PULMONARY SURFACTANT PROTEINS

Abstract: Novel proteins useful for enhancing pulmonary surfactant activity, methods for obtaining said proteins and compositions containing one or more of the proteins. The proteins of this invention include two separate proteins characterized by molecular weights of about 35 kd and two separate proteins characterized by molecular weights of about 5.5-9 kd.

Description: / PULMONARY SURFACTANT PROTEINS Field of the Invention This invention relates to proteins isolated from human lung lavage, methods for obtaining said proteins and uses thereof. Background of the Invention Throughout this application various publications are refer¬ enced. Full citations for these publications may be found at the end of the specification. The disclosure of these publications are hereby incorporated by reference in order to more fully describe the _state of the art to which this invention pertains. Hyaline Membrane Disease (HMD) and Respiratory Distress Syndrome (RDS) are synonymous terms denoting the clinical condition of pulmonary dysfunction in premature infants. The disease is attributable to the absence of surface active material (surfactant) which lines the air-alveolar interface in the lung and prevents collapse of the alveoli during respiration. Current therapy is predominantly supportive. However, recent clinical trials indicate that one promising therapy is the instillation of bovine-derived surfactant into the lungs of the neonate. Surface tension in the alveoli of the lung is lowered by a lipoprotein complex called pulmonary surfactant. This complex consists of phospholipid and 5-10% protein (King, 1982) . The protein fraction of the surfactant is composed of nonserum and serum proteins. The major surfactant associated protein is reportedly a 35,000 dalton nonserum, sialoglycoprotein (Shelly et al., 1982? Bhattacharyya et al, 1975; Sueishin and Benson 1981; King et al, 1973, Katyal & Singh, 1981) . This protein reportedly seems to be important for the normal function of the pulmonary surfactant (King et. al. , 1983; Hawgood et.al., 1985). It is present in reduced amounts in amniotic fluid samples taken shortly before the birth of infants who subsequently develop respir¬ atory distress syndrome (Katyal and Singh, 1984; Shelly et al., 1982; King et al. , 1975). Recently the biosynthesis of a 35,000 dalton protein in normal human lung tissue was studied and in an in vitro translation reaction, proteins of 29 and 31 kDa were identified as the primary translation products (Floros et al., 1985). A 35kDa protein also accumulates in the lungs of patients with alveolar protein- osis (Battacharyya and Lynn, 1978, Battacharyya and Lynn, 1980a) . This protein has the same electrophoretic mobility, immunological determinants and peptide mapping as the 35kDa protein from normal human broncho-alveolar lavage material (Phelps et al., 1984; Whitsett et al., 1985). In addition to the above mentioned proteins, the presence in rat lungs of a number of lowermolecular weight surfactant- associated proteins has recently been reported. See D. L. Wang, A. Chandler and A. B. Fisher, Fed. Proc. 44(4): 1024 (1985) , Abstract No. 3587 (ca. 9000 dalton rat protein) and S. Katyal and G. Singh, Fed. Proc. 44(6): 1890 (1985), Abstract No. 8639 (10,000 - 12,000 dalton rat protein). Finally, a Feb. 6, 1985 press release from California Biotechnology Inc. reports the cloning and "detailed manip¬ ulation" of "the gene encoding human lung surfactant pro¬ tein." However, the press release does not characterize that protein or describe the "detailed manipulations." Two other reports of possible surfactant-related proteins have also been published recently, namely, J.A. Whitsett et al. , 1986, Pediatr. Res. 2J 460 and A. Takahashi et al., 1986, BBRC 135 : 527 . The present invention relates to a new group of proteins recovered and purified from lung lavage of patients with alveolar proteinosis, methods for obtaining the proteins, corresponding recombinant proteins, antibodies to the proteins for use in diagnostic products, compositions containing the novel proteins, and methods for using the compositions, e.g. in the treatment of infants afflicted with conditions such as Respiratory Distress Syndrome (RDS) , as a drug delivery vehicle in the administration of other therapeutic materials to the lungs or other organs and in the treatment of adult RDS, which can occur during cardiopulmonary operations or in other situations when the lungs are filled with fluid and natural pulmonary surfactant production and/or function ceases. While it is possible that one or more of the proteins described hereinafter is similar or identical to proteins discussed in the above- mentioned papers, the exact relationship of the protein of this invention to prior proteins cannot at present be confirmed given the inadequacies of the priordisclosures with respect to amino acid or nucleotide sequence data, surfactant activity of prior proteins and the like. Summary of the Invention This invention relates to novel proteins useful for enhancing pulmonary surfactant activity, methods for obtaining said proteins and compositions containing one or more of the proteins. The proteins of this invention include the following: 1. A protein characterized by a molecular weight of about 35 kd and by being encoded for by the DNA sequence depicted in Table 1. 2. A protein characterized by a molecular weight of Table 1 DNA and Protein Sequence of 35K PSP Clone IA GAA TTCCGCAGAG ACCCAAGCAC CTGGAGGCTC -3 0 30- 31C 220 338 34 β TGTGTG T GGG TCGCTGfiTTT CTTGGAGCCT GAAAAGRRGG AGCAGCGACT GGACCCAGAO CC ftTG MET 357 372 387 -G3 CTG TGC CCT CTG GCC CTC RCC CTC ATC TTG ftTG GCA GCC TCT GGT GCT GCG fro L eu Cys Pro Leu Ala Leu Thr Leu lie Lou MET Ala Ala Ser Gly Ala Ala <n3_. 417 433 447 TGC -' GTG AflG GAC GTT TGT GTT GGA AGC CCT GGT ATC CCC CGC RCT CCT GGA Cys Glu Val Lys Asp Val Cy3 Vαl Gly Ξβr Pro Gly Ilo Pro Gly Thr Pro Gly 462 477 492 337 TZZ Cεt GGC CTG CCA GGC R3G GRC GGG RGA GAT GGT GTC ARA GGA GRC CCT GGC Sεr His Sly Leu Pro Gly Arg Asp Sly firg Aap Gly Val Lys Gly Asp Pro Gly 522 337 532 CCT CCA GGC CZZ ATG GGT CCG CCT GGA GAA ACA CCA TGT CCT CCT GGG RAT AAT Pro P>-o Gly Pro MET Gly Pro Pro Gly Glu Thr Pro Cys Pro Pro Gly Ran 3 n 5S7 582 397 612 GGS CTG CCT GSfi GCC CCT GGT GTC CCT GGA GAG C3T GGA GAG ABS GGG GAG CCT Gly Leu Pre. Gly Ala Pro Gly Val Pro Gly Glu. Arg Gly Glu Lye Gly Glu Pro £27 642 637 GGC GfiG RGA GGC CCT CCA GGG CTT CCA GCT CAT CTA GAT GAG GAB CTC CAA GCC Gly Glu Arg Gly Pro Pro Gly Leu Pro Ala Hie Leu Amp Glu Glu Leu Gin Alo Table 1 (cont ' d . _7_ --7 702 717 ACA CTC CCC GAC TTC AGA CAT CflA ATC CTG CAG ACA ABB GSA GCC CTC AGT CTG T h r- Leu His Asp Phe Arg Him Gin lie Leu Gin Thr Arg Gly Ala Lou Setr Lou 732 747 762 777 CAG GGC TCC ATA RTG RCA GTA GSA GAG AAG OTC TTC TCC AGC AAT GGG CAG TCC Gin Gly Ser Ilo MET Thr Val Gly Glu Lye Val Phβ Ser Sor Aβn Gly Gin Gor 792 Θ07 622 ATC ACT TTT GAT GCC ATT CAG GAG GCA TGT GCC AGA GCA GGC GGC CGC ATT GCT l e Thr Phe Asp Ala lie Gin Glu Ala Cys Ala Arg Ala Gly Gly Arg Ilo Ala 837 832 667 062 GTC CCA AGG AAT CCA GAG GAA AAT GAG GCC ATT GCA RGC TTC GTG AAG AAG TfiC Val Pro fli-g Asi-i Pr * 61u Glu Amn Glu Ala lie Ala Sor Phβ Val Lya Lyn Tyr 897 912 927 fifiC ACA TAT GCC TAT GTA GGC CTG ACT GAG GGT CCC AGC CCT GGA GAC TTC CGC Ran Thr Tyr flla Tyr_Val Gly Leu Thr Glu Gly Pro Ear Pro Gly ftβp Phβ ArQ 942 957 972 937 TfiC TCA GBT GGG ACC CCT GTA AAC TAC ACC RAC TC3 TAC CGA GGG GA3 CCT GCA Tyn. Ser flop Gly Thr Pro Val Amn Tyr Thr Amn Trp Tyr Arg Gly Glu Pro Ale 1002 1017 lβ32 1047 GGT CGG GGA AAA GfiG CAG TGT GTG GAG ATG TAC ACA GAT GGG CAB TGG AAT GAC Gly Arg Gly Lye Glu Gin Cye Val O u NET Tyr Thr Asp Gly Gin Trp Aon Aep 1862 1077 .._ .1099 . 1109 AGG AAC TGC CTG TAC TCC CGA CTS ACC ATC TGT SAG TTC TGA GABGCATTTO QGCCATGG3A Arg flεn Cys Leu Tyr Ser Arg Leu Thr lie Cyo Glu Phβ . 1119 1129 1139 1149 1139 H69 1179 CAGGGAGGAT CCTGTCTGGC CTTCAQTTTC CATCCCCAGG ATCCACTTGG TCTGTBAGAT GCTACAACTC 1189 CCTTTCAACAGAATTC 87/02037 about 35 kd and by being encoded for by the DNA sequence depicted in Table 2. 