Note: Descriptions are shown in the official language in which they were submitted.
1 134080
RECOMBINANT ALVEOLAR SURFACTANT PROTEIN
Technical Field
The invention relates to the field of
recombinant protein production. More specifically it
relates to the production of alveolar surfactant protein
(ASP) which is useful in the management of certain
respiratory diseases.
Background Art
The human lung is composed of a large number of
small sacs or alveoli in which bases are exchanged
between the blood and the air spaces of the lung. In
healthy individuals, this exchange is mediated by the
presence of a protein containing surfactant complex which
is synthesized in the microsomal membranes of type II
alveolar cells. In the absence of adequate levels of
this complex, a lung cannot properly function--i.e., the
alveoli collapse during exhalation, and cannot be
subsequently re-inflated by inhaling. Thus, the
untreated inability to synthesize this complex may result
in death or in sever physical damage.
The best documented instance of inadequate
surfactant complex levels occurs in premature infants and
infants born after complicated pregnancies, and is widely
known as respiratory distress syndrome (RDS). A widely
publicized form of this syndrome has been designated
hyaline membrane disease, or idiopathic RDS. RDS is
currently the leading cause of infant mortality
and morbidity in the United States and in other developed
countries, and substantial efforts have been directed to
diagnosis and treatment. Current treatment has focused
on mechanical (pressure) ventilation which, at best, is
X T
1344580
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an invasive stop-gap measure that often results in damage
to the lung and other deleterious side effects, including
complications such as bronchopulmonary dysplasia,
interstitial emphysema and pneumothorax. Mental
retardation has also resulted on occasion when this
treatment was used (Hallman, M., et al, Pediatric Clinics
of North America (1982) 29:1057-1075).
Limited attempts have been made to treat the
syndrome by surfactant substitution. This would be a
method of choice, as, in general, only one administration
is required, and the potential for damage is reduced.
For example, Fujwara, et al, Lancet (1980) 1:55-used a
protein-depleted surfactant preparation derived from
bovine lungs; the preparation is effective by
immunogenic. Hallman, M., et al, Pediatrics (1983)
71:473-482 used a surfactant isolate from human amniotic
fluid to treat a limited number of infants with some
success. U.S. Patent 4,312,860 to Clements discloses an
artificial surfactant which contains no protein and is
said to be useful in this approach although no data are
shown. In short, surfactant substitution has not been
widely used clinically.
The preferred surfactant substitute would be
the lung surfa~~tant complex itself. This complex is
composed of apoprotein, two phospholipids (dipalmitoyl
phosphocholine (DPPC) and phosphatidyl-glycerol (PG))
which are present in major amount, several lipid
components preaent in only very minor amount, and calcium
ions. The apoprotein contains proteins having molecular
weights of the order of 32,000 daltons and very
hydrophobic proteins of the order of about l0,000 daltons
(King, R.J. et al. Am J Physiol (1973) 224:788-795). The
32,000 dalton protein is glycosylated and contains
hydroxyproline"
major reason for the limited progress in
surfactant rep~_acement therapy has been the lack of
--- -3- I340 ~8D
availability of the protein portion of the complex.
Replacement therapies have focused on attempts to use the
lipid components alone, and it appears that the
performance of such treatment can be markedly improved by
addition of the apoprotein (Hallman, M., et al, Pediatric
Clinics of North American (1982) (supra)). At present,
however, these proteins are available only form normal
adult human lung, and from amniotic fluid. Even
efficient isolation procedures would not provide an
adequate supply. Thus, it would be desirable to have
available a method for producing practical quantities of
apoprotein for use alone or in conjunction with the
saturated phospholipid portion of the complex.
Disclosure of Invention
The invention provides a means for obtaining
the apoprotein portion of the lung surfactant complex in
quantity and under conditions which permit optimization
of its features. The remaining components of the
complex, dipal.mitoyl phosphocholine and phosphatidyl-
glycerol, along with calcium ions are already readily
available. The availability of required quantities of
manipulable apoprotein both makes possible research
efforts to optimize the form of complex useable in
therapy, and opens the possibility for routine
replacement therapy of respiratory distress syndrome.
4 i34080
Thus, in one aspect, the invention relates to
recombinantly produced mammalian alveolar surfactant
protein (ASP). These proteins are mixtures of relatively
high molecular weight, relatively water soluble proteins
of about 32 k.d (32K ASP) and of lower molecular weight,
hydrophobic proteins of about 10-20 kd (10K ASP). Both
proteins encourage formation of surface tension lowering
films when complexed with phospholipid in the presence of
calcium ion. The invention further relates to DNA
sequences encoding mammalian ASP, including human and
canine 32K and 10K ASP, to expression vectors suitable
for production of these proteins, to recombinant host
cells transformed with these vectors, and to methods of
producing the recombinant ASPs and their precursors. In
other aspect the invention relates to pharmaceutical
compositions containing human ASP and to methods of
treating RDS 'using them.
In order to facilitate a description of another
aspect of the invention by reference to the appended
drawings, Figures 1, 2, and 3 thereof show DNA sequences
encoding canine 32K ASP, canine 10K ASP and human 32K ASP
respectively. A more complete description of these
Figures and other Figures of the drawings are provided
hereinafter.
In accordance with an aspect of the invention
an alveolar surfactant protein (ASP) in substantially
pure form is provided. The substantially pure ASP is
selected from the group consisting of:
a) human ASP having the amino acid sequence
encoded by the DNA shown in Figure 3 as exon II-IV DNA
and that portion of exon I DNA encoding mature ASP amino
acid sequence,, or by the naturally occurring allelic
variants thereof:
b) canine ASP having the amino acid sequence
encoded by the DNA sequence shown as encoding the mature
ASP in Figure 1 or by the naturally occurring allelic
variants thereof
130580
4a
wherein for the ASP of paragraphs a) and b),
one or more of the encoded proline residues is
optionally substituted for by a hydroxyproline residue;
and
c) the canine ASP encoded by the DNA sequence
shown as encoding mature ASP in Figure 2 and by the
naturally occurring allelic variants thereof.
Brief Description of the Drawings
Figure 1 shows the DNA sequence encoding
canine 32K ASP, along with the deduced amino acid
sequence.
Figure 2 shows DNA sequence determined for a
cDNA encoding canine 10K ASP along with the deduced
amino acid sequence.
Figure 3 shows the nucleotide sequence of the
human 32K ASP gene and the deduced amino acid sequence.
Figure 4 is an autoradiograph of S-Met
labeled, secreted proteins from CHO cells transfected
with ~:gHS-15.
Figure 5 shows the sequence of the 3' terminal
portion of human ASP cDNA contained in pHS-6.
