gtt att att tgt get tac caa aac ggt aaa gat

[0497] 143

[0498] Leu Val Glu Leu Ala Val Ile Ile Cys Ala Tyr Gln Asn Gly Lys Asp

[0499] 35 40 45

[0500] ttg caa gag caa gat ttc aag gaa ttg aag gag ttg ttg gaa aga act

[0501] 191

[0502] Leu Gln Glu Gln Asp Phe Lys Glu Leu Lys Glu Leu Leu Glu Arg Thr

[0503] 50 55 60

[0504] ttg gaa aga gct ggt tgt gct ttg gat gat att gtt gct gat ttg ggt

[0505] 239

[0506] Leu Glu Arg Ala Gly Cys Ala Leu Asp Asp Ile Val Ala Asp Leu Gly

[0507] 70 75

[0508] ttg gaa gag ttg ttg ggt tct att ggt gtt tct act gga gat atc atc

[0509] 287

[0510] Leu Glu Glu Leu Leu Gly Ser Ile Gly Val Ser Thr Gly Asp Ile Ile

[0511] 85 90 95

[0512] caa ggt ttg tac aag ttg ttg aag gag ttg aag atc gat gaa act gtt

[0513] 335

[0514] Gln Gly Leu Tyr Lys Leu Leu Lys Glu Leu Lys Ile Asp Glu Thr Val

[0515] 100 105 110

[0516] ttt aac gct gtt tgt gat gtt act aag aaa atg ttg gat aac aag tgt

[0517] 383

[0518] Phe Asn Ala Val Cys Asp Val Thr Lys Lys Met Leu Asp Asn Lys Cys

[0519] 115 120 125

[0520] ttg cca aag atc ttg caa gga gat ttg gtt aag ttc ttg gat ttg aag

[0521] 431

[0522] Leu Pro Lys Ile Leu Gln Gly Asp Leu Val Lys Phe Leu Asp Leu Lys

[0523] 130 135 140

[0524] tac aag gtt tgt atc gaa ggt gga gat cca gaa ttg att att aag gat

[0525] 479

[0526] Tyr Lys Val Cys Ile Glu Gly Gly Asp Pro Glu Leu Ile Ile Lys Asp

[0527] 145 150 155

[0528] ttg aag atc atc ttg gag aga ttg cct tgt gtt ttg ggt ggt gtt ggt

[0529] 527

[0530] Leu Lys Ile Ile Leu Glu Arg Leu Pro Cys Val Leu Gly Gly Val Gly

[0531] 160 165 170 175

[0532] ttg gat gat ttg ttt aaa aac atc ttc gtt aag gat ggt att ttg tct

[0533] 575

[0534] Leu Asp Asp Leu Phe Lys Asn Ile Phe Val Lys Asp Gly Ile Leu Ser

[0535] 180 185 190

[0536] ttc gaa ggt att get aag cct ttg gga gat ttg ttg att ttg gtt ttg

[0537] 623

[0538] Phe Glu Gly Ile Ala Lys Pro Leu Gly Asp Leu Leu Ile Leu Val Leu

[0539] 195 200 205

[0540] tgt cct aat gtc aag aat atc aat gtt tca tca gag aac ctt tac ttt

[0541] 671

[0542] Cys Pro Asn Val Lys Asn Ile Asn Val Ser Ser Glu Asn Leu Tyr Phe

[0543] 210 215 220

[0544] cag gga gcg gcc gc

[0545] 685

[0546] Gln Gly Ala Ala

[0547] 225

[0548] <210>9

[0549] <211>4219

[0550] <212> DNA

[0551] <213> Artificial Sequence

[0552] <220>

[0553] <223> pPBUFCYea-LvRsnl expression vector resulting from the insertion of the SEQ ID NO:9 into the SEQ ID NO:10

[0554] <400>9

[0555] agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60

[0556] gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120

[0557] tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180

[0558] agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240

[0559] acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300

[0560] tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360

[0561] agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420

[0562] gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480

[0563] ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 540

[0564] cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600

[0565] ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660

[0566] ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720

[0567] gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780

[0568] atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840

[0569] actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900

[0570] caacttgaga agatcaaaaa acaactaatt attcgaaacg atg aga ttt cct tca 955

[0571] Met Arg Phe Pro Ser

[0572] 1 5

[0573] att ttt act gct gtt tta ttc gca gca tcc tcc gca tta gct gct cca 1003

[0574] Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser Ala Leu Ala Ala Pro

[0575] 10 15 20

[0576] gtc aac act aca aca gaa gat gaa acg gca caa att ccg gct gaa gct 1051

[0577] Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln Ile Pro Ala Glu Ala

[0578] 25 30 35

[0579] gtc atc ggt tac tca gat tta gaa ggg gat ttc gat gtt gct gtt ttg 1099

[0580] Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe Asp Val Ala Val Leu

[0581] 40 45 50

[0582] cca ttt tcc aac agc aca aat aac ggg tta ttg ttt ata aat act act 1147

[0583] Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu Phe Ile Asn Thr Thr

[0584] 60 65

[0585] att gcc agc att gct gct aaa gaa gaa ggg gta tct ctc gag aaa aga 1195

[0586] Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val Ser Leu Glu Lys Arg