3. A protein encoded for by the DNA sequence of Table 6 or by a DNA sequence capable of hybridizing thereto and characterized by a molecular weight of about 5.5- 9 d; and 4. A protein characterized by a molecular weight of about 6 kd and an amino acid composition as set forth in Table 4. Detailed Description of the Invention The proteins of this invention were obtained by subjecting pulmonary lavage material from an alveolar proteinosis patient to a combination of separation techniques followed by chromatographic purification. More specifically, the lavage material was centrifuged, and the protein-containing pellet so obtained was washed with buffer and extracted with a solvent such as 1-butanol to remove lipids and lipid- associated proteins. The butanol extract was set aside and treated as described below. The 1-butanol-insoluble material was then washed, redissolved in buffer and purified chroma- tographically. Two proteins were thus obtained which are characterized by a molecular weight of about 35 kd. One of the 35 kd proteins is encoded for by the DNA sequence depicted in Table 1; the second 35 kd protein is encoded for by the DNA sequence depicted in Table 2. Butanol-soluble proteins were obtained by cryoprecipitation. More specifi¬ cally, storage of the 1-butanol extract at -20°C yielded a precipitate which was purified chromatographically to yield a protein characterized by an apparent molecular weight of about 6 kd (as determined by SDS-PAGE) and the observed amino acid composition set forth in Table 3. A second 6 kd (as determined by SDS-PAGE)protein was obtained by concentrating the supernatant to dryness and purifying the residue chromatographically. The observed amino acid 7 TABLE 2 DNA and Protein Sequence of 35K PSP Clone 6A GA A T T CCGGA GACCCAAGCA CCTGGAGGCT CTOTGTOTCO Q TCCCTGAOT TTCTTCGAGC CTGRAABGAP ββ 9_» ,0 ° 12* AGAGCRGCGA CTGGfiCCCRG AGCC ATG TGB CTG TGC CCT CTG GCC CTC ARC CTC ATC MPT Trp- LCΛ Cy- Pro eu Ala Leu Agn Leu Ilo 133 154 169 TTG ATG GCA βCC TCT GGT GCT GCG TGC GAA GTG AAG GAC GTT TGT CTT GGA CGC Leu MET Ala Ala Ser Gly Ala Ala Cyβ Glu Val Ly3 R-p Val Cyo Val Gly Sor 18* 153 214 229 CCT GGT ATC CCC GGC ACT CCT GGA TCC CAC GCC CTG CCA GGC ACG GAC G33 RGA Pro Gly He Pro Gly Thr Pro Gly Ser H.9 Gly Lou Pro Gly Arg Asp Gly firg 244 253 274 239 GRT GGT CTC SRA GSR GAC CCT GGC CCT CCA GGC CCC ATG GGT CCG CCT GGA GfiA RSD Gly Leu Lys Gly Asp Pro Gly Prr Pro Gly Pro MET Gly Pro Pro Gly Glu 324 319 334 RTG CCA TGT CCT CCT GGfl AAT GAT GGG CTG CCT GGA GCC CCT GGT ATC CCT GGA MET Pro Cys Pro Pro Gly flsn Asp Gly Leu Pro Gly Ala Pro Gly He Pro Gly 343 364 379 394 GA3 TGT GGA GfiG AAG GGG GAG CCT GGC GAG AGG GGC CCT CCA GGG CTT CCA GCT Glu Cyβ Gly Glu Lys Gly Glu Pro Gly Glu Arg Gly Pro Pro Gly Lou Pro Ala 409 424 439 CAT CTA GAT GAG GAG CTC CAA GCC ACA CTC CAC GAC TTT AGA CAT CAA ATC CTG His Leu Asp Glu Glu Lou Gin Ala Thr Leu His Aep Phe Arg Hlo Bin Ilo Leu 434 469 464 499 CAG ACA AGG GGA GCC CTC AGT CTG CAG GGC TCC ATA ATG ACG GTA GGA GAG AAG Glr. Thr Arg Gly Ala Lou Sor Leu Gin Gly Ser Ilo MET Thr Val Gly Glu Lye j 314 529 544 359 GTC TTC TCC AGC AAT GGG CAG TCC ATC ACT TTT GAT GCC ATT CAG GAG GCA TGT V..1 Phe Ger Get- Asn Gly Gin Cor He Thr Pho Asp Ala Ilo Gin Glu Al* Cy 8 TABLE 2 fCONTINUED. 574 5B9 604 GCC AGA GCA GGC GGC CGC ATT GCT GTC CCA AGG RAT CCA GAG GAA AAT GAG GCC fil . Arg Ala Gly Gly Arg lie Ala Val Pro Arg Asn Pro Glu Glu Asn Glu Ala 613 634 649 664 ~~ T GCA ASC TTC GTG RfiG RAG TAC AAC ACA TAT GCC TAT GTA GGC CTG ACT GAG He Ala Ser Phe Val Lys Ly3 Tyr Asn Thr Tyr Ala Tyr Val Gly Leu Thr Glu 679 694 7C9 GGT CCC RSC CCT GGA GAC TTC CGC TRC TCA GAC GGG RCC CCT GTA ARC TRC ACC Gly Pro Ser Pro Gly Asp Phe Arg TyVSer Asp Gly Thr Pro Val Asn Tyr* Thr 724 739 754 769 ARC TGG TAC CGA GGG GAG CCC GCA GGT CGG GGA AAA GAG CA3 TGT GTG GAG ATG _ Aen rp TyV Arg Gly Glu Pro Ala Gly Arg Gly Lye Glu Gin Cy- Val Glu MET 734 799 814 829 TAC ACA GAT GGG CAG TGG AAT GAC AGG ARC TGC CTG TAC TCC CGA CTG ACC ATC Tyr Thr Asp Gly Gin Trp Asn Asp Arg Aen Cys Leu Tyr Ser Arg Leu Thr He 831 861 871 861 891 TGT GAG TTC TGA GAGGCATTTA GGCCATGGGA CAGGGAGGAC GCTCTCTG3C CTTCGGCCTC Cys Glu Phe 9ι31 911 921 931 941 CATCCTGAGS CTCCACTTGG TCTGTGAGAT GCTAGAACTC CCTTTCAACA TABLE 3 TABLE 4 Asp/Asn 3.06 2.7 Thr 1.18 2.0 Ser 2.55 2.1 Glu/Gln 5.97 1.6 Pro 7.64 6.3 Gly 7.38 22.9 Ala 9.13 3.3 Cys 9.14 0.95 Val 10.13 5.5 Met 3.46 3.4 He 6.46 4.8 Leu 16.23 17.3 Tyr 2.31 3.3 Phe 1.55 6.3 His .34 2.9 Lys 1.62 3.6 Arg 7.88 1.94 (calculated based on M =10,000 daltons; ave residue MW=110) lø εa 3 GOOTTCCGGT GCC OTG GCT GOG TCO COC CTG CTG COG TGG CTG CTG CTG CTG CTG MET Ola Glu Ser His Leu Leu Gin Trp Leu Leu Leu Leu Leu TGC COO GOG TGT GOG GOC OTC GTC COC OTC CTT OOC OOG OTG GCC OOG GOG GCC Cys Gin Glυ Cys Glυ Osp lie Val His lie Leu Osn Lys MET Ola Lys Glu Ola £03 £98 313 OTT TTC COG GOC OCG OTG OGG OOG TTC CTG GOG COG GOG TGC OOC GTC CTC CCC lie Phe Gin Osp Thr MET Org Lys Phe Leu Glu Gin Glu Cys Os Val Leu Pro 3£8 34__ 358 373 TTG OOG CTG CTC OTG CCC COG TGC OOC COO GTG CTT GOC GOC TOC TTC CCC CTG π 448 463 478 i GGC CTG TGC 000 TCC CGG COG CCO GOG CCO GOG COG GOG CCO GGG OTG TCO GOC Gly Leu Cys Lys Ser Org Gin Pro Glu Pro Glu Gin Glu Pro Gly MET Ser Osp CCC CTG CCC 000 CCT CTG CGG GOC CCT CTG CM GOC CCT CTG CTG GOC OOG CTC Pro Leu Pro Lys Pro Leu Org Osp Pro Leu Pro Osp Pro Leu Leu Osp Lys Leu 553 568 583 GTC CTC CCT GTG CTG CCC GGG GCC CTC COG GCG OGG CCT GGG CCT COC OCO COG Val Leυ Pro Val Leυ Pro Gly Ola Leu Gin Ola Rrg Pro Gly Pro His Thr Gin 598 613 6£8 64C GOT CTC TCC GOG COG COO TTC CCC OTT CCT CTC CCC TOT TGC TGG CTC TGC OGG 718 733 748 GTG GCC COG GTG TGC CGC GTG GTR CCT CTG GTG GCG GGC GGC OTC TGC COG TGC Val Ola Gin Val Cys Org Val Val Pro Leυ Val Ola Gly Gly He Cys Gin Cys CTG GCT GOG CGC TOC TCC GTC OTC CTG CTC GOC OCG CTG CTG GGC CGC OTG CTG Leυ Ola Glυ Org Tyr Ser Val He Leu Leu Osp Thr Leu Leυ Gly Org MET Leu 8£3 838 853 CCC COG CTG GTC TGC CGC CTC GTC CTC CGG TGC TCC OTG GOT GOC OGC GCT GGC Pro Gin Leu Val Cys Org Leu Val Leυ Org Cys Ser MET Osp Osp Ser Ola Gly 868 883 898 913 CCO OGG TCG CCG OCO GGO GOO TGG CTG CCG CGO GOC TCT GOG TGC COC CTC TGC OTG CTC COG GCC TGT GTT GGC TCC TGG CTG GOC OGG GOO OOG TGC OOG COO TTT MET Leυ Gin Ola Cys Val Gly Ser Trp Leu Osp Org Glu Lys Cys Lys Gin Phe 1033 1048 1063 1078 GTG GOG COG COC OCG CCC COG CTG CTG OCC CTG GTG CCC OGG GGC TGG GOT GCC Val Glu Gin His Thr Pro Gin Leu Leυ Thr Leυ Val Pro Org Gly Trp Osp Ola 1093 1108 11£3 COC OCC OCC TGC COG GCC CTC GGG GTG TGT GGG OCC OTG TCC OGC CCT CTC COG His Thr Thr Cys Gin Ola Leu Gly Val Cys Gly Thr MET Ser Ser Pro Leυ Gin 113Θ 1153 1166 1176 1186 1196 I l 1£76 1£86 1£96 1306 1316 13£6 1336 TGOOOOGOOG CCTCOGCTCC COCOCCGCCC TCCTCOCCTC CCTTCCTCGG COGTCOCTTC COCTGGTGGO 1346 1356 1366 1376 1386 1396 1406 CCOCGGGCCC CCOGCCCTGT GTCGGCCTTG TCTGTCTCOG CTCOOCCOCO GTCTGOCOCC OGOGCCCOCT IHIG 14£6 1436 1446 1456 1466 1476 TCCOTCCTCT CTGGTGTGOG GCOCOGCGOG GGCOGCOTCT GGOGGOGCTC TGCOGCCTCC OCOCCTOCCO I486 1496 1506 1516 15£6 1536 1546 l 16£6 1636 1646 1656 1666 1676 1686 OTOOOOOOTG GGOOCOTOCO.GOOCTCTOOO OGOTOGOCOT COGOOOTTGT TOOGTTOOGC TTTTTCOOOO 1696 17ø6 1716 17£6 1736 1746 1756 OOTCOGCOOT TCCCCOGCGT OGTCOOGGGT GGOCOCTGCO CGCTCTGGCO TGOTGGGOTG GCGOCCGGGC 1766 1776 1786 1796 1806 1816 18£6 OOGCTTTCTT CCTCGOGOTG CTCTGCTGCT TGOGOGCTOT TGCTTT6TT0 OGOTOTOOOO OGGGGTTTCT 1836 1846 1856 1866 1876 1886 1896 TTTTGTCTTT CTGT00GGT6 GOCTTCCOGC TTTTGOTTGO OOGTCCTOGG GTGOTTCTOT TTCTGCTGTG OCCOOTGCTO OTOOOGTCCT TTCTCTTTT 0000000000 0000000000 OOCGGOOTTC Deduced protein sequence of 6Kd PSP protein is underlined 14 composition of the latter 6kd protein is set forth in Table 4. The two approximately 6kd proteins differ significantly from each other with respect to amino acid composition, as well as from the protein described by Tanaka, Chem. Pharm. Bull. 311:4100 (1983). Additionally, the N-terminal peptide sequence of the cold butanol-insoluble 6 kd protein was determined (Table 5) . For the sake of simplicity, both low molecular weight PSP proteins are referred to hereinafter as "6k** proteins based on their approximate apparent molecular weights as determined by conventional SDS-PAGE. It should