.. ..,,
x .w
''' S- 134080
FigL.re 6 shows DNA sequence determined for a
cDNA encoding human 10K ASP along with the deduced amino
acid sequence.
Figure 7 shows the relevant junction and coding
sequences of the expression vector pASPc-SV(10).
Figure 8 shows the relevant junction and coding
sequences of the expression vector pASPcg-SV(10).
Figure 9 shows the relevant junction and coding
sequences of the expression vector
pMT-Apo:gHS(HinfI/EcoRI).
Figure 10 shows the results of SDS PAGE
conducted on 35S labeled supernatant proteins, with and
without treatment with endo F, secreted by pMT-
Apo:gHS(HinfI/EcoRI) transfected CHO cells.
Figure 11 shows the results of SDS PAGE
immunoblotted with labeled human antiASP conducted can
supernatant proteins, with and without treatment with
endo F, secreted by pMT-Apo:gHS(HinfI/EcoRI) transfected
CHO cells.
Figure 12 shows the results of in vitro
determination of the ability of ASP to enhance surface
tension lowering by phospholipids.
Modes of Carrying Out the Invention
A. Definitions
As used herein, "alveolar surfactant protein
(ASP)" refers to apoprotein associated with the lung
surfactant complex and having ASP activity as defined
hereinbelow. The ASP of a11 species examined appears to
comprise one or more components of relatively high
molecular weight (of the order of 32 kd) designated
herein "32K ASP" and one or more quite hydrophobic
components of relatively low molecular weight (of the
order of 10-2C kd) designated herein "lOK ASP". (King.
.z.
~
~-.
-6-
R.J., et al, J Appl Phvsiol (1977) 42:483-49l;
Phizackerley, P.J.R., Biochem J (1979) 183:731-736.)~~~ ~~
These terms refer to the native sequences and to
equivalent modifications thereof. For example, human
32K ASP has the amino acid sequence shown in Figure 3;
ASP proteins of approximately 32 kd derived from other
species such as dogs, monkeys, or other mammals have
substantial degrees of homology with this sequence (see
Figure 1 in connection with the canine ASP). Additional
sequence for other specific 32K ASP (canine) and 10K ASP
(human and canine) is disclosed hereinbelow.
The recombinant ASP proteins of the invention
' have amino acid sequences corresponding to those of the
native proteins. It is understood that limited
modifications may, however, be made without destroying
activity, and that only a portion of the entire primary
structure may be required. For example) the human ASP
32K recombinant protein of the invention has an amino
acid sequence substantially similar to that shown in
Figure 3, but minor modifications of this sequence which
do not destroy activity also fall within the definition
of 32K human ;ASP and within definition of the protein
claimed as such, as further set forth below. Also
included within the definition are fragments of the
entire sequence of Figure 3 which retain activity.
As is the case for a11 proteins, the ASP
proteins can occur in neutral form or in the form of
basic or acid addition salts depending on its mode of
preparation, ~or, if in solution, upon its environment.
It is well understood that proteins in general, and,
theref ore, any ASP, in particular) may be found in the
form of its acid addition salts involving the free amino
groups, or basic salts formed with free carboxyls.
Pharmaceutically acceptable salts may, indeed, enhance
13~U~80
the functionality of the protein. Suitable
pharmaceutically acceptable acid addition salts include
those formed from inorganic acids such as, for example.
hydrochloric or sulfuric acids, or from organic acids
such as acetic or glycolic acid. Pharmaceutically
acceptable bases include the alkali hytoxides such as
potassium or sodium hydroxides, or such organic bases as
piperidine, glucosamine, trimethylamine, choline) or
caffeine. In addition, the protein may be modified by
combination with other biological materials such as
lipids and saccharides, or by side chain modification)
such as acetylation of amino groups, phosphorylation of
hydroxyl side chains, or oxidation of sulfhydryl groups
of other modification of the encoded primary sequence.
Indeed, in its native form, the 32K ASP is a
glycosylated protein, and certain of the encoded proline
residues have been converted to hydroxyptoline. It is
also found in association with the phospholipds DPPC and
PG. Included within the definition of any ASP herein
are glycosylated and unglycosylated forms, hydroxylated
and non-hydtoxylated forms, the apoprotein alone, or in
association with lipids, and, in short, any composition
of an amino acid sequence substantially similar to that
of the native sequences which retains its ability to
facilitate the exchange of gases between the blood and
lung air spaces and to per~it re-inflation of the
alveoli.
It is further understood that minor
modifications of primary amino acid sequence may result
in proteins which have substantially equivalent or
enhanced activity as compared to the native sequences.
These modifications may be deliberate, as through
site-directed mutagenesis, or may be accidental, such as
through mutation of hosts which are ASP producing
i3405~80~ ..._
organisms. A11 of these modifications are included as
long as the ASP activity is retained.
"ASP activity" for a protein ie defined as the
ability, when combined with lipids either alone or in
combination with other proteins, to exhibit activity in
the in vivo assay of Robertson, B. Luna (1980)
158:57-68. In this assay, the sample to be assessed is
administered through an endotrachial tube to fetal
rabbits or lambs delivered prematurely by Caesarian
section. (These "preemies" lack their own ASP, and are
supported on a ventilator.) Measurements of lung
compliance, blood gases and ventilator pressure provide
indices of activity. Preliminary assessment of activity
may also be made by an in vitro assay, for example that
of King, R. J., et al, Am J Phvsiol (1972) 223:715-726,
or that illustrated below of Hawgood) et al) which
utilizes a straightforward measurement of surface
tension at a .air-water interface when the protein is
mixed with a phospholipid vesicle preparation.
"Ope.rably linked" refers to a juxtaposition
wherein the components are configured so as to perform
their usual function. Thus, control sequences operably
' linked to coding sequences are capable of effecting the
expression of the coding sequence.
"Control sequence" refers to a DNA sequence or
sequences whit h ate capable) when properly ligated to a
desired coding sequence, of effecting its expression in
hosts compatible with such sequences. Such control
sequences include promoters in both procaryotic and
eucaryotic hosts, and in procaryotic organisms also
include ribosome binding site sequences) and, in
eucaryotes, termination signals. Additional factors
necessary or helpful in effecting expression may
subsequently be identified. As used herein, "control
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_g_
sequences" simply refers to whatever DNA sequence may be
required to effect expression in the particular host
used.
"Cells" or "recombinant host cells" or "host
cells" are often used interchangably as will be clear
from the context. These terms include the immediate
subject cell, and, of course, the progeny thereof. It
is understood that not a11 progeny are exactly identical.....,.
to the parental cell, due to chance mutations or
differences in environment. However, such altered
progeny are included when the above terms are used.
