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 chromatographically . two proteins were thus obtained which are characterized by a molecular weight of about 35 kd . those proteins are described in greater detail in in published international application wo 86 / 02037 . butanol - soluble proteins were obtained by cryoprecipitation . more specifically , storage of the 1 - butanol extract at - 20 ° c . yielded a precipitate which was purified chromatographically to yield a protein characterized by a predominant band having 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 composition of the latter 6 kd protein is set forth in table 2 . the two low molecular weight proteins of this invention 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 4 ). as previously mentioned , for the sake of simplicity , both low molecular weight psp proteins are referred to hereinafter as &# 34 ; 6k &# 34 ; proteins based on the approximate apparent molecular weights of their predominant protein bands as determined by conventional sds - page . it should be understood , however , that the actual molecular weights of these protein bands are presumably in the range of ˜ 5 -˜ 9 kilodaltons . the fact that these proteins can now be obtained in pure form by the above - described methods made 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 . by way of example , genes encoding the two 35 kd proteins were cloned as described in detail in wo 86 / 02037 . additionally , oligonucleotide probes based on the n - terminal sequence of the cold butanol - insoluble 6k protein ( see table 4 ) were synthesized and were used to screen a cdna library prepared from human lung mrna ( toole et al ., 1984 ) as described in greater detail in example 2 , below . several clones which hybridized to the probes were identified . based on hybridization intensity one clone was selected , subcloned into m13 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 ., 1984 , 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 1 . table 4______________________________________ ## str35 ## ______________________________________ (-) = not determined ( positions 8 , 11 & amp ; 12 were unidentified ) 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 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 under stringent conditions may be expressed in host cells or cell lines by conventional expression methods to produce &# 34 ; recombinant &# 34 ; proteins having surfactant or surfactant enhancing activity . with respect to the cloned approximately 6k protein , this invention encompasses vectors containing a heterologous dna sequence encoding the characteristic peptide sequence ile through cys corresponding to nucleotides a - 656 through c - 757 of the sequence shown in table 1 , i . e ., ikriqamipkgalavavaqvcrvvplvaggicqc . one such vector contains the nucleotide sequence . ## str36 ## other vectors of this invention contain a heterologous dna sequence encoding at least a portion of the characteristic peptide sequence substantially as depicted in the underlined peptide region of table 6 , i . e ., fpiplpycwlcralikriqamipkgalavavaqvcrvvplvaggicqclaerysvilldtllgrml . one such vector contains the dna sequence substantially as depicted in the underlined nucleotide sequence of table 1 , i . e ., ## str37 ## another exemplary vector contains a heterologous dna sequence , such as the nucleotide sequence depicted in table 1 , which encodes the full - length peptide sequence of table 1 . dna inserts for such vectors which comprise a dna sequence shorter than the full - length cdna of psp6k - 17 - 3 , depicted in table 1 , 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 &# 34 ; recombinant &# 34 ; 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 . 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 , and the like may be obtained from natural sources or synthesized by known procedures . see kaufman , proc . natl . acad . sci . 82 : 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 , 36 : 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 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 microbial 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 dipalmitoylphosphatidyl choline or with mixtures of such fatty acids or lipids which may be obtained from commercial sources or by conventional methods , or with 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 mg of total lipid . it is contemplated that certain subcombinations of one or more of the proteins of this invention with one or more of the proteins described in wo 86 / 02037 and compositions containing such subcombinations may be especially useful in the treatment of patients with particular clinical indications . pulmonary lavage ( 50 ml ) from an alveolar proteinosis patient was centrifuged at 10 , 000 × g for 5 min . the pellet was collected and washed 5 times in 20 mm tris hcl , 0 . 5m 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 extract so obtained 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 chcl 3 : meoh ( 2 : 1 , v / v ), applied to sephadex lh20 columns and eluted with chcl 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 6n hcl at 110 ° c . for 22 hrs followed by chromatography on a beckman model 63000 amino acid analyzer . n - terminal sequence was determined on an applied biosystems 470a sequencer molecular weights were determined on 10 - 20 % gradient sds polyacrylamide gels . 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 4 an oligonucleotide probe was synthesized . the 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 synthesized . these pools contained either 384 or 192 different sequences . a cdna library from human lung m 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 mer 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 . 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 4 was obtained , confirming that the isolated clone coded for the approximately 6 kd 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 oompletely 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 1 ), the second began at nucleotide 148 of clone 17 . sequence of the 5 &# 39 ; end of a third clone confirmed the sequence of the 5 &# 39 ; end of clone 17 . the clones are identical throughout the coding region and differ only at two positions in the 3 &# 39 ; untranslated region . as those of ordinary skill in this art will appreciate , the cloned gene may be conveniently obtained by excision from psp6k - 17 - 3 ( atcc no . 40245 ) or may be recloned using sequence information provided herein in table 1 . 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 . 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( 1984 ) biochem . biophs . acta , 791 - 226 - 238 . 18 . shelley , s . a ., balis , j . u ., 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 . 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 .
this invention relates to novel proteins useful for enhancing pulmonary surfactant activity , methods and materials for obtaining said proteins and compositions containing one or more of the proteins .