be understood, however, that the actual molecular weights of these proteins are in the range of 5.5-9 kilodaltons. The fact that the four proteins can now be obtained in pure form by the above-described methods now makes it possible for one to apply conventional methods to elucidate the amino acid composition and sequence of the proteins; to prepare oligonucleotide probes based on the elucidated peptide sequences; to identify genomic DNA or cDNA encoding the proteins by conventional means, e.g., via (a) hybridization of labeled oligonucleotide probes to DNA of an appropriate library (Jacobs et al., 1985), (b) expression cloning (Wong et al., 1985) and screening for surfactant enhancing activity or (c) immunoreactivity of the expressed protein with antibodies to the proteins or fragments thereof; and to produce corresponding recombinant proteins using the identified genomic DNA or cDNA and conventional expression technology i.e. by culturing genetically engineered host cells such as microbial, insect or mammalian host cells containing the DNA so identified, for instance, transformed with the DNA or with an expression vector containing the DNA. 15 By way of example, tryptic fragments of one of the two 35 kd proteins were prepared and sequenced. Oligonucleo¬ tide probes were synthesized based on the elucidated peptide sequence of the tryptic fragments and were used to screen a lambda gtlO cDNA library made from human lung mRNA. Numerous clones were identified which hybridized to the probes. DNAs from two of these positive clones (PSAP-1 and PSAP-2) were subcloned into M13 for DNA sequencing, thus generating the clones MPSAP-1A and MPSAP-6A. The nucleotide sequence for the cDNA clones encoding each of the two 35kd surfactant proteins was thereby elucidated and is presented above in Tables 1 and 2, respectively. The sequences of subclones encoding the two 35 kd proteins are similar to each other but not identical. The sequence differences result in restriction fragment polymoirphism between the two clones with respect to the coding region recognized by the restriction enzyme Pstl. Considerably more nucleotide variation between the two clones was found in their 3' untranslated regions. Plas ids PSP35K-1A-10 and PSP35K-6A-8 were constructed by inserting the approximately 940-950 nucleotide EcoRI fragments depicted in Tables 1 and 2, respectively, into the EcoRI site of plas id SP65 (see infra) . PSP35K-1A-10 contains the polylinker site adjacent to the EcoRI site at cDNA position 1, while PSP35 -6A-8 contains the polylinker site adjacent to the EcoRI site at cDNA position 947. PSP35K-1A-10 and PSP35K-6A-8 have been deposited with the American Type Culture Collection (ATCC) , Rockville, MD under accession Nos. ATCC 40243 and 40244, respectively. Additionally, oligonucleotide probes based on the N-terminal sequence of the cold butanol-insoluble 6K protein (See Table 5) were synthesized and were used to screen a cDNA library prepared from human lung RNA (Toole et al., 1984) as described in greater detail in Example 4, below. Several clones which hybridized to the probes were identified. 16 Based on hybridization intensity one clone was selected, subcloned into 13 and sequenced. Plasmid PSP6K-17-3 was constructed by inserting the cloned cDNA so identified as an EcoRI fragment into the EcoRI site of plasmid SP65 (D.A. Melton et al., 19S4, Nucleic Acids Res. , 12:7035-7056) . PSP6K-17-3 has been deposited with the ATCC under accession No. ATCC 40245. The nucleotide sequence of the cloned cDNA insert is shown in Table 6« TABLE 5 1 5 10 F P I P L P Y (-) W L (-) (-) A L (-) = Not determined positions 8, 11 and 12 were unidentified 17 As those skilled in the art will appreciate, the cDNA insert in PSP6K-17-3 contains an open reading frame encoding a protein having a molecular weight of over 40kd. It is presently believed that the primary translation product is further processed, e.g., by Type II pneumocytes (Alveolar Type II cells) , to yield the approximately 6K protein. It is contemplated that the cloned cDNA, portions thereof or sequences capable of hybridizing thereto may be expressed in host cells or cell lines by conventional expression methods to produce "recombinant" proteins having surfactant or surfactant enhancing activity. With respect to the cloned approximately 6 protein, this invention encompasses vectors containing a heterologous DNA sequence encoding the characteristic peptide sequence lie through Cys corresponding to nucleotides A-656 through C- 757 of the sequence shown in Table 6, i.e., IKRIQAMIPKGAL- AVAVAQVCRVVPLVAGGICQC. One such vector contains the nucleotide sequence ATC AAG CGG ATC CAA GCC ATG ATT CCC AAG GGT GCG CTA GCT GTG GCA GTG GCC CAG GTG TGC CGC GTG GTA CCT CTG GTG GCG GGC GGC ATC TGC CAG TGC Other vectors of this invention contain a heterologous DNA sequence encoding the characteristic peptide sequence substantially as depicted in the underlined peptide region of Table 6, i.e., FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRW- PLVAGGICQCLAERYSVILLDTLLGRML. One such vector contains the DNA sequence substantially as depicted in the underlined nucleotide sequence of Table 6, i.e.. 18 TTC CCC ATT CCT CTC CCC TAT TGC TGG CTC TGC AGG GCT CTG ATC AAG CGG ATC CAA GCC ATG ATT CCC AAG GGT GCG CTA GCT GTG GCA GTG GCC CAG GTG TGC CGC GTG GTA CCT CTG GTG GCG GGC GGC ATC TGC CAG TGC CTG GCT GAG CGC TAC TCC GTC ATC CTG CTC GAC ACG CTG CTG GGC ATG CTG Another exemplary vector contains a heterologous DNA sequence, such as the nucleotide sequence depicted in Table 6, which encodes the full-length peptide sequence of Table 6. DNA inserts for such vectors which comprise a DNA sequence shorter than the full-length cDNA of PSP6K-17-3, depicted in Table 6, may be synthesized by known methods, e.g. using an automated DNA synthesizer, or may be prepared from the full-length cDNA sequence by conventional methods such as loop-out mutagenesis or cleavage with restriction enzymes and ligation. Vectors so prepared may be used to express the subject proteins by conventional means or may be used in the assembly of vectors with larger cDNA inserts. In the former case the vector will also contain a promoter to which the DNA insert is operatively linked and may additionally contain an amplifiable and/or selectable marker, all as is well known in the art. The proteins of this invention may thus be produced by recovering and purifying the naturally-occuring proteins from human pulmonary lavage material as described herein. Alternatively, the corresponding "recombinant" proteins may be produced by expression of the DNA sequence encoding the desired protein by conventional expression methodology using microbial or insect or preferably, mammalian host cells. Suitable vectors as well as methods for inserting therein the desired DNA are well known in the art. Suitable host cells for transfection or transformation by such vectors and expression of the cDNA are also known in the art. 19 Mammalian cell expression vectors, for example, may be synthesized by techniques well known to those skilled in this art. The components of the vectors such as the bacterial replicons, selection genes, enhancers, promoters r and the like may be obtained from natural sources or synthesized by known procedures. See Kaufman, Proc. Natl. Acad. Sci. J32.: 689-693 (1985) . Established cell lines, including transformed cell lines, are suitable as hosts. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants are also suitable. Candidate cells need not be genotypically deficient in the selection gene so long as the selection gene is dominantly acting. The host cells preferably will be established mammalian cell lines. For stable integration of vector DNA into chromosomal DNA, and for subsequenct amplification of integrated vector DNA, both by conventional methods, CHO (Chinese hamster Ovary) cells are generally preferred. Alternatively, the vector DNA may include all or part of the bovine papilloma virus genome (Lusky et al.. Cell. 3_6:391-401 (1984) and be carried in cell lines such as C127 mouse cells as a stable episomal element. Other usable mammalian cell lines include HeLa, COS-1 monkey cells, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines and the like. Cell lines derived from Alveolar Type II cells may be preferred in certain cases such as expression of the 6K protein (alone or with one or more other proteins of this invention) using the cDNA insert from PSP6K-13-7 or a fragment thereof. Stable transformants then are screened for expression of the product by standard immunological or enzymatic assays. The presence of the DNA encoding the proteins may be detected 20 by standard procedures such as Southern blotting. Transient expression of the DNA encoding the proteins during the several days after introduction of the expression vector DNA into suitable host cells such as COS-1 monkey cells is measured without selection by activity or immunological assay of the proteins in the culture medium. In the case of bacterial expression, the DNA encoding the protein may be further modified to contain preferred codons for bacterial expression as is known in the art and preferably is operatively linked in-frame to a nucleotide sequence encoding a secretory leader polypeptide permittng bacterial secretion of the mature variant protein, also as is known in the art. The compounds expressed in mammalian, insect or icrobial host cells may then be recovered, purified, and/or characterized with respect to physicochemical, biochemical and/or clinical parameters, all by known methods. One or more of the proteins of this invention may be combined with a pharmaceutically acceptable fatty acid or lipid such as dipal itoylphosphatidyl choline or with mixtures of such fatty acids or lipids which may be obtained from commercial sources or by conventional methods, orwith natural surfactant lipids to provide a formulated pulmonary surfactant composition. Natural surfactant lipids may be extracted by known methods from lung lavage, e.g. bovine or human lung lavage. Typically the weight ratios of total lipids to total proteins in the composition will be about 20:1 to about 100:1. At the levels currently being tested in clinical trials, one dose of the surfactant composition corresponds to 1-2 mg of total protein and 98-99 g. of total lipid. 21 It is contemplated that certain subcombinations of (1) the 35 kd protein encoded for by the nucleotide sequence of Table 1, (2) the 35 kd protein encoded for by the nucleotide sequence of Table 2, (3) the 6 kd protein encoded by the cDNA sequence of Table 6 and having the amino acidcomposition set forth in Table 3 and (4) the 6 kd protein having the amino acid composition set forth in Table 4 may be especially useful in the treatment of patients with particular clinical indications. Thus, this invention specifically contemplates the following subcombinations and compositions containing such subcombinations: (a) proteins (1), (2), (3) or (4) ; (b) proteins (1) and (2) (c) proteins (1) and (3) (d) proteins (1) and (4) (e) proteins (2) and (3) (f) proteins (2) and (4) (g) proteins (1), (2), and (3); (h) proteins (2), (3), and (4)' (i) proteins (3) and (4) ; (j) proteins (1), (2), and (4); and (k) proteins (1), (3) and (4) At present compositions containing proteins (3) and/or (4) are preferred. 22 EXPERIMENTAL EXAMPLES Example 1 Isolation and Characterization of the 35Kd Surfactant Associated Proteins \ Pulmonary lavage (50 ml) from an alveolar proteinosis patient was centrifuged at 10,000 x g for 5 min. The pellet was collected and washed 5 times in 20 mm Tris HCl, 0.5 M NaCl, pH 7.4. The lipids and lipid-associated proteins were extracted from the washed pellet by shaking with 50 ml 1-butanol for 1 hr at room temperature. The butanol-insoluble material was collected by centrifugation, washed with distilled water and dissolved in 50 mM sodium phosphate, pH 6.0 and 6M guanidine HCl. The protein was applied to a Vydac C4 reverse phase column and eluted with a gradient of acetonitrile: 2-propanol (2:1,v:v) containing 0.1% trifluoro- acetic acid. The major protein peak eluting at 50% B was collected and evaporated to dryness. The proteins present were analyzed by SDS-PAGE (Laemmli, 1970) . Alkylation and Tryptic Mapping The protein so obtained(approx. 50ug) was taken up and reduced in 200mM Tris, ImM EDTA, 6M guanidine HCl, 20mM DTT, pH8.5 at 37°C for 2 hrs. Solid iodacetamide was added to a final concentration of 60mM and the reaction incubated at 0°C for 2 hrs under argon in the dark. The reaction was stopped and the reagents removed by dialysis into 0.1M NH4HCO 3 , 50mM 2-mercaptoethanol, pH7.5 followed by further dialysis into lOOmM NH4HCO3, pH7.5. The alkylated protein was digested with trypsin (3% trypsin by weight) at 37°C for 16 hrs and the digest chromatographed over a C18 Vydac Reverse phase HPLC column (4.6x250mm). 23 The tryptic peptides were eluted with a linear gradient of 95% acetonitrile and 0.1% TFA, collected and subjected to N-terminal Edman degradation using an Applied-Biosystems Model 470A protein sequencer. The PTH-amino acids were analyzed by the method of Hunkapillar and Hood (1983) . Sequence data so obtained for tryptic fragments T19, T26 and T28 is presented below in Table 7. Example 2 Isolation and Characterization of the Low Molecular Weight Lipid Associated Proteins The butanol extract obtained in Example 1 was stored at -20°C causing precipitation of one of the low MW proteins. The precipitate was collected by centrifugation and dried under vacuum. The butanol layer containing butanol-soluble protein was evaporated to dryness. The precipitated cold butanol insoluble protein and the cold butanol-soluble protein were then purified in parallel by the same method as follows. Each crude protein was separately dissolved in CHCI3 : MeOH (2:1, v/v) , applied to Sephadex LH20 columns and eluted with CECl 3 :MeOH (2:1). The proteins were then analyzed by SDS-PAGE. Fractions containing the protein were pooled and evaporated to dryness. Amino acid composition was determined by hydrolysis in 6 N HCl at 110°C for 22 hrs followed by chromatography on a Beck an model 63000 amino acid analyzer. F-terminal sequence was determined on an Applied Biosystems 470A sequencer. Molecular weights were determined on 10-2 % gradient SDS polyacrylamide gels. 24 TABLE 7 Peptide Observed Sequence T19 Asp Val Cys Val Gly Ser Hyp Gly lie Hyp Gly Thr Hyp Gly Ser His Gly Leu Pro Gly T26 Ala Leu Ser Leu Gin Gly Ser He MET Thr Val (-) (-) Lys T28 Asn Pro Glu Glu Asn Glu Ala He Ala (-) Phe Val Lys Hyp" = ydroxyproline 25 Example 3 Screening of the cDNA Library and Sequencing of Clones for the 35Kd Proteins Based on the amino acid sequence of tryptic fragment T28, (Table 7) an oligonucleotide probe was synthesized. The probe consisted of four pools of 20 mers and each pool contained 32 different sequences. The sequences of the 20 mers are depicted in Table 8. A cDNA library from human lung mRNA was prepared as described in Toole et al., (19B4) and screened with the total mixture of the four pools using tetra ethyl am oniumchloride as a hybridization solvent (Jacobs et al. , 1985) Between 0.5-1% of the phage clones were positive with this probe. DNA from two of these clones were subcloned into M13 for DNA sequence analysis. By using Pool II as a sequencing primer, the nucleotide sequence corresponding to tryptic fragment T26 was identified in both clones, confirming that the isolated clones code for the major protein species found in the partially purified 35kd protein from lavage material of alveolar proteinosis patients (see above) . The two clones differed in nucleotide sequence at three positions out of 250 nucleotides. Both cloneswere completely sequenced by generating an ordered set of deletions with Bal 31 nuclease, recloning into other M13 vectors and sequencing via the dideoxynucletide chain termination procedure (Viera and Messing, 1982; Sanger et al., 1977). One clone corresponded to a full-lenth copy of the type referred to as IA (Table 1) , the second to an incomplete copy of the type referred to as 6A (Table 2). By using an oligonucleotide specific for type 6A, a full-length clone of this type was identified. The 5' EcoRI fragment of the 26 TABLE 8 Four 20-mer