B. General Description
The methods illustrated below to obtain DNA
sequences encoding ASP are merely for purposes of
illustration and are typical of those that might be
used. However, other procedures may also be employed.
as is understood in the art.
B.1. The Nature of the Surfactant Comvlex
The .alveolar surface of lung has been studied
extensively by a number of techniques, and by a number
of groups. It appears that the basement membrane of the
alveolus is composed of type i and type II alveolar
Z5 cells, of which the type II cells comprise approximately
3i of the sur:Eace. The type II cells are responsible
for the exocrine secretion of materials into a lining
fluid layer covering the basement membrane, which
materials decrease the surface tension between the
liquid of the lining and the gas phase of the contained
volume. The fluid layer, then, is comprised of water
derived from the blood plasma of the alveolar
capillaries, and the surfactant secretions of the type
II cells.
13~o~go
The type II cells, themselves, contain 60-100
pg of protein and about 1 pg of lipid phosphorus per
cell where the ratio between type II cell DPPC and PG
phosphorus :Ls about 8 to 1. Studies of the apoprotein
5 components have been based on pulmonary lavage from
various species, and have been shown to comprise two
major proteins, as discussed above, of approximate
molecular weights 10-20 kd and of 32 kd (Kikkawa, Y., et
al, Laboratory Investigation (l983) i:122-139.) It is
10 not clear whether the apoproteins are bound to the
phospholipid component (King, R.J., et al, AM Rev Respir
Dis (1974) k0_:273) or are not (Shelly, S.A., et al, J
Lipid Res (7.975) 16:224).
It: has been shown that the higher molecular
weight protHin obtained by pulmonary lavage of dogs, and
separated by gel electrophoresis is composed of 3 major
components of molecular weight 29,000, 32,000, and
36,000 daltans. The 32,000 dalton protein was used to
obtain sequence data, as set forth below; however, a11 3
of these proteins have identical N-terminal sequences,
and there is evidence that they differ only in degree of
glycosylation. Digestion of the 36 kd and 32 kd bands
with endoglycosidase F, which removes carbohydrate side
chains, results in products which co-migrate with the 29
kd component:. The mobility of the 29 kd component is
unaffected by this treatment. It has also been shown
that the 32 kd fraction aggregates into dimers and
trimers.
The smaller molecular weight proteins are
extracted with more difficulty, but these, too, appear
to be mixtures (Phizackerley, et al (supra); description
below).
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B.2. Cloning of Coding Sequences for Canine
and Human ASP Proteins
The entire canine and human ASP 32K protein
encoding sequences have been cloned, and are available
for expression in a variety of host cells as set forth in
9IC below. In addition, DNA sequences encoding several of
the lower molecular weight proteins from both human and
canine sources have also been obtained.
The canine 32K sequence was obtained from a
cDNA library prepared from mRNA isolated from adult
canine lung, by probing with two sets of synthetic
oligonucleotides, one prepared to accommodate a11 the
possible sequences encoding amino acids 1-5 of the N-
terminal sequence and the other amino acids 7-11 of that
sequence, as well as a single 15-mer encoding the amino
acids 1-5, selected on the basis of mammalian codon
preference. Immobilized cDNA from the library
constructed in E. coli was probed using these
oligonucleotide sets. False positives were minimized by
requiring hybridization to more than one set.
Successfully hybridizing clones were sequenced, and one
was shown to contain the correct N-terminal sequence.
The cDNA insert from the successful clone,
excised with PstI, was then used as a probe of the
original canine cDNA library, to obtain two additional
clones containing inserts encoding other regions of the
ASP which, together with this probe, span 844 nucleotides
containing the complete coding sequence of canine 32K
ASP. The entire nucleotide sequence of the three
appropriate inserts, and the deduced 256 amino acid
sequence are shown i_n Figure 1.
This same originally retrieved N-terminal
encoding fragment used above was also used as a probe to
obtain fragments from a human genomic library in ~, phage
Charon 28. The entire coding sequence for human ASP 32K
protein was found to be contained in a single phage
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plaque, and to be contained within 2 contiguous BamHI
fragments, a 'i'1.2 kb and a 3' 3.5 kb fragment. The
pertinent portions of these fragments, encoding human
ASP, and containing 3 introns, are shown in Figure 3; the
deduced amino acid sequence of human ASP, contains 228
amino acids, a:nd is preceded by a signal sequence of at
least 25 amino acids. The human 32K ASP cDNA
corresponding to the full length protein was also
obtained by probing human cDNA libraries derived from
human fetal anal adult lung mRNA.
Extensive homology exists between the canine
and human 32K amino acid sequences.
Similar strategies were followed in obtaining
cDNA encoding human and canine 10K ASP. The canine lung
cDNA library described above was probed with two
synthetic oligomer mixtures designed to correspond to the
N-terminal amino acid sequence of a 16.5 kd (on unreduced
gels) canine protein, and clones hybridizing to both
probes were recovered and sequenced. One of these
clones, which contained canine ASP encoding sequence, was
used to probe a cDNA library prepared in bacteriophage
gtl0 from mRNA isolated from adult human lung to obtain a
human lOK ASP encoding clone.
B.3. Expression of ASP
As the nucleotide sequences encoding the
various human and canine ASP are now available, these may
be expressed in a variety of systems as set forth in 9IC.
If procaryotic systems are used, an intronless coding
sequence should be used, along with suitable control
sequences. The cDNA clones for any of the above ASP
proteins may be excised with suitable restriction
13~40~84
-13-
enzymes and ligated into procaryotic vectors for such
expression. For procaryotic expression of ASP genomic
DNA, the DNA should be modified to remove the introns)
either by site-directed mutagenesis) or by retrieving
corresponding portions of cDNA and substituting them for
the intron-containing genomic sequences. The intronless
coding DNA is then ligated into expression vectors for
procaryotic a:xpression.
As exemplified below, ASP encoding sequences
may also be used directly in an expression system
capable of processing the introns, usually a mammalian
' host cell culture. To effect such expression, the
genomic sequences can be ligated downstream from a
controllable mammalian promoter which regulates the
expression of these sequences in CHO cells.
B.4. Protein Recover
The ASP protein may be produced either as a
mature protein or a fusion protein, or may be produced
along with a signal sequence in cells capable of
processing this sequence for secretion. It is
advantageous to obtain secretion of the protein) as this
minimizes the difficulties in purification; thus it is
preferred to express the human ASP gene which includes
the codons for native signal sequence in cells capable~~~~~~~
of appropriate processing. It has been shown that
cultured mammalian cells are able to cleave and process
heterologous mammalian proteins containing signal
'sequences, and to secrete them into the medium
(McCormick, F., et al) Mol Cell Biol (1984) 4:l66).