[0587] 75 80 85

[0588] gag gct gaa gct gca gga ttg ttg gaa gga ttt ttg gtc gga ggt ggt 1243

[0589] Glu Ala Glu Ala Ala Gly Leu Leu Glu Gly Phe Leu Val Gly Gly Gly

[0590] 90 95 100

[0591] gtc cct ggt cct ggt aca gca tgt ttg act aag gca ttg aaa gac agt 1291

[0592] Val Pro Gly Pro Gly Thr Ala Cys Leu Thr Lys Ala Leu Lys Asp Ser

[0593] 105 110 115

[0594] gga gac ttg ttg gtt gag ttg gct gtt att att tgt gct tac caa aac 1339

[0595] Gly Asp Leu Leu Val Glu Leu Ala Val Ile Ile Cys Ala Tyr Gln Asn

[0596] 120 125 130

[0597] ggt aaa gat ttg caa gag caa gat ttc aag gaa ttg aag gag ttg ttg 1387

[0598] Gly Lys Asp Leu Gln Glu Gln Asp Phe Lys Glu Leu Lys Glu Leu Leu

[0599] 135 140 145

[0600] gaa aga act ttg gaa aga gct ggt tgt gct ttg gat gat att gtt gct 1435

[0601] Glu Arg Thr Leu Glu Arg Ala Gly Cys Ala Leu Asp Asp Ile Val Ala

[0602] 150 155 160 165

[0603] gat ttg ggt ttg gaa gag ttg ttg ggt tct att ggt gtt tct act gga 1483

[0604] Asp Leu Gly Leu Glu Glu Leu Leu Gly Ser Ile Gly Val Ser Thr Gly

[0605] 170 175 180

[0606] gat atc atc caa ggt ttg tac aag ttg ttg aag gag ttg aag atc gat 1531

[0607] Asp Ile Ile Gln Gly Leu Tyr Lys Leu Leu Lys Glu Leu Lys Ile Asp

[0608] 185 190 195

[0609] gaa act gtt ttt aac gct gtt tgt gat gtt act aag aaa atg ttg gat 1579

[0610] Glu Thr Val Phe Asn Ala Val Cys Asp Val Thr Lys Lys Met Leu Asp

[0611] 200 205 210

[0612] aac aag tgt ttg cca aag atc ttg caa gga gat ttg gtt aag ttc ttg 1627

[0613] Asn Lys Cys Leu Pro Lys Ile Leu Gln Gly Asp Leu Val Lys Phe Leu

[0614] 215 220 225

[0615] gat ttg aag tac aag gtt tgt atc gaa ggt gga gat cca gaa ttg att 1675

[0616] Asp Leu Lys Tyr Lys Val Cys Ile Glu Gly Gly Asp Pro Glu Leu Ile

[0617] 230 235 240 245

[0618] att aag gat ttg aag atc atc ttg gag aga ttg cct tgt gtt ttg ggt 1723

[0619] Ile Lys Asp Leu Lys Ile Ile Leu Glu Arg Leu Pro Cys Val Leu Gly

[0620] 250 255 260

[0621] ggt gtt ggt ttg gat gat ttg ttt aaa aac atc ttc gtt aag gat ggt 1771

[0622] Gly Val Gly Leu Asp Asp Leu Phe Lys Asn Ile Phe Val Lys Asp Gly

[0623] 265 270 275

[0624] att ttg tct ttc gaa ggt att gct aag cct ttg gga gat ttg ttg att 1819

[0625] Ile Leu Ser Phe Glu Gly Ile Ala Lys Pro Leu Gly Asp Leu Leu Ile

[0626] 280 285 290

[0627] ttg gtt ttg tgt cct aat gtc aag aat atc aat gtt tca tca gag aac 1867

[0628] Leu Val Leu Cys Pro Asn Val Lys Asn Ile Asn Val Ser Ser Glu Asn

[0629] 295 300 305

[0630] ctt tac ttt cag gga gcg gcc gcc agc ttt cta gaa caa aaa ctc atc 1915

[0631] Leu Tyr Phe Gln Gly Ala Ala Ala Ser Phe Leu Glu Gln Lys Leu Ile

[0632] 310 315 320 325

[0633] tca gaa gag gat ctg aat agc gcc gtc gac cat cat cat cat cat cat 1963

[0634] Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His His His His His His