Oligonucleotide Pools For Cloning 35K PSP Proteins T A A GCCTCGTTTTCTTCNGGGTT GCCTCGTTTTCCTCNGGGTT T A A GCCTCGTTCTCTTCNGGGTT T A A GCCTCGTTCTCCTCNGGGTT 27 lambda gtlO cDNA clone was subcloned into M13 and sequenced as above by using specific olignucleotides as primers. This sequence is presented in Table 2. The two clones differ within the coding region at 7 nucleotides which led to amino acid changes and at 6 nucleotides which resulted in silent changes. These changes result in restriction fragment polymorphism between the two clones within the coding region for the restriction enzyme Pstl. Clone 6A has 2 Pstl sites at the nucleotide position 454 and 478 (Table 2) and clone IA has 3 Pstl sites at 454, 478 and 756 (Table 1) . Additional DNA sequencing of each clone at the 3• untranslated region revealed a large lkb untranslated region and consid¬ erably more nucleotide variation between MPSAP-IA and MPSAP-6A. DNA Binding and Hybrid Selection Very dilute (10-15 ug/ml) single stranded DNA from either subclone MPSAP-IA or MPSAP-1B was applied to nitrocellulose paper (10 ug/cm ) under vacuum (Kafatos et al., 1979). MPSAP-IA represents the M13 subclone of a 0.9kb ECORI fragment in one orientation in M13. MPSAP-1B represents the same fragment cloned in the opposite orientation in M13. Each filter (1cm 2 ) was cut into nine equal size pieces and each piece was used in a 20-30ul hybridization reaction. Each reaction containedhuman lungRNA (5mg/ml) , 50% deionized formamide (Fluka AG Chemical Corp.) , lOmM PIPES [(Piperazine-N, N'-bis) (2-ethanesulfonic acid)] pH 6.4 and 0.4M NaCl (Miller et al., 1983). The source and preparation of the RNA have been reported previously (Floros et al., 1985) . Each hybridization reaction was routinely incubated at 50°C for 3 hrs. At the end of the incubation period each filter was washed for five minutes with 1ml 1XSSC (.15M NaCl, 0.015M sodium citrate, 0.5% SDS at 60°C five times. Then it was washed for five minutes with 1ml of 2mM 28 EDTA, pH 7.9 at 60°C three times. The selected RNA was eluted by boiling for one minute in 300ul of ImM EDTA pH 7.9 and lOug of yeast tRNA (Boehringer, Mannheim). The precipitated RNA was translated, immunoprecipitated and subjected to one and two dimensional gel electrophoresis as described in Floros et al., 1985. Example 4 Screening of the cDNA Library and Sequencing of Clones for the 6Kd Proteins Based on the first six amino acids of the sequence shown in Table 5 an oligonucleotide probe was synthesized. The 0 probe consisted of six pools of 17 mers. Three of the pools each contained 128 different sequences, and three of the pools each contained 64 different sequences.Based on the first seven amino acids two pools of 20 mers were synthe¬ sized. These pools contained either 384 or 192 different sequences. A cDNA library from human lung mRNA was prepared as described in Toole et al., (1984) and screened with the total mixture of the six pools using tetramethylammoniumchloride as a hybridization solvent (Jacobs et al., 1985). Approximately 100,000 phage were screened, and 100 phage which hybridized to the probe were plaque purified. The phage were then pooled into groups of 25 and screened with the individual 17 mer and 20 er pools. Six phage which hybridized most intensely to one of the 20 mer oligonucleotide probes and one of the corresponding 17 mer pools (pool 1447 containing 128 different sequences) were plaque purified. The 17 mer pool 1447 was divided into four pools of 32 different sequences and hybridized to a dot blot of DNA prepared from these phage. 29 Based on the hybridization intensity, DNA from one of these six phage were subcloned into M13 for DNA sequence analysis. A sequence corresponding in identity and position to the amino acids shown in Table 5 was obtained, con¬ firming that the isolated clone coded for the approximately 6kd cold butanol-insoluble protein found in the lavage material of alveolar proteinosis patients (see above) . The first clone obtained was presumed to be an incomplete copy of the mRNA because it lacked an initiating methionine, and was used to isolate longer clones. Two clones were completely sequenced by generating an ordered set of deletions with Bal 31 nuclease, recloning into other M13 vectors and sequencing via the dideoxynucleotide chain termination procedure (Viera and Messing, 1982; Sanger et al., 1977). One clone corresponded to a full-length copy of the type referred to as 17 (Table 6) , the second began at nucleotide 148 of clone 17. Sequence of the 5' end of a third clone confirmed the sequence of the 5' end of clone 17. The clones are identical throughout the coding region and differ only at two positions in the 3 ■ untranslated region. 30 REFERENCES 1. Bhattacharyya, S.N., and Lynn, W.S. (1978) Biochem. Biophys. Acta 537, 329-335 2. Bhattacharyya, S.N., and Lynn, W. S. (1980) Biochem. Biophys. Acta 625, 451-458 3. Bhattacharyya, S.N., Passero, M.A. , DiAugustine, R.P. , and Lynn, W. S. (1975) J. Clin. Invest. 55, 914-920 4. Floros, J., Phelps, D.S., and Taeusch, W.H. (1985) J. Biol. Chem. 260, 495-500 5. Hawgood, S., Benson, B. J. , and Hamilton, Jr. R. L. (1985) Biochemistry 24, 184-190 6. Hunkapiller, M. W. and Hood, L. E. (1983) Methods in Enzymology 91, 486- 7. Jacobs, K. , Shoemaker, C. , Rudersdorf, R. , Neil, S. D. , Kaufman, R. J., Mufson, A., Seehra, J., Jones, S. S., Hewick, R. , Fritsch,E. E., Kawakita, M. , Shimizu, T., and Miyake, T. (1985) Nature (Lond.) 313, 806-810. 8. Kafatos, E. , Jones, W. C. , and Efstratiadis, A. (1979) Nucleic acid Rest. 7, 1541-1552. 9. Katyal, S. L. , Amenta, J. S., Singh, G. , and Silverman, J. A. (1984) Am. J. Obstet. Gynecol. 148, 48-53. 10. Katyal, S. L. and Singh, G. (1981) Biochem. 31 Biophys. Acta 670, 323-331. 11. King, R. J. , Carmichael, M. C. , and Horowitz, P.M. (1983) J. Biol. Chem. 258, 10672-10680. 12. King, R. J. (1982) J. Appl. Physiol. Exercise Physiol. 53, 1-8. 13. King. R. J., Klass, D. J., Gikas, E. G., and Clements, J. A. (1973) Am. J. Physiol. 224 , 788-795. 14. King, R. J. , Ruch, J. , Gikas, E. G. , Platzker, A. C. G. , and Creasy, R. K. (1975) J. of Applied Phys. 39, 735-741. 15. Laemmli, U. K. (1970) Nature (Lond.) 227, 680-685. 16. Miller, J. S., Paterson, B. M. , Ricciardi, R. P., Cohen, Land Roberts, B. E. (1983) Methods inEnzymology lOlp. 650-674. 17. Phelps, D. S., Taeusch, W. H. , Benson, B. , and Hawgood, S. (1984) Biochem. Biophs. Acta, 791-226-238. 18. Shelley, S. A., Balis, J. U. f Paciga, J. E., Knuppel, R. A., Ruffolo, E. H. , and Bouis, P. J. (1982) Am. J. Obstet. Gynecol. 144, 224-228. 19. Sigrist, H. , Sigrist-Nelson, K. , and Gither, G. (1977) BBRC 74, 178, 184. 32 20. Sueishi, K. , and Benson, G. J. (1981) Biochem. Biophys. Acta 665, 442-453. 21. Toole, J. J. , Knopf, J. L. , Wozney, J. M. , Sultzman L. A., Bucker, J. L. , Pittman, D. D. , Kaufman, R. J. , Brown, E., Shoemaker, C. , Orr, E. C. , Amphlett, G. W. , Foster, W. G., Coe, M. L. , Knutson, G. L. , Eass, D. N. , Hewick, R. M. (1984) Nature (Lond.) 312, 342-347. 22. Whitsett, J. A., Hull, W. , Ross, G. , and Weaver, T. (1985) Pediatric Res. 19, 501-508. 23. Wong, G.G. et al., 1985, Science, 228:810-815