When secreted into the medium, the ASP protein
is recovered using standard protein purification
techniques. The purification process is simplified,
because relatively few proteins are secreted into the
~3~o~so
-14-
medium, and th.e majority of the secreted protein will,
therefore, already be ASP. However, while the procedures
are more laborious, it is within the means known in the
art to purify this protein from sonicates or lysates of
cells in which it is produced intracellularly in fused or
mature form.
B.5. Assay for ASP Activity
In vitro methods have been devised to assess
the ability of ASP proteins to function by reducing
surface tension (synonymous with increasing surface
pressure) to generate a film on an aqueous/air interface.
Studies using these methods have been performed on the
isolated native 10K canine ASP. (Benson, B.J., et al Prog
Resp Res (l984) l8:83-92; Hagwood, S., et al,
Biochemistry (1985) 24:184-l90.)
Tanaka, Y, et al, Chem Pharm Bull (1983)
31:4100-4l09 disclose that a 35 kd protein obtained from
bovine lung enhanced the surface spreading of DPPC;
Suzuki, Y., J Lipid Res (1982) 23:62-69; Suzuki, Y., et
al, Prog Resp Res (1984) l8:93-100 showed that a 15 kd
protein from pig lung enhanced the surface spreading of
the lipid-protein complex from the same source.
Since the function of the surfactant complex in
vivo is to create a film at the air/aqueous interface in
order to reduce surface tension, the ability of ASP
proteins to enhance the formation of the film created by
the spread of lipid or lipoprotein at such a surface in
an in vitro model is clearly relevant to its utility.
B.6. Administration and Use
The ;purified proteins can be used alone and in
combination in pharmaceutical compositions appropriate
for administration for the treatment of respiratory
134080
_ 15_
distress syndrome in infants or adults. The
compositions and protein products of the invention are
also useful in treating related respiratory diseases
such as pneumonia and bronchitis. For use in such
treatment, either of the components, but preferably the
32K component, either alone or, even more preferably, in
combination with the 10K component of human ASP is
combined with natural or synthetic lipids to reconstruct
a surfactant complex. The complex contains about 50% to
almost 100% (wt/wt) lipid and 50% to less than 1% ASP;
preferably ASP is 5%-20% of the complex. The lipid
portion is pl:eferably 80%-90% (wt/wt) DPPC with the
remainder unsaturated phosphatidyl choline, phosphatidyl
glycerol, tri:acylglycerols, palmitic acid or mixtures
thereof. The complex is reassembled by mixing a
solution of ASP with a suspension of lipid lipsomes, of
by mixing the lipid protein solutions directly in the
presence of detergent or an organic solvent. The
detergent or solvent may then be removed by dialysis.
While it is possible to utilize the natural
lipid component from lung lavage in reconstructing the
complex) and to supplement it with appropriate amounts
of ASP proteins, the use of synthetic lipids is clearly
preferred. First, there is the matter of adequate
supply, which. is self-evident. Second) purity of
preparation and freedom from contamination by foreign
proteins, including infectious proteins, which may
reside in the lungs from which the natural lipids are
isolated, are assured only in the synthetic
preparations. Of course, reconstitution of an effective
complex is more difficult when synthetic components are
used.
While the 32K human ASP may be used alone as
the protein component of the compositions, the
r~,
1340580
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combination of 32K and 10K proteins is preferred. The
protein ratio is typically in the range of 80:Z0 for
32K:lOK group. The 32K protein may be added directly to
an aqueous suspension of. phospholipid vesicles in an
aqueous solution; because it is so hydrophobic, the lOK
protein is added to the lipids in an organic solvent,
such as chloroform, the solvents evaporated, and the
vessicles re-formed by hydration.
The compositions containing the complex are
preferably those suitable for endotracheal
administration, i.e., generally as a liquid suspension,
as a dry powder "dust" or as an aerosol. For direct
endotracheal administration, the complex is,suspended. in,~_..
a liquid with suitable excipients such as, for example)
water, saline, dextrose, or glycerol and the like. The
compositions may also contain small amounts of non-toxic
auxiliary substances such as pH buffering agents, for
example, sodium acetate or phosphate. To prepare the
"dust", the complex, optionally admixed as above, is
lyophylized, and recovered as a dry powder.
If to be used in aerosol administration, the
complex is supplied in finely divided form along with an
additional surfactant and propellent. Typical
surfactants which may be administered are fatty acids
and esters, however, it is preferred, in the present
case, to utilize the other components of the surfactant
complex, DPPC and PG. Useful propellents are typically
gases at ambient conditions, and are condensed under
pressure. Lower alkanes and fluorinated alkanes, such
as Freon) may be used. The aerosol is packaged in a
container equipped with a suitable valve so that the
ingredients may be maintained under pressure until
released.
134080
-17-
The surfactant complex is administered, as
appropriate tc> the dosage form, by endotracheal tube, by
aerosol administration, or by nebulization of the
suspension or dust into the inspired gas. Amounts of
complex between about 0.1 mg and 10 mg are administered
in one dose. For use in newly born infants, one
administration is generally sufficient. For adults,
sufficient reconstituted complex is administered to
replace demonstrated levels of deficiency (Hallman, M.,
et al, J Clinical Investigation (1982) 70:673-682).
C. Standard Methods
Most of the techniques which are used to
transform cells, construct vectors, extract messenger
RNA, prepare cDNA libraries, and the like are widely
practiced in the art, and most practitioners are familiar
with the standard resource materials which describe
specific conditions and procedures. However, for
convenience, the following paragraphs may serve as a
guideline.
C.1. Hosts and Control Seauences
Both procaryotic and eucaryotic systems may be
used to express the ASP encoding sequences; procaryotic
hosts are the most convenient for cloning procedures.
Procaryotes most frequently are represented by various
strains of E. coli; however, other microbial strains may
also be used. Plasmid vectors which contain replication
sites and control sequences derived from a species
compatible with the host are used; for example, E. coli
is typically transformed using derivatives of pBR322, a
plasmid derived from an E. coli species by Bolivar, et
al, Gene (1977) 2:95. pBR322 contains genes for
i340580
... _18_
ampicillin and tetracycline resistance, and thus provides
additional markers which can be either retained or
destroyed in constructing the desired vector. Commonly
used procaryot.ic control sequences which are defined
herein to include promoters for transcription initiation,
optionally with an operator, along with ribosome binding
site sequences, include such commonly used promoters as
the beta-lactamase (penicillinase) and lactose (lac)
promoter systems (Chang, et al, Nature (l977) 198:l056
and the tryptcphan (trp) promoter system (Goeddel, et al
Nucleic Acids Res (1980) 8:4057 and the lambda derived PL
promoter and N-gene ribosome binding site (Shimatake, et
al, Nature (1981) 292:128).