[0635] 330 335 340

[0636] tgagtttgta gccttagaca tgactgttcc tcagttcaag ttgggcactt acgagaagac 2023

[0637] cggtcttgct agattctaat caagaggatg tcagaatgcc atttgcctga gagatgcagg 2083

[0638] cttcattttt gatacttttt tatttgtaac ctatatagta taggattttt tttgtcattt 2143

[0639] tgtttcttct cgtacgagct tgctcctgat cagcctatct cgcagctgat gaatatcttg 2203

[0640] tggtaggggt ttgggaaaat cattcgagtt tgatgttttt cttggtattt cccactcctc 2263

[0641] ttcagagtac agaagattaa gtgagacctt cgtttgtgcg gatcccccac acaccatagc 2323

[0642] ttcaaaatgt ttctactcct tttttactct tccagatttt ctcggactcc gcgcatcgcc 2383

[0643] gtaccacttc aaaacaccca agcacagcat actaaatttt ccctctttct tcctctaggg 2443

[0644] tgtcgttaat tacccgtact aaaggtttgg aaaagaaaaa agagaccgcc tcgtttcttt 2503

[0645] ttcttcgtcg aaaaaggcaa taaaaatttt tatcacgttt ctttttcttg aaattttttt 2563

[0646] ttttagtttt tttctctttc agtgacctcc attgatattt aagttaataa acggtcttca 2623

[0647] atttctcaag tttcagtttc atttttcttg ttctattaca acttttttta cttcttgttc 2683

[0648] attagaaaga aagcatagca atctaatcta aggggcggtg ttgacaatta atcatcggca 2743

[0649] tagtatatcg gcatagtata atacgacaag gtgaggaact aaaccatggc caagttgacc 2803

[0650] agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac 2863

[0651] cggctcgggt tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac 2923

[0652] gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg 2983

[0653] gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac 3043

[0654] ttccgggacg cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag 3103

[0655] ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga 3163

[0656] cacgtccgac ggcggcccac gggtcccagg cctcggagat ccgtccccct tttcctttgt 3223

[0657] cgatatcatg taattagtta tgtcacgctt acattcacgc cctcccccca catccgctct 3283

[0658] aaccgaaaag gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt 3343

[0659] tatgttagta ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg 3403

[0660] cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa 3463

[0661] ggctttaatt tgcaagctgg agaccaacat gtgagcaaaa ggccagcaaa aggccaggaa 3523

[0662] ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 3583

[0663] caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 3643

[0664] gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 3703

[0665] cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta 3763

[0666] tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 3823

[0667] gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 3883

[0668] cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 3943

[0669] tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 4003

[0670] tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 4063

[0671] caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 4123

[0672] aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 4183

[0673] cgaaaactca cgttaaggga ttttggtcat gagatc 4219

[0674] <210>10

[0675] <211>341

[0676] <212> PRT

[0677] <213> Artificial Sequence

[0678] <220>

[0679] <223> amino acid sequence of the modified version of the Lv-Rsn-1 surfactant protein encoded by the nucleotide sequence SEQ ID NO:9, which in turn is contained in the SEQ ID NO:11.

[0680] <400>10

[0681] Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

[0682] 5 10 15

[0683] Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

[0684] 20 25 30

[0685] Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

[0686] 35 40 45

[0687] Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

[0688] 55 60

[0689] Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

[0690] 70 75 80

[0691] Ser Leu Glu Lys Arg Glu Ala Glu Ala Ala Gly Leu Leu Glu Gly Phe

[0692] 85 90 95

[0693] Leu Val Gly Gly Gly Val Pro Gly Pro Gly Thr Ala Cys Leu Thr Lys

[0694] 100 105 110

[0695] Ala Leu Lys Asp Ser Gly Asp Leu Leu Val Glu Leu Ala Val Ile Ile

[0696] 115 120 125

[0697] Cys Ala Tyr Gln Asn Gly Lys Asp Leu Gln Glu Gln Asp Phe Lys Glu

[0698] 130 135 140

[0699] Leu Lys Glu Leu Leu Glu Arg Thr Leu Glu Arg Ala Gly Cys Ala Leu

[0700] 145 150 155 160

[0701] Asp Asp Ile Val Ala Asp Leu Gly Leu Glu Glu Leu Leu Gly Ser Ile

[0702] 165 170 175

[0703] Gly Val Ser Thr Gly Asp Ile Ile Gln Gly Leu Tyr Lys Leu Leu Lys

[0704] 180 185 190

[0705] Glu Leu Lys Ile Asp Glu Thr Val Phe Asn Ala Val Cys Asp Val Thr

[0706] 195 200 205

[0707] Lys Lys Met Leu Asp Asn Lys Cys Leu Pro Lys Ile Leu Gln Gly Asp

[0708] 210 215 220

[0709] Leu Val Lys Phe Leu Asp Leu Lys Tyr Lys Val Cys Ile Glu Gly Gly

[0710] 225 230 235 240

[0711] Asp Pro Glu Leu Ile Ile Lys Asp Leu Lys Ile Ile Leu Glu Arg Leu

[0712] 245 250 255

[0713] Pro Cys Val Leu Gly Gly Val Gly Leu Asp Asp Leu Phe Lys Asn Ile

[0714] 260 265 270

[0715] Phe Val Lys Asp Gly Ile Leu Ser Phe Glu Gly Ile Ala Lys Pro Leu

[0716] 275 280 285

[0717] Gly Asp Leu Leu Ile Leu Val Leu Cys Pro Asn Val Lys Asn Ile Asn

[0718] 290 295 300

[0719] Val Ser Ser Glu Asn Leu Tyr Phe Gln Gly Ala Ala Ala Ser Phe Leu

[0720] 305 310 315 320

[0721] Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His

[0722] 325 330 335

[0723] His His His His His

[0724] 340