Claims: 33 What is claimed is: 1. A purified protein useful for enhancing pulmonary surfactant activity characterized by a molecular weight of about 35 , 000 daltons and by being encoded for a DNA sequence selected from the group consisting of: a) GAA TTCCGCAGAG ACCCAAGCAC CTGGAGGCTC 29 0 - J OO 310 320 330 34 Q TGTG T GTGGG TCGCTGATTT CTTGGAGCCT GPAAAGfiAGG AGCAGCGACT GGACCCACAG CC ATG ΠET 3Ξ7 372 337 -33 CTG T3C CCT CTG CCC CTC ACC CTC ATC TTG ATG GCA GCC TCT GGT GCT CCG ~~ a Lou Cys Pro Leu Ala Leu Thr * Leu Ho Lou MET Ala Ala Ser Gly Ala Ala _C-Z 417 432 447 TGC GCA GTG AAG GAC GTT TGT GTT GGA AGC CCT GGT ATC CCC GGC ACT CCT GGA Cys Glu Val Lys Aop Val Cys Vαl Gly Ser Pro Gly Ilo Pro Gly Thr Pro Gly 462 477 492 Ϊ37 TCC CZC GGC CTG CCA GGC AGG GAC GGG AGA GAT GGT GTC AAA GΞA GAC CCT GGC Ssr His Gly* Leu Pro Gly Arg Aop Gly Arg Asp Gly Vαl Lys Gly Asp Pro Gly Ξ22 337 552 CCT CCA GGC CCC ATG GGT CCG CCT GSA GAA ACA CCA TGT CCT CCT GGG AAT AAT Pro P o Gly Pro MET Gly Pro Pro Gly* Glu Thr Pro Cys Pro Pro Gly Asn Aon -£>7 5Θ2 397 612 GGG CTG CCT GSA GCC CCT GGT GTC CCT GGft GAG C3T GGA GAS AAG GGG GAG CCT Gly Leu Pro Gly Ala Pro Gly Val Pro Gly Glu. Arg Gly Glu Lys Gly Glu Pro e27 £ 2 637 GGC G A G PGA GGC CCT CCA GGG CTT CCA GCT CAT CTA GAT GAG GAG CTC CAA GCC Gly Glu Arg Gly Pro Pro Gly Leu Pro Ala Hie Leu A«p Olu Glu Leu Gin Ala 34 claim 1 cont. C72 -.37 703 717 A CA CTC CAC GAC TTC AGA CAT CAA ATC CTQ CAG ACA AOG GSA CCC CTC AGT CTG Thr L eu His Asp Pho Arg Hie Gin Ilo Leu Gin Thr Arg Gly Ala Lou Sor Leu 732 747 762 777 CAG GGC TCC ATA ATG ACA GTA GGA GAG AAG OTC TTC TCC AGC AAT G3G CAG TCC Gin Gly Ser Ilo MET Thr Val Gly Glu Lye Val Pho Sor Sor Aon Gly Oln 5or 792 807 β22 ATC ACT TTT GAT GCC OTT CAG GAG GCP TGT GCC AGA GCA GGC GGC CGC ATT GCT lie Thr Phe Asp Ala lie Gin Glu Ala Cys Ala Arg Ala Gly Gly Arg lie Ala 837 632 G67 0S2 GTC CCA AGG AAT CCA GAG GAA AAT GAG GCC ATT GCA AGC TTC GTG AAG AAG TAC Val Pro Arg Aen Pre- Glu Glu Aen Glu Ala Ilo Ala Sor Pho Val Lya Lyo Tyr 397 912 927 AAC ACA TAT GCC TAT GTA GGC CTG ACT GAG GGT CCC AGC CCT GGA GAC TTC CGC Aan Thr Tyr Ala Tyr_Val Gly Lou Thr Glu Gly Pro Sor Pro Gly βep Phe Arg 942 937 972 907 TAC TCA GAT GGG ACC CCT GTA AAC TAC ACC AAC TOO TAC CGA CGG CAG CCT GCA Tyπ, Ser Anp Gly Thr Pro Val Aen Tyr Thr Aen Trp Tyr Rrg Gly Glu Pro Ala 1002 1017 1032 1347 GGT CGG GGA AAA GAG CAG TGT GTG GAG ATG TAC AGO GAT GGG CAG TG3 AAT GAC Gly Arg Gly Lys Glu Gin Cye Val Glu WET Tyr Thr Aβp Gly Gin Trp Aen Aep 1062 1077 ... .1099 . 1109 AGG AAC TGC CTG TAC TCC CGA CTO ACC »TC TGT 0A3 TTC TGA GSGGCATTTO GGCCATGG3Λ Arg Pan Cye Lou Tyr Ser Arg Leu Thr 11* Cyβ Slu Phβ . 1119 1129 1139 1149 1139 1169 1179 CAGGGAGGAT CCTGTCTGGC CTTCAQTTTC CATCCCCAGQ ATCCACTTGG TCTGTGAGAT GCTACAACTC 1189 CCTTTCAACAGAATTC 34A. claim 1 cont. and G A ATT CCSGA GACCCAAGCA GCTCGAOGCT CTOTGTOTCO QTCCCTCAQT TTCTTCCAGC CTGSAOAGAO b. aa ~~ - O 124 _*G~3CAGCGA CTG3ACCCAG AGCC ATG TGG CTG TGC CCT CTG GCC CTC AAC CTC OTC MET Trp-Leu Cys Prrj Let Ala Leu Asn Leu lie 139 1S4 169 TTG ATG GCA GCC TCT GGT GCT GCG TGC GAA GTG AAG GAC GTT TGT OTT G3A CGC Leu MET Ala Ala Ser Gly Ala Ala Cyo Glu Vαl Lys Asp Val Cyo Val 6Iy Sor 13* 193 214 229 CCT GGT ATC CCC GGC ACT CCT GOA TCC CAC GCC CTG CCA GGC RCG GAC GGG RGA Pro Gly lie Pro Gly Thr Pro Gly Ser His Gly Lou Pro Gly Arg Asp Gly Arg 244 253 274 239 GAT GGT CTC ASA GSA GAC CCT GGC CCT CCA GGC CCC ATG GGT CCG CCT GGA GAA P SD uly Leu Lys Gly Asp Pro Gly Prr Pro Gly Pro WET Gly Pro Pro Gly Glu 304 319 334 ATG CCA TGT CCT CCT GGA AAT GAT GGG CTG CCT GSA GCC CCT GGT ATC CCT GGA rET Pro Cys Pro Pro Gly Asn Asp Gly Leu Pro Gly Al Pro Gly He Pro Gly 343 364 379 394 GA3 T3T GGA GAS AAG GGG GAG CCT GGC GAG AGG GGC CCT CCA GGG CTT CCA GCT Glu Cys Gly Glu Lys Gly Glu Pro Gly Glu Arg Gly Pro Pro Gly Lou Pro Ala ' 409 424 439 CAT CTA GAT GAG GAG CTC CAA GCC ACA CTC CAC GAC TTT AGA CAT CAA OTC CTG His Leu Asp Glu Glu Lou Gin Ala Thr Leu His Aep Pho Arg Hie Gin Ilo Leu 43 469 434 499 CAG ACA AGG GGA GCC CTC AGT CTG CAO GGC TCC ATA OTG ACG GTA GGA GAG AAG Glr. Thr- Arg Gly Ala Lou Sor Lou G n Gly Ser Ilo MET Thr Val Gly Glu Lye =14 329 344 359 C-TC TTC TCC AGC OAT GGG CAG TCC OTC ACT TTT GAT GCC ATT CHC1 GAG GCO TOT v.il Phe __pι- Set* Oβn Oly Oln Dor Ilo Thr Pho flap Ala Ilo Uln Glu Ola Cyn claim 1 cnnr.. 3 ^ B 374 389 604 GCC PGA GCA GGC GGC CGC OTT GCT GTC CCA AGG OAT CCO CAG OAA AAT GAG GCC d l - Org Ola Gly Gly Arg lie Ala Val Pro Arg Aen Pro Glu Glu Osn Glu Ala 813 634 643 664 ► • IT' GCO OGC TTC GTG AAG AAG TAC AAC ACA TAT GCC TAT GTA GGC CTG ACT GAG lie Ala Ser Pho Val Lys Lys Tyr Aon Thr Tyr Olα Tyr Val Gly Leu Thr Glu 673 694 709 GGT CCC AGC CCT GGA GAC TTC CGC TAC TCA GAC GGG OCC CCT GTA AAC TAC ACC Gly Pro Ser Pro Gly Asp Pho Arg T~ ~tSer Aop Gly Thr Pro Val Aan Tyr Thr 724 739 754 769 AAC TGG TAC CGA GGG GAG CCC GCA GGT CGG GGA AAA GAG CA3 TGT GTG GAG OTG Asn Trp TyV Org Gly Glu Pro Ola Gly Arg Gly Lys Glu Gin Cys Val Glu MET 734 799 314 323 TAC PICA GOT GGG CAG TGG OAT GAC OGG OOC TGC CTG TAC TCC CGA CTG ACC OTC Tyr Thr Osp Gly Gin Trp Oβn Osp Org Aen Cys Leu Tyr Ser Org Leu Thr He 351 361 371 331 891 TGT GAG TTC TGA GAGGCOTTTA GGCCATGGGA COGGGOGGAC GCTCTCTG3C CTTC3GCCTC Cys Glu Phe 9.31 911 921 931 941 COTCCTGAGG CTCCACTTGG TCTGTGAGAT GCTAGAACTC CCTTTCAOCO 2 . "- A purified protein useful for enhancing pulmonary surfactant activity which is characterized by a molecular weight of about 5.5-9 kilodaltons and encoded by the following DNA sequence: 10 28 43 GAATTCCGGT GCC ATG GCT GAG TCA CAC CTG CTG COG TGG CTG CTG CTG CTG CTG MET Ala Glu Ser His Leu Leu Gin Trp Leu Leu Leu Leu Leu CCC ACG CTC TGT GGC CCA GGC ACT GCT GCC TGG ACC ACC TCA TCC TTG GCC TGT Pro Thr Leu Cys Gly Pro Gly Thr Ala Ala Trp Thr Thr Ser Ser Leu Ala Cys 34C claim 2 cont. ll β 133 143 16 GCC COG GGC CCT GAG TTC TGG TGC CAA AGC CTG GAG CAA GCA TTG CAG TGC AGA Ala Gin Gly Pro Glu Phe Trp Cys Gin Ser Leu Glu Gin Ala Leu Gin Cys Arg 173 193 208 GCC CTA GGG COT * TGC CTO CAG GAA GTC TGG GGO CAT GTG GGO GCC GAT GAC CTO Ola Leu Gly His Cys Leu Gin Glu Val Trp Gly His Val Gly Ola Osp Osp Leu 223 233 253 268 TGC COO GAG TGT GAG GAC ATC GTC CAC ATC CTT AAC AAG ATG GCC AAG GOG GCC Cys Gin Glu Cye Glu Asp lie Val His He Leu Asn Lys MET Ala Lys Glu Ola 233 ' 238 313 OTT TTC COG GOC ACG ATG OGG ~'G TTC CTG GAG CAG GflG TGC AAC GTC CTC CCC He Phe Bin Osp Thr MET Ot-g Lys Phe Leu Glu Gin Glu Cys Osn Val Leu Pro 323 343 358 373 TTG OOΞ CTG CTC OTG CCC COG TGC AAC CAA GTG CTT GAC GAC TAC TTC CCC CTG Leu Lys Leu __<.ι MET Pro Gin Cys Osn Gin Val Leu Osp Osp Tyr Phe Pro Leu 283 4ι33 418 433 ετc ATC GOC TOC rrc COG OAC CAG ACT GAC TCA OOC GGC OTC TGT ATG CAC CTG Val He Osp Tyr Phe Gin Osn Gin Thr Asp Ser Osn Gly He Cys MET His Leu 448 463 478 GGC CTG TGC OAfl TCC CGG CAG CCA GAG CCA GAG CAG GAG CCA GGG ATG TCA GOC Gly Leu Cys Lys Ser Org Gin Pr»o Glu Pro Glu Gin Glu Pro Gly MET Ser Osp 433 508 523 533 CCC CTG CCC AOA CCT CTG CGG GAC CCT CTG CCA GAC CCT CTG CTG GAC OOG CTC Pro Leu Pro Lys Pro Leu Arg Asp Pro Leu Pro Asp Pro Leu Leu Asp Lys Leu 553 563 583 GTC CTC CCT GTG CTG CCC GGG GCC CTC CAG GCG AGG CCT GGG CCT COC OCO COG Val Leu Pro Val Leu Pro Gly Ola Leu Gin Ola Org Pro Gly Pro His Thr Gin 34D claim 2 cont. 593 613 623 643 GOT CTC TCC GAG CAG CAA TTC CCC OTT CCT CTC CCC TAT TGC TGG CTC TGC OGG Osp Leu Ser* Glu Gin Gin Phe Pro He Pro Leu Pro Tyr Cys Trp Leu Cys Org 658 673 ' 638 783 GCT CTG OTC AAG CGG ATC CAA GCC ATG ATT CCC AAG GGT GCG CTO GCT GTG GCO Ala Leu He Lys flrg He Gin Ola WET He Pro Lys Gly Ola Leu Ola Va Ola 713 733 748 GTG GCC COG GTG TGC CGC GTG GTO CCT CTG GTG GCG GGC GGC ATC TGC COG TGC Val Ola Gin Val Cys Org Val Val ' Pro Leu Val Ola Gly Gly He Cys Gin Cys 7~ - 778 793 β θ CTG GCT GOG CGC TAC TCC GTC OTC CTG CTC GAC OCG CTG CTG GGC CGC OTG CTG Leu Ola Glu Arg Tyr Ser Val He Leu Leu Osp Thr Leu Leu Gly Org MET Leu BΞ3 833 S5 CCC COG CTG GTC TGC CEC CTC GTC CTC CGG TGC TCC OTG GAT GOC OGC ECT GGC ro Gin Leu Val Cys Org Leu Val Leu Org Cys Ser MET Osp Osp Ser O la Gl y θ £8 BΘ3 838 913 CC' OGG TCG -CCG OCO GGO GOO TGG CTG CCG CGO GOC TCT GOG TGC CAC CTC TGC Pro flrg Ser Pro T h r Gly Glu Trp Leu Pro Org Osp Ser Glu Cys His Leu Cy s S_B 343 358 373 O TG TCC GTG ACC OCC COG GCC GGG AAC OGC OGC GAG CAG GCC OTO CC~ CflG GC O M ET Ser V al T h r T h r Gin Ola Gly Osn Ser Ser Glu Gin Ola He Pro Gin Ola 3B8 100 1018 OTG CTC CAG GCC TGT GTT GGC TCC TGG CTG GAC OGG GOO OOG TGC O O G COO TTT MET Leu Gin Ala Cys Val Gly Ser Trp Leu Osp Org Glu Lys Cys Lys Gin P h e 1023 1043 1063 1079 GTG G O G C O G COC OCG CCC COG CTG CTG OCC CTG GTG CCC OGG GGC TG G GO T GCC Val Glu Gin His T h r Pro Gin Leu Leu Thr Leu Val Pro Arg Gly Trp Asp Ola SUBSTITUTE SHEET 8 / 34E claim 2 cont. 1033 * 1108 1123 - CAC OCC OCC TGC CAG GCC CTC GGG GTG TGT GGG ACC ATG TCC OGC CCT CTC COG Hrs Thr Thr Cys Gin Ola Leu Gly Val Cys Gly Thr MET Set- Set- Pro Leu Gin 1133 1153 1166 1176 1136 1136 TGT OTC COC OGC CCC GAC CTT TGOTGAGAOC TCOGCTGTCC AGCTGCAAAG GAAAAGCCOO Cys He His Ser- Pro Asp Leu 1206 1216 1226 1236 1246 1256 1266 GTGOGOCGGG CTCTGGGACC ATGGTGOCCO GGCTCTTCCC CTGCTCCCTG GCCCTCGCCO GCTGCCOGGC 1276 1286 1236 1306 1316 1326 1236 TEOOKOGOi- CCTCOGCTCC COCOCCGCCC TCCTCOCCTC CCTTCCTCGG COGTCOCTTC COCTGGTGGO 13H6 1356 1366 1376 1386 1336 14ø6 CCOCGGGCCC CCOGCCCTGT GTCGGCCTTG TCTGTCTCOG CTCOOCCOCO GTCTGOCOCC OGOGCCCOCT 1-*16 14£6 1436 1446 1456 1466 1476 TCCOTCCTCT CTGGTGTGOG GCOCOGCGOG GGCOGCOTCT GGOGGOGCTC TGCOGCCTCC OCOCCTOCCO I486 1496 1506 1516 1526 1536 1546 CGOCCTCCCO GGGCTGGGCT COGGAflflflflC COGCCOCTGC TTTOCOGGOC OGGGGGTTGO OGCTGOGCCC 1556 1566 1576 1536 1596 1606 1616 CGCCTCOCOC CCOCCCCCOT GCACTCAAAG OTTGGATTTT OCOGCTOCTT GCfiOTTCflflfl OTTCOGOOGO 1626 1636 - 1646 1656 1666 1676 1686 OTOOOflOOTG GGOOCOTOCfl.GOACTCTOOO OGATAGACAT CAGAAATTGT TAAGTTAAGC TTTTTCOAOA 163S 1706 1716 1726 1736 1746 1756 AATCAGCAAT TCCCCAGCGT OGTCAAGGGT GGACACTGCA CGCTCTGGCA TGATGGGATG GCGOCCGGGC 1766 1776 1736 1796 1806 1316 1826 OOGCTTTCTT CCTCGAEATG CTCTGCTGCT TGAGOGCTAT TGCTTTGTTO OGOTOTOOOO OGGGGTTTC 1336 1346 1356 1866 1876 1886 1896 TTTTGTCTTT CTGTAOGGTG GOCTTCCOGC TTTTGOTTGO OOGTCCTOGG GTGOTTCTOT TTCTGCTGTG 1906 1916 1926 1936 1346 1956 1966 OTTTOTCTGC TGAOAGCTCA GCTGGGGTTG TGCOOGCTOG GGOCCCOTTC CTGTGTOOTO COOTGTCTGC 1976 1986 3996 . 