In addition to bacteria, eucaryotic microbes,
such as yeast, may also be used as hosts. Laboratory
strains of Saccharomyces cerevisiae, Baker's yeast,~are
most used although a number of other strains are commonly
available. Vectors employing, for example, the 2 ~,
origin of replication of Broach, J.R., Meth Enz (l983)
10l:307, or other yeast compatible origins of
replications (see, for example, Stinchcomb, et al, Nature
(1979) 282:39; Tschempe, et al, Gene (1980) 10:l57 and
Clarke, L, et al, Meth Enz (l983) 101:300) may be used.
Control sequences for yeast vectors include promoters for
the synthesis of glycolytic enzymes (Hess, et al, J Adv
Enzyme Reg (1968) 7:149; Holland, et al, Biochemistry
(1978) 17:4900). Additional promoters known in the art
include the promoter for 3-phosphoglycerate kinase
(Hitzeman, et al, J Biol Chem (1980) 255:2073), and those
for other glycolytic enzymes. Other promoters, which
have the additional advantage of transcription controlled
by growth conditions are the promoter regions for alcohol
dehydrogenase 2, isocytochrome C, acid phosphatase,
degradative enzymes associated with nitrogen metabolism,
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.-
and enzymes responsible for maltose and galactose
utilization. It is also believed terminator sequences
are desirable at the 3' end of the conding sequences.
Such terminators are found in the 3' untranslated region
following the coding sequences in yeast-derived genes.
It is also, of course, possible to express
genes encoding polypeptides in eucaryotic host cell
cultures derived from multicellular organisms. See, for
example, Tissue Cultures, Academic Press, Cruz and
Patterson, editors (l973). These systems have the
additional advantage of the ability to splice out introns
and thus can be used directly to express genomic
fragments. Useful host cell lines include VERO and HeLa
cells, and Chinese hamster ovary (CHO) cells. Expression
vectors for such cells ordinarily include promoters and
control sequences compatible with mammalian cells such
as, for example, the commonly used early and late
promoters fron. Simian Virus 40 (SV 40) (Fiers, et al,
Nature (l978) 273:113), or other viral promoters such as
those derived from polyoma, Adenovirus 2, bovine papiloma
virus, or avian sarcoma viruses. The controllable
promoter, hMTII (Karin, M., et al, Nature (l982) 299:797-
802) may also be used. General aspects of mammalian cell
host system transformations have been described by Axel;
U.S. Patent No. 4,399,2l6 issued 16 August 1983. It now
appears, also that "enhancer" regions are important in
optimizing expression; these are, generally sequences
found upstream or downstream of the promoter region in
non-coding DNF. regions. Origins of replication may be
obtained, if needed, from viral sources. However,
integration into the chromosome is a common mechanism for
DNA replication in eucaryotes.
~~~o~sv
r-. -20-
C.2. Transformations
Depending on the host cell used, transformation
is done using standard techniques appropriate to such
cells. The calcium treatment employing calcium chloride,
as described by Cohen, S.N., Proc Natl Acad Sci (USA)
(l972) 69:2110, or the RbCl2 method described in
Maniatis, et al, Molecular Cloning: A Laboratory Manual
(l982) Cold Spring Harbor Press, p. 254 may be used for
procaryotes or other cells which contain substantial cell
wall barriers. For mammalian cells without such cell
walls, the calcium phosphate precipitation method of
Graham and van der Eb, Virology (1978) 52:546, optionally
as modified by Wigler, M., et al, Cell (l979) 16:777-785
may be used. Transformations into yeast may be carried
out according to the method of Van Solingen, P., et al, J
Bact (1977) 130:946 or of Hsiao, C.L., et al, Proc Natl
Acad Sci (USA) (l979) 76:3829.
Probing cDNA or Genomic Libraries
cDNA or genomic libraries are screened using
the colony hybridization procedure. Each microtiter
plate is replicated onto duplicate nitrocellulose filter
papers (S & S type BA-85) and colonies are allowed to
grow at 37~C fo r 14-16 hr on L agar containing 15 ~g/ml
tetracycline. The colonies are lysed with loo SDS and
the DNA is fixed to the filter by sequential treatment
for 5 min with 500 mM NaOH/1.5 M NaCl, then 0.5 M Tris
HC1 (pH 8.0)/1.5 M NaCl followed by 2 x standard saline
citrate (SSC). Filters are air dried and baked at 80~C
for 2 hr.
For nick-translated probe, the duplicate
filters are prehybridized at 42~C for 16-18 hr with 10 ml
per filter of DNA hybridization buffer (50o formamide
lr
-21- 134080
(40% formamide if reduced stringency), 5 x SSC, pH 7.0,
5x Denhardt's solution (polyvinylpyrrolidine, plus Ficoll
and bovine serum albumin; 1 x = 0.02% of each), 50 mM
sodium phosphate buffer at pH 7.0, 0.2% SDS, 50 ~g/ml
yeast tRNA, and 50 ~g/ml denatured and sheared salmon
sperm DNA).
Samples are hybridized with nick-translated DNA
probes at 42~C for 12-36 hr for homologous species and
37~C for heter~~logous species contained in 5 ml of this
same DNS hybridization buffer. The filters are washed
two times for 30 min, each time at 50~C, in 0.2 x SSC,
0.1% SDS for homologous species hybridization, and at 50~C
in 3 x SSC, 0.1% SDS for heterologous species
hybridization. Filters are air dried and
autoradiographed for 1-3 days at -70~C.
For synthetic (15-30 mer) oligonucleotide
probes, the duplicate filters are prehybridized at 42~C
for Z-8 hr with 10 ml per filter of oligo-hybridization
buffer (6 x SSC, 0.1% SDS, 1 mM EDTA, 5x Denhardt's,
0.05% sodium pyrophasphate and 50 ~,g/ml denatured and
sheared salmon sperm DNA).
The samples are hybridized with kinased
oligonucleotide probes of 15-30 nucleotides under
conditions which depend on the composition of the
oligonucleotide. Typical conditions employ a temperature
of 30-42~C for 24-36 hr with 5 ml/filter of this same
oligo-hybridization buffer containing probe. The filters
are washed two times for 15 min at 23~C, each time with 6
x SSC, 0.1% SDS and 50 mM sodium phosphate buffer at pH
7, then are washed once for 2 min at the calculated
hybridization temperature with 6 x SSC and 0.1% SDS, air
dried, and are autoradiographed at -70~C for 2 to 3 days.
-22-
C.4. cDNA Library Production
Double-stranded cDNA is synthesized and
prepared for insertion into the plasmid vector pBR322
using homopolymeric tailing mediated by calf thymus
terminal transferase (Sutcliffe, J.G., Nucleic Acid Res
(1978) 5:2721-2732). First strand cDNA is synthesized by
the RNA-dependent DNA polymerase from Avian
Myeloblastosis Virus, by priming with oligo (dT) 12-18
on 5 ~.g mRNA. The RNA template is then liberated from
the nascent DNA strand by denaturation at l00~C for 5 min,
followed by chilling on ice. Second strand DNA is
synthesized by using the large fragment of DNA polymerase
I of E. coli, relying on self-priming at the 3'-end of
the first strand molecule, thereby forming a double-
stranded hairpin DNA. These molecules are blunt-ended at
the open-ended termini, and the hairpin loop is cleaved
open with S1 nuclease from Asperqillus oryzae. S1
nuclease digestion of the double-stranded cDNA takes
place in 300 mM NaCl, 30 mM NaOAc, pH 4.5, 3 mM ZnCl2 for
30 min at 37~C with 600 units enzyme. The cDNA is
extracted with phenol: chloroform, and small
oligonucleotides are removed by three ethanol
precipitations in the presence of ammonium acetate. This
is done as follows: a half volume of 7.5 M ammonium
acetate and two volumes ethanol are added to the cDNA
solution, which is precipitated at -70~C. The blunt-
ended, double-stranded cDNA is then fractionated by size
using gel filtration through a column (0.3 x 14 cm)
SepharoseT"" 4B (Pharmacia Fine Chemicals, Piscataway, NJ)
or by ultracentrifugation in 5-20% glycerol gradient
followed by fractionation of the gradient. cDNA roughly
greater than the desired length, e.g., 300 base pairs is
retained and recovered by precipitation with 70o ethanol.
i34(1~8p
-23-
Short (10-30 nucleotides) polymeric tails of
deoxycytosine are added to the 3' termini of the cDNA
using a reaction containing 0.2 M potassium cacodylate,
25 mM Tris, pH 6.9, 2 mM dithiothreitol, 0.5 mM CoCl2,
200 mM cDTP, 400 ~.g/ml BSA, and 40 units calf thymus
terminal deoxynucleatide transferase for 5 min at 22~C.
The reaction is extracted with phenol:chloroform, and
small oligonucleotides are removed with three ethanol
precipitations in the presence of ammonium acetate.
The dC-tai.led cDNA is annealed with pBR322
which has been cleaved with PstI and tailed with oligo
dG: 2.5 ~g pBR322-dG DNA is annealed with the cDNA at a
vector concentration of 5 ~g/ml, and the hybrids are
transferred into E. coli MC1061 by the CaCl2-treatment
described by Casabadan, M., et al, Mol Biol (1980)
138:l79-207.
C.S. Vector Construction
Construction of suitable vectors containing the
desired coding and control sequences employs standard
legation and restriction techniques which are well
understood in the art. Isolated plasmids, DNA sequences,
or synthesized oligonucleotides are cleaved, tailored,
and relegated in the form desired.
Site specific DNA cleavage is performed by
treating with the suitable restriction enzyme (or
enzymes) under conditions which are generally understood
in the art, and the particulars of which are specified by
the manufacturer of these commercially available
restriction enzymes. See, e.g., New England Biolabs,
Product Catalog. In general, about 1 ~.g of plasmid or
-24-
DNA sequence i.s cleaved by one unit of enzyme in about 20
~,l of buffer solution; in the examples herein, typically,
an excess of restriction enzyme is used to insure
complete dige~~tion of the DNA substrate. Incubation
times of about. one hour to two hours at about 37~C are
workable, although variations can be tolerated. After
each incubation, protein is removed by extraction with
phenol/chloroform, and may be followed by ether
extraction, anal the nucleic acid recovered from aqueous
fractions by ~~recipitation with ethanol. If desired,
size separation of the cleaved fragments may be performed
by polyacylamide gel or agarose gel electrophoresis using
standard techniques. A general description of size
separations is found in Methods in Enzymology (1980)
65:499:560.
Restriction cleaved fragments may be blunt
ended by treating with the large fragment of E. coli DNA
polymerase I (Klenow) in the presence of the four
deoxynucleotid.e triphosphates (dNTPs) using incubation
times of about 15 to 25 min at 20 to 25~C in 50 mM Tris pH
7.6, 50 mM NaCl, 6 mM MgCl2, 6 mM DTT and 5-10 ~,M dNTPs.
The Klenow fragment fills in at 5' sticky ends but chews
back protruding 3' single strands, even though the four
dNTPs are present. If desired, selective repair can be
performed by supplying only one of the, or selected,
dNTPs within the limitations dictated by the nature of
the sticky ends. After treatment with Klenow, the
mixture is extracted with phenol/chloroform and ethanol
precipitated. Treatment under appropriate conditions
with S1 nuclease or Bal-31 results in hydrolysis of any
single-stranded portion.
Synthetic oligonucleotides are prepared by the
method of Efimov, V.A., et al (Nucleic Acids Res (1982)
-24a- 1340y0
...
6875-6894), anal can be prepared using commercially
available automated oligonucleotide synthesizers.
Kinasing of single strands prior to annealing or for
labeling is achieved using an excess, e.g., approximately
10 units of polynucleotide kinase to 1
-~ 1340~8~
_25_.
nmole substr~~te in the presence of 50 mM Tris, pH 7.6)
mM MgCl2, 5 mM dithiothreitol, 1-2 mM ATP, 1.7
pmoles Y32P-ATP (2.9 mCi/mmole), 0.1 mM spermidine)
0.1 mM EDTA.
5 Ligations are performed in 15-50 ul volumes
under the following standard conditions and
temperatures 20 mM Tris-C1 pH 7.5, 10 mM MgCl2, 10
mM DTT, 33 ug/ml BSA, 10 mM-50 mM NaCl, and either 40
u.M ATP, 0.01-0.02 (Weiss) units T4 DNA lipase at 0~C
10 (for "sticky end" ligation) or 1 mM ATP, 0.3-0.6 (Weiss)
units T4 DNA lipase at 14~C (for "blunt end" ligation).
Intermolecular "sticky end" ligations are usually
performed at 33-100 ug/ml total DNA concentrations
(5-100 nM tot:al end concentration). Intermolecular
blunt end lidations (usually employing a 10-30 fold
molar excess of linkers) are performed at 1 ~.~M total
ends concentc:ation.