2øø6 2016 2026 OCCflATGCTA OTOOOGTCCT OTTCTCTTTT OROOAOOOOA OOAOOOOOOO OACGGOOTTC Deduced protein sequence of 6Kd PSP protein is underlined P. IN . _- S__TI UTE .' ~ *K 7/02037 34G 3 . A p urified protein useful for en h ancing pu l monary sur f actant activity characterized by a mo l ecu l ar weig h t o f a b out 5 .9-9 kilodaltons and an amino acid composition substantially as follows: Asp/Asn 2.7 Thr 2.0 Ser 2.1 Glu/Gln 1.6 Pro 6.3 Gly 22.9 Ala 3.3 Cys 0.95 Val 5.5 Met 3.4 lie 4.8 Leu 17.3 Tyr 3.3 Phe 6.3 His 2.9 Lys 3.6 Arg 1.94 (calculated based on M =10 , 000 daltons ; ave residue MW=110) 34H 4. A recombinant DNA molecule encoding a protein of claim 1. 5. A recombinant DNA molecule encoding a protein sequence of cl aim 2 or a DNA molecule capable of hybridizing th e ret o , which encodes a prote in having pulmonary surfactant enhancing activity. 6. A host cell or cell line transformed with the DNA of claim 4 or claim 5. 7. A method for producing a protein useful for enhancing pulmonary surfactant activity which comprises culturing a cell or cell line of claim 6. 8 . A protein useful for enhancing pulmonary surfactant activity produced by the method of claim 7. 9 . A pulmonary surf actant compos ition comprising an effective surfactant enhancing amount of one or more of the proteins of claim 1 , 2 , 3 , or 8 and a pharmaceutically acceptable fatty acid and/or lipid. 1 0 . A ve ctor conta in ing a heterologous DNA sequence encoding a peptide comprising the sequence : I KRI QAMI PKGALAVAVAQVCRWPLVAGGI CQC 11. A vector containing the heterologous DNA sequence : ATC AAG CGG ATC CAA GCC ATG ATT CCC AAG GGT GCG CTA GCT GTG GCA GTG GCC CAG GTG TGC CGC GTG GTA CCT CTG GTG GCG GGC GGC ATC TGC CAG TGC 12 . A vector containing the heterologous DNA sequence encoding a peptide comprising the sequence: SUBSTITUTE SHEET 7/02037 341 FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRWPLVAGGICQCLAERY- SVILLDTLLGRML 13. A vector containing the heterologous DNA sequence: TTC CCC ATT CCT CTC CCC TAT TGC TGG CTC TGC AGG GCT CTG ATC AAG CGG ATC CAA GCC ATG ATT CCC AAG GGT GCG CTA GCT GTG GCA GTG GCC CAG GTG TGC CGC GTG GTA CCT CTG GTG GCG GGC GGC ATC TGC CAG TGC CTG GCT GAG CGC TAC TCC GTC ATC CTG CTC GAC ACG CTG CTG GGC ATG CTG A __ _MME—.NrSDlJE—.DU C t-Ll_A_l _I_MiTISι_J [received by / tthhee IInntteerrnnaational Bureau on 25 February 1987 (25.02.87): o rriiggiinnaall ι claims 1-14 replaced by amended claims 1-13 (1 1 pages)] 1. A purified protein useful for enhancing pulmonary surfactant activity characterized by a molecular weight of about 35,000 daltons and by being encoded for a DNA sequence selected from the group consisting of: a) GAA TTCCGCAGAG ACCCAAGCAC CTGGAGGCTC 290 300 310 320 330 340 TGTGTG T G GG TCGCTGATTT CTTGGAGCCT GAAAOGAAGG AGCOGCGACT GGACCCACAO CC OTG ΠET 357 372 337 ■ ~_3 CTG TGC CCT CTG CCC CTC OCC CTC OTC TTG OTG GCO GCC TCT GGT GCT GCG "Trp Leu Cys Pro Leu Ala Leu Thr Leu He Lou MET Ala Ala Sor Gly Oiα Ola _ι?2 417 432 447 TGC GflO GTG OOG GOC GTT TGT GTT GGO OGC CCT GGT OTC CCC GGC OCT CCT GGO Cys Glu Val Lys flap Val Cy- Val Gly Ser Pro Gly Ilo Pro Gly Thr Pro Gly 462 477 492 537 TCC COC GGC CTG CCA GGC AGG GAC GGG AGO GAT GGT GTC AAO G30 GOC CCT GGC Ser His Gly Leu Pro Gly Arg Aap Gly Arg flap Gly Val Lys Gly Asp Pro Gly 522 537 552 CCT CCO GGC CCC ATG GGT CCG CCT GGA GAA ACA CCA TGT CCT CCT GGG AAT AAT Pro Pro Gly Pro MET Gly Pro Pro Gly Glu Thr Pro Cys Pro Pro Gly Aan Osn =67 582 597 612 GGG CTG CCT GGO GCC CCT GGT GTC CCT GG« GAG CGT GGO GOG OHB GGG GAG CCT Gly Leu ~r _, Gly Ala Pro Gly Val Pro Gly Glu. Arg Gly Glu Lys Gly Glu Pro e£7 642 657 GGC GAG AGA GGC CCT CCA GGG CTT CCA GCT CAT CTA GAT GAG GAG CTC COO GCC Gly Glu Arg Gly Pro Pro Gly Leu Pro Ala H.β Leu Asp Glu Glu Lou Gin Ala claim 1 cont. 672 - ' 7 7β2 7l7 O C O CTC COC GOC TTC AGA COT CAA ATC CTO CAG OCO AOG GGA GCC CTC AGT CTG T hr - Leu His Osp Pho Org His Gin He Leu Gin Thr Arg Gly Ala Lou Sor Lou ' 732 747 762 777 CAG GGC TCC ATA ATG CCA GTA GGA GAG AOG OTC TTC TCC AGC AAT GGS CAG TCC Gin Gly Sor Ho KET Thr Val Gly Glu Lye Vat Pho Sor Sor Aen Gly Oln Sor 792 307 Θ22 ATC ACT TTT GAT GCC OTT CAG GAG GCA TGT GCC AGA GCA GGC GGC CGC ATT GCT He Thr Phe Asp Ala He Gin Glu Ala Cys Ala Arg Ala Gly Gly Arg Ilo Ola 837 352 C67 382 GTC CCA AGG OAT CCA GAG GAA AAT GAG GCC ATT GCA OGC TTC GTG AAG AAG TAC Val Pro Arg Part Pre- Glu Glu Aen Glu Ala Ho Ola Sor Phβ Val Lys Lyo Tyr 397 912 927 AAC ACA TAT GCC TOT GTA GGC CTG ACT GAG GGT CCC AGC CCT GGfl GAC TTC CGC Asn Thr- Tyr Ala T r_Val Gly Leu Thr- Glu Gly Pro Sor* Pro Gly Aβp Phβ Arg 942 957 972 907 TAC TCA GAT GGG ACC CCT GTA AAC TAC ACC RAC TGG TAC CGA CGG GAG CCT GCA Tyu. Ser Asp Gly Thr- Pro Val Oβn Tyr* Thr Osn Trp Tyr flrg Gly Glu Pro Ala 1002 1817 1032 1047 GGT CGG GGA AAA GAG CAS TGT GTG GAG ATG TAC OCfl GAT GGG CAG TGG AAT GAC Gly Arg Gly Lys Glu Gin Cys Val Glu IΕT Tyr Thr Asp Gly Gin Trp flβ Asp 1062 1077 .1099 . 1109 AGG AAC TGC CTG TAC TCC CGA CTG ACC ATC TGT OAG TTC TGA GRGGCOTTTfl GGCCATGGGft Arg Asn Cyβ Lou Tyr Sor Org Leu Thr II* Cyo Glu Phβ . 1119 1129 1139 1149 1159 1169 1179 CAGGGAGGAT CCTGTCTGGC CTTCAGTTTC CATCCCCAGQ OTCCACTTGG TCTGTGAGAT GCTACAACTC 1189 CCTTTCAACAGAATTC claim 1 cont. and V .. GA ATT CCSGA GOCCCOADCA GCTGGAOC.CT CTOTGTOTCO Q TCCCTGOOT TTCTTGCAGC CTGOOOβGOA ββ 90 10 124 i S Gi ϊ GCAGCGA CTG3ACCCAG AGCC OTD TGG CTG TGC CCT CTG GCC CTC AAC CTC OTC MET Trp- eu Cyo Prr? Leu Ala Leu Asn Leu Ilo 139 134 169 TTS ATG GCA GCC TCT GGT GCT GCG TGC GAA GTG AAG GAC GTT TGT CTT GGO C3C Leu MET Ala Ala Ser Gly Ola Ala Cyo Glu Val Lys Asp Val Cyo Val Gly Sor IB* 199 214 229 CCT GGT OTC CCC GGC ACT CCT GGA TCC CAC GCC CTG CCA GGC OCG GAC GGG RGA Pro Gly lie Pro Dly Thr Pro Gly Ser His Gly Lou Pro Gly Org Osp Gly Org 244 253 274 239 GOT GGT CTC ASA GGO GAC CCT GGC CCT CCA GGC CCC ATG GGT CCG CCT GGA GAA Pso Gly Leu Lys Gly Asp Pro Gly Prr Pro Gly Pro MET Gly Pro Pro Gly Glu 304 319 334 ATG CCA TGT CCT CCT GGA OAT GAT GGG CTG CCT GGA GCC CCT GGT ATC CCT GGO l-ET Pro Cys Pro Pro Gly Osn Osp Gly Leu Pro Gly Ala Pro Gly He Pro Gly _• 349 364 379 394 G03 TGT GGO GAG AAG GGG GAG CCT GGC GAG AGG GGC CCT CCA GGG CTT CCO GCT Glu Cys Gly Glu Lys Gly Glu Pro Gly Glu Arg Gly Pro Pro Gly Lou Pro Ala / 409 424 439 CAT CTO GAT GAG GOG CTC CAA GCC ACA CTC CAC GAC TTT AGA CAT CAA OTC CTG His Leu Asp Glu Glu Lou Gin Ala Thr Lou His Aβp Phe Arg Hlo Gin Ho Leu 3 69 4β4 499 CS3 ACA OGG GGA GCC CTC OGT CTG CAQ GGC TCC ATA OTB ACG GTA GGO GAG AAG Gin Thr Arg Gly Ala Lou Sor Lou Gin Gly Sor Ho MET Thr Val Gly Glu Lya « t 514 529 544 359 GTC TTC TCC AGC OAT GGG CAG TCC OTC ACT TTT GOT GCC OTT C«G GOG GCO TGT .il Phe Orr Get- Osn Gly Gin Sor Ho Thr Pho Asp Ola Ilo Gin Glu Ola Cyv claim 1 cnnt. 374 589 604 GCC AGA GCA GGC GGC CGC OTT GCT GTC CCA AGG RAT CCA CAG GAA OAT GAG GCC Ola Org Olα Gly Gly Org He Ola Val Pro flrg flmn Pro Glu Glu Osn Glu Olα 613 334 649 664 o - GCO OGC TTC GTG OAG RAG TAC RAC OCA TAT GCC TAT GTA GGC CTG ACT GAG He Ala Ser Pho Val Lys Lys Tyr Aαn Thr Tyr Ola Tyr ' Val Gly Lou Thr Glu 679 694 709 GGT CCC AGC CCT GGO GAC TTC CGC TAC TCA GAC GGG RCC CCT QTA RAC TAC ACC Gly Pro Sor Pro Gly Asp Phe Org TV-Ser flop Gly Thr Pro Val Asn Tyr Thr 724 739 754 769 OOC TGG TAC CGO GGG GAG CCC GCA GGT CGG GGA AAA GAG CAG TGT GTG GAG OTG Asn Trp Tyr- flrg Gly Glu Pro Ala Gly Arg Gly Lys Glu Gin Cys Val Glu MET 794 799 814 823 TAC ACA GAT GGG CAG TGG OAT GAC OGG AAC TGC CTG TAC TCC CGA CTG OCC OTC Tyr Thr Osp Gly Gin Trp Osn Osp Arg Osn Cyo Leu Tyr Ser Arg Leu Thr He SSI 361 871 381 391 TGT GAG TTC TGA GAGGCATTTO GGCCATGGGA CAGGGRGGflC GCTCTCTG3C CTTC3GCCTC Cys Glu Phe . 