In vector construction employing "vector
fragments", t:he vector fragment is commonly treated with
bacterial alkaline phosphatase (BAP) or calf intestinal
alkaline phosphatase (CIP) in order to remove the 5'
phosphate and prevent religation of the vector.
Digestions are conducted at pI-I 8 in approximately 150 mM
Tris, in the presence of Na+ and Mg+2 using about 1
unit of BAP or CIP per ug of vector at 60~ for about
one hour. In order to recover the nucleic acid
fragments, the preparation is extracted with
phenol/chloroform and ethanol precipitated.
Alternatively, religation can be prevented in vectors
which have been double digested by additional
restriction enzyme digestion of the unwanted fragments.
For portions of vectors derived from cDNA or
genomic DNA which require sequence modifications, site
specific primer directed mutagenesis is used. This~is
,.-
,.
1340580
-26-
conducted using a primer synthetic oligonucleotide
complementary to a single stranded phage DNA to be
mutagenized Except for limited mismatching) representing
the desired mutation. Briefly, the synthetic
oligonucleoti.de is used as a primer to direct synthesis
of a strand complementary to the phage) and the
resulting double-stranded DNA is transformed into a
phage-supporting host bacterium. Cultures of the
transformed bacteria are plated in top agar, permitting
plaque formation from single cells which harbor the
phage.
Theoretically. 50% of the new plaques will
contain the F~hage having, as a single strand, the
mutated form; 50% will have the original sequence. The ~~~-
resulting plaques are hybridized with kinased synthetic
primer at a temperature which permits hybridization of
an exact match, but at which the mismatches with the
original strand are sufficient to prevent
hybridization. Plaques which hybridize with the probe
are then picked) cultured. and the DNA recovered.
Details of site specific mutation procedures ate
described below in specific examples.
C.6. Verification of Construction
In the constructions set forth below, correct
ligations for plasmid construction ate confirmed by
first transforming E. coli strain MC1061 obtained from
Dr. M. Casadaban (Casadaban, M., et al, J Mol Biol
(1980) 138:179-207) or other suitable host with the
ligation mixture. Successful transformants are selected
by ampicillin, tetracycline or other antibiotic
resistance,or using other markets depending on the mode
of plasmid construction) as is understood in the art.
Plasmids from the transformants are then prepared
13~A~80
-27-
according to the method of Clewell) D. B., et al, Proc
Natl Acad Sci. USA (1969) 62:1159, optionally following
chloramphenicol amplification (Clewell, D. B., J
Bacteriol (1972) 110:667). The isolated DNA is analyzed
by restriction and/or sequenced by the dideoxy method of
Sanger, F., et al, Proc Natl Acad Sci (USA) (l977)
74:5463 as further described by Messing, et al, Nucleic
Acids Res (1981) 9:309, or by the method of Maxam, et
al, Methods i.n EnzymoloQV (1980) 65:499.
C.7. Hosts Exemplified
Host. strains used in cloning and expression
herein are as follows:
For cloning and sequencing, and for expression
of construction under control of most bacterial
promoters, E. coli strain MC1061 was used.
For M13 phage recombinants, E. coli strains
susceptible to phage infection) such as E. coli strain
JM101 are employed.
The cells used for expression are Chinese
hamster ovary (CHO) cells.
D. Cloninu and Expression of ASP
Both. canine and human ASP proteins were
obtained in purified form. Canine cDNA was used to
provide probes for the human ASP genomic and cDNA
library.
D.1. Purification of Canine ASP
D.l.a. Isolation of the Surfactant Complex
Lung surfactant complex was prepared from
canine lungs obtained from exsanguinated canines. A11
procedures) including the lavage, were performed at 4~C
and the isolated material was stored at -15~C.
134080
_28_
The lungs were degassed and lavaged 3 times
with one liter per lavage of 5 mM Tris-HC1, 100 mM NaCl,
pH 7.4 buffer. The Ca+2 concentration of this buffer
was less than 5 x 10 6 M (Radiometer F2112 Ca:
Radiometer A/S, Copenhagen. Denmark). The pooled lung
washings were spun at 150 x gay for 15 min (Sorval~M
RC2-H) to remove cellular material. The supernatant was
then spun at 20,000 x gav for 15 hr (Beckman L3-90)
using a type 15 rotor (Beckman Instruments), and the
resulting pellet was dispersed in buffer containing 1.64
M sodium bromide. After equilibration for 1 hr, the
suspension was spun at 100,000 x gav for 4 hr (Beckman
L5-50B) in ~~ SW28 rotor (Beckman Instruments). The
pellicle was resuspended in buffer and spun at 100,000 x
gav for 1 hr (Beckman L5-50B). This pellet containing
the complex was resuspended in double distilled water.
D.l.b. Extraction of Livid and 10 kd Protein
Pellet resuspended in water at a concentration
of 10-15 mg phospholipid/ml was injected into a 50-fold
volume excess of n-butanol (Sigrist, H.) et al, Biochem
Biovhys Res Commun (l977) 74:178-184) and was stirred at
room temperature for 1 hr. After centrifugation at
10,000 x gav foc 20 min (Sorval RC2-B), the, pellet, _,_.
which contains the 32K ASP is recovered for further
purification as described below. The supernatant, which
is a single phase, contains the lipids and the lower
molecular weight proteins. To obtain the lipids, the
supernatant was dried under vacuum at 40~C and the
lipids were extracted (Folch, J., et al) J Biol Chem
(1957) 226:497-509).
To obtain the hydrophobic protein. the
supernatant was subjected to rotovap to remove the
butanol) and further dried by addition of ethanol
,~, 1340580
-29-
followed by rotovap. The dried residue was suspended in
redistilled chloroform containing 0.1 N HCl, and
insoluble material removed by centrifugation.
The resulting solution~was chromatographed over
an LH-20 column (Phatmacia) and developed in
chloroform. (LH-20 is the hydroxypropyl derivative of
SephadexT"G-50: it is a hydrophobic gel which is inert to
organic solvents.) The proteins are excluded; .""
lipids/phospholipids elute from the included volume.
Protein is recovered from the void volume
fractions by evaporation of the chloroform under
nitrogen) and then subjected to sizing on polyacrylamide
gels. When run under non-reducing conditions, three
bands of 16.5 kd) 12 kd, and 10 kd were obtained; under
reducing conditions, a single broad band of 10-12 kd was
found .
The 16.5 kd and 12 kd bands from the
non-reduced gels were subjected to N-terminal analysis
by Edman degradation) to give the following sequences:
For 16.5 kd: ?-Pro-Ile-Pro-Leu-Pro-Tyr-Cys-Trp-Leu-Cys-
Arg-Thr-Leu-Ile-Lys-Arg-Ile-Gle-Ala-Met-Ile-
Pro-Lys-Gly-Val-Leu-Ala-Val-Thr- ? -Gly-Gln-
For 12 kd: Ile-Pro-Cys-Phe-Pro-Ser-Ser-Leu-Lys-Arg-Leu-
Leu-Ile-Ile-Val-Trp
D.l.c. Protein Fractionation and Verification
as ASP 32K Protein
The precipitate from the n-butanol extraction
above was dried under nitrogen and washed twice in 20 ml
of buffer containing ZO mM octyl-ti-D-glucopyranoside.
After centrifugation at 100.000 x gav for 1 hr
w,.,_,~e
1340580
-30-
(Beckman L5--50B)( the pellet was dispersed in 0.3 M
lithium diiodosalicylate, 0.05 M, pyridine (pH 8.4) on
ice, diluted with an equal volume of water, and mixed
with a volume of n-butanol equal to the aqueous phase.
A total of 9 n--butanol-water partitions were performed
to lower the detergent concentration in the aqueous
phase. The final lower, aqueous phase containing the
protein was lyophilized for 15 hr, taken up in 2 ml of
buffer and spun at 100,000 x gav (Beckman L5-50B) to
remove any remaining insoluble material. The lithium
diiodosalicylate concentration in the final sample,
calculated from an extinction coefficient of 4 x 103
at 323 nm (Marchesi) V. T. and Andrews, E. P., Science
(1971) 174:1247-1248), was less than 10 u.M.
The thus purified canine ASP 32K apoprotein was
reconstituted with surfactant lipids purified as above.
The reconstituted material had surface activity as
measured by the surface balance and its in vivo
biological activity was demonstrated by inspiration into
fetal rabbits maintained on a ventilator.
D.l.d. Further Protein Purification
The protein fraction obtained in the previous
subparagraph 'was reduced by incubation with 50 mM DTT in
1% SDS, 50 mM '1'ris-HC1. 1 mM EUTA pH 7.5 at 37~C for 1
hr, aklylated with 100 mM iodoacetamide (Sigma) at 0~C
for 30 min, and subjected to polyacrylamide gel
eletrophoresis by the procedure of Laemmli, U. K.,
Nature (1970) 227:680-685. The proteins were visualized
by soaking the gel in 4 M sodium acetate solution and
the 32K band was sliced out with a razor blade, and
electroluted by the protocol of Hunkapiller, M. W., et
al, Methods in EnzymoloQY (1983) 91:227-235, New York.
-31- 1340580
Academic Press, using the CBS Scientific (Del Mar)
California) electrolution device.
The eluted protein was lyophilized and its
N-terminal amino acid sequence was determined from one
nanomole of protein using the Applied Biosysteme 4?OA
gas-phase sequences (Applied Biosystems Inc.. Foster
City) CA) in accordance with the instructions of the
manufacturer. PTH amino acids were identified with a
Beckman 334T HPLC, using a 0.46 x 25 cm IBM CN-column.
The gradient: applied was as indicated is Hunkapiller. N.
W., and Hood) L. E., Methods in Enzvmoloav (l983)
9l:486-492, New York. Academic Press, with the following
modifications: Instead of a binary gradient system a
ternary gradient system was used in which acetonitrile
and methanol wets pumped by separate pumps and the ratio
of the two varied with time over the course of the
gradient, with appropriate modification of the gradient
program: instead of the Permaphase ETHr guard column.
a "5 x 0.46 cm IBM*CN" analytical "mini-column", was
used; and the column was heated to Z8~C) rather than to
32~C.
The N-terminal amino acid sequence was:
1 5 10
Ile-Glu-Asn-Asn-Thr-Lys-Asp-Val-Cys-Val-Gly-Asn-
15 20 ~ 25
Hyp-Gly-Ile-Hyp-Gly-Thr-Hyp-Gly-Ser-His-Gly-Leu-Hyp-Gly-
Arg-?-Gly-Arg-?--Gly-Val.
"Hyp" indicates the modified amino acid
30 hydroxyproline.
Amino acid composition data for the canine 32K
protein show a hydroxyproline content consistent with
the hydroxylation of proline residues in the deduced
sequence (see below) which appear in the collagen-like
* trade-mark
Ca
1340580
-32-
Pattern Gly-X-Hyp. As this pattern is also shown in the
human N-terminal sequence it is probable, by analogy to
the canine data, that similarly disposed prolines in the
human sequence are hydroxylated.
Informatian regarding processing was obtained
by purification and sequencing of collagenase treated
canine ASP.
Purified canine ASP was digested with bacterial
collagenase (Worthington, Freehold NJ) at a l:l
enzyme:substrate ratio in 5 mM Tris pH 7.4-5 mM CaCl2 at
37~C. That produced a 22 kd limit digest product as
analyzed on SDS gels. This 22 kd band was electroeluted
from a gel and subjected to amino.acid sequence analysis
as described above. Two amino acids were identified at
each cycle, indicating the collagenase treatment had
produced two peptides which remain linked by a disulfide
bridge. From the cDNA clone sequence it can be
demonstrated that the two sequences correspond to amino
acids 78-l10 and 203-231 in the intact molecule. The
sequences obtained are:
2 4 6 8 10 12 14
16 18 20 22 24 26 28
Gly-Pro-Hyp-Gly-Leu-Pro-Ala-Ser-Leu-Asp-Glu-Glu-Leu-Gln-
Gly-Lys-Glu-Gln-Cys-Val-Glu-Met-Tyr-Thr-Asp-Gly-Gln-Trp-
Thr-Thr-Leu-His-Asp-Leu-Arg-His-Gln-Ile-Leu-Gln-Thr-Met-
Asn-Asn-Lys-Asn-Cys-Leu-Gln-Tyr-Arg-Leu-Ala-Ile-Cys-Glu-
30 32
Gly-Val-Leu-Ser-Leu
Phe
and demonstrate that translation is complete, and that
the C-terminus of the protein is intact.
1340580
-33-
D.l.e. Isolation of Human ASP
Human 32K and lower molecular weight ASP was
prepared following the procedure described above for
canine proteins from a patient suffering from alveolar
proteinosis (a syndrome which results from the presence
of excess surfactant in the lung).
The 32K ASP has the N terminal sequence:
1 5 10
Glu-'Jal-Lys-Asp-Val-Cys-Val-Gly-Ser-Hyp-Gly-Ile-
15
Hyp-c:,ly-Thr-Hyp