931 911 921 931 941 COTCCTGAGG CTCCACTTGG TCTGTGAGAT GCTAGAACTC CCTTTCAOCO 2. * • A purified protein useful for enhancing pulmonary surfactant activity which is characterized by a molecular weight of about 5.5-9 kilodaltons and encoded by the following DNA sequence: 10 28 43 GAATTCCGGT GCC ATG GCT..GAG TCA CAC CTG CTG CAG TGG CTG CTG CTG CTG CTG MET Ala Glu Ser His Leu Leu Gin Trp Leu Leu Leu Leu Leu 58 73 33 103 CCC ACG CTC TGT GGC CCA GGC ACT GCT GCC TGG OCC OCC TCO TCC TTG GCC TGT Pro Thr Leu Cy* Gly Pro Gly Thr Ala Ala Trp Thr Thr Ser Ser Leu Ala Cys claim 2 cont. ll β 133 l_,8 163 GCC COG GGC CCT GAG TTC TGG TGC CAA AGC CTG GAG CAA GCA TTG CAG TGC AGA Ala Gin Gly Pro Glu Phe Trp Cys Gin Ser Leu Glu Gin Ala Leu Gin Cys Org 178 193 208 GCC CTO GGG CAT ' TGC CTA CAG GAA GTC TGG GGO COT GTG GGA GCC GAT GAC CTA Ala Leu Gly His Cys Leu Gin Glu Val Trp Gly His Val Gly Ala Asp Asp Leu 223 238 253 268 TGC CAA GAG TGT GAG GOC ATC GTC CAC ATC CTT AAC AAG ATG GCC AAG GOG GCC Cys Gin Glu Cy* Glu Asp He Val His He Leu Osn Lys MET Ola Lys Glu Ola 283 " 298 313 OTT TTC COG GOC ACG OTG OGG OAG TTC CTG GAG CAG GAG TGC OOC GTC CTC CCC He Phe Gin Osp Th MET Org Lys Phe Leu Glu Gin Glu Cys Osn Val Leu Pro 328 343 358 373 TTG OOG CTG CTC OTG CCC COG TGC OOC COO GTG CTT GOC GOC TOC TTC CCC CTG Leu Lys Leu Leu MET Pro Gin Cys Osn Gin Val Leu Osp Osp Tyr Phe Pro Leu 388 403 418 433 GTC OTC GOC TOC TTC COG OOC COG ACT GAC TCO OOC GGC OTC TGT OTG CAC CTG Val He Osp Tyr Phe Gin Osn Gin Thr Osp Ser Osn Gly He Cys MET His Leu 443 463 478 GGC CTG TGC OAO TCC CGG CAG CCO GOG CCO GOG CAG GOG CCO GGG OTG TCfl GOC Gly Leu Cys Lys Ser flrg Gin Pr»o Glu Pro Glu Gin Glu Pro Gly MET Ser Osp 493 508 523 538 CCC CTG CCC 000 CCT CTG CGG GAC CCT CTG CCO GAC CCT CTG CTG GOC OAG CTC Pro Leu Pro Lys Pro Leu Org Asp Pro Leu Pro Asp Pro Leu Leu Asp Lys Leu 553 568 533 GTC CTC CCT GTG CTG CCC GGG GCC CTC CAG GCG AGG CCT GGG CCT COC OCA COG Val Leu Pro Val Leu Pro Gly Ala Leu Gin Ala Org Pro Gly Pro His Thr Gin claim 2 cont. 538 613 623 643 GOT CTC TCC GRG CAG CAA TTC CCC ATT CCT CTC CCC TOT TGC TGG CTC TGC OGG Asp Leu Ser Glu Gin Gin Phe Pro He Pro Leu Pro Tyr Cys Trp Leu Cys Arg 658 673 • 688 703 GCT CTG OTC AAG CGG ATC CAA GCC ATG ATT CCC OAG GGT GCG CTO GCT GTG GCO Ola Leu He Lys flrg lie Gin Ola MET He Pro Lys Gly Ala Leu Ala Val Ola 713 733 748 GTG GCC CAG STG TGC CGC GTG GTA CCT CTG GTG GCG GGC GGC ATC TGC CAG TGC Val Ola Gin Val Cys flrg Val Val Pro Leu Val Ala Gly Gly He Cys Gin Cys 763 778 793 808 CTG GCT GAG CGC TfiC TCC GTC ATC CTG CTC GAC ACG CTG CTG GGC CGC ATG CTG Leu Ala Glu flrg Tyr Ser Val He Leu Leu Osp Thr Leu Leu Gly flrg MET Leu 8E3 838 853 CCC CAG CTG GTC TGC CGC CTC GTC CTC CGG TGC TCC OTG GOT GAC OGC GCT GGC Pro Gin Leu Val Cys flrg Leu Val Leu flrg Cys Ser MET Asp Asp Ser Ala Gly B68 883 838 31 ' flGG TC -CCG ACA GGA Gflfl TGG CTG CCG CGA GAC TCT GflG TGC COC CTC TGC Pro fl rg Ser r* T h r Gly Glu Trp Leu Pro Org Osp Ser Glu Cys His Leu Cys 92B 943 958 973 O TG TCC GTG ACC ACC CAG GCC GGG OAC OGC OGC GAG COG GCC OTO CCO C O G GCO MET Ser Val T h r Thr Gin Ola Gly Osn Ser Ser Glu Gin Ola He Pro Gin Ala 3S3 1003 101S ATG CTC CAG GCC TGT GTT GGC TCC TGG CTG GAC AGG GAA AAG TGC AAG CAA TTT MET Leu Gin Ala Cys Val Gly Ser Trp Leu Osp Arg Glu Lys Cys Lys Gin P h e 1 ( 533 1048 10G3 1 8 GTG GflG CAG COC ACG CCC CAG CTG CTG OCC CTG GTG CCC OGG GGC TGG GAT GCC Val Glu Gin His T h r Pro Gin Leu Leu Thr Leu Val Pro Arg Gly Trp Asp Ala claim 2 cont . 1093 ' 1103 1123 * COC OCC OCC TGC COG GCC CTC GGG GTG TGT GGG OCC OTG TCC OGC CCT CTC COG His Thr Thr Cys Gin Ala Leu Gly Val Cys Gly Thr MET Ser Ser Pro Leu Gin 1138 1153 1166 1176 1186 1196 TGT ATC CAC AGC CCC GOC CTT TGOTGAGAOC TCOGCTGTCC AGCTGCAOAG GOflflflGCCflfl Cys He His Ser Pro Osp Leu liiZiG 1216 1226 1236 1246 12Ξ6 1266 GTGOGOCGGG CTCTGGGOCC ATGGTGOCCO GGCTCTTCCC CTGCTCCCTG GCCCTCGCCO GCTGCCOGGC 1276 1286 1236 1306 1316 1326 1336 TGOAOOGORG CCTCAGCTCC COCOCCGCCC TCCTCOCCTC CCTTCCTCGG COGTCOCTTC COCTGGTGGO 1346 1356 1366 1376 1336 1336 1406 CCOCGGGCCC CCOGCCCTGT GTCGGCCTTG TCTGTCTCOG CTCOflCCflCfl GTCTGOCOCC OGOGCCCOCT 1Ή16 1426 1436 1446 1456 1466 1476 TCCOTCCTCT CTGGTGTGOG GCOCOGCGOG GGCOGCflTCT GGflGGOGCTC TGCOGCCTCC OCOCCTOCCO 1486 1496 1506 1516 1526 1536 1546 CGOCCTCCCO GGGCTGGGCT COGGOAflflflC CAGCCACTGC TTTACAGGAC AGGGGGTTGA OGCTGOGCCC 1556 1566 1576 1536 1596 1606 1616 CGCCTCOCflC CCACCCCCAT GCACTCAAAG ATTGGATTTT ACAGCTACTT GCAATTCAAO ATTCAGAAGO 1626 1636 • 1646 1656 1666 1676 1686 AT AOOAATG GGAACOTOCA.GAACTCTAAA A6ATAGACAT COGAAATTGT TAAGTTAAGC TTTTTCOOOfl 1696 1706 1716 1726 1736 1746 1756 AOTCAGCOAT TCCCCAGCGT AGTCAAGGGT GGACACTGCA CGCTCTGGCO TGOTGGGOTG GCGOCCGGGC claim 2 cont. 1765 1776 1736 1796 IS06 1816 1826 AAGCTTTCTT CCTCGAGATG CTCTGCTGCT TGAGAGCTAT TGCTTTGTTA AGATATAOOfl AGGGGTTTCT 1836 1846 1856 1866 1876 1886 1836 TTTTGTCTTT CTGTAAGGTG GACTTCCAGC TTTTGATTGA AAGTCCTA6G GTGATTCTAT TTCTGCTGTG 1306 1916 1926 1936 1946 1956 1366 ATTTATCTGC TGAAAGCTCA GCTGGGGTTG TGCOAGCTOG GGACCCATTC CTGTGTAATA CAATGTCTGC 1976 1936 1936 2006 2016 2026 ACCRAT6CTA flTflflflGTCCT OTTCTCTTTT AAflflflOflflflfl OflflAAflflflAA AACGGAATTC Deduced protein sequence of 6 d PSP protein is underlined 3 . A purified protein useful for enhancing pulmonary surfactant activity characterized by a molecular weight of about 5 .9-9 kilodaltons and an amino acid composition substantially as follows : Asp/Asn 2.7 Thr 2.0 Ser 2.1 Glu/Gln 1.6 Pro 6.3 Gly 22.9 Ala 3.3 Cys 0.95 Val 5.5 Met 3.4 He 4.8 Leu 17.3 Tyr 3.3 Phe 6.3 His 2.9 Lys 3.6 Arg 1.94 (calculated based on MW=10 , 000 daltons ; ave residue MW=110) 4. A recombinant BKA molecule encoding a protein, of claim 1. 5. A recombinant DNA molecule encoding a protein sequence of claim 2 or a DNA molecule capable of hybridizing thereto, which encodes a protein having pulmonary surfactant enhancing activity. 6. A host cell or cell line transformed with the DNA of claim 4 or claim 5. 7. A method for producing a protein useful for enhancing pulmonary surfactant activity which comprises culturing a cell or cell line of claim 6. 8. A protein useful for enhancing pulmonary surfactant activity produced by the method of claim 7. 9. A pulmonary surfactant composition comprising an effective surfactant enhancing amount of one or more of the proteins of claim 1, 2 r 3 , or 8 and a pharmaceutically acceptable fatty acid and/or lipid. 10. A vector containing a heterologous DNA sequence encoding a peptide comprising the sequence: IKRIQAMIPKGALAVAVAQVCRWPLVAGGICQC 11. A vector containing the heterologous DNA sequence: ATC AAG CGG ATC O-A GCC ATG ATT CCC AAG GGT GCG CTA GCT GTG GCA GTG GCC CAG GTG TGC CGC GTG GTA CCT CTG GTG GCG GGC GGC ATC TGC CAG TGC 12. A vector containing the heterologous DNA sequence encoding a peptide comprising the sequence: FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRWPLVAGGICQCLAERY- SVILLDTLLGRML 13. A vector containing the heterologous DNA sequence: TTC CCC ATT CCT CTC CCC TAT TGC TGG CTC TGC AGG GCT CTG ATC AAG CGG ATC CAA GCC ATG ATT CCC AAG GGT GCG CTA GCT GTG GCA GTG GCC CAG GTG TGC CGC GTG GTA CCT CTG GTG GCG GGC GGC ATC TGC CAG TGC CTG GCT GAG CGC TAC TCC GTC ATC CTG CTC GAC ACG CTG CTG GGC ATG CTG

Inventors: FLOROS JOANNA, JACOBS KENNETH A, PHELPS DAVIS S, STEINBRINK D RANDALL, TAEUSCH H WILLIAM JR, FLOROS, JOANNA, JACOBS, KENNETH, A., PHELPS, DAVIS, S., STEINBRINK, D., RANDALL, TAEUSCH, H., WILLIAM, JR.
Applicants: BRIGHAM & WOMENS HOSPITAL, GENETICS INST, BRIGHAM AND WOMEN'S HOSPITAL, GENETICS INSTITUTE, INC.
Application year: 1986
Main classification: C07H21
IPC codes: A61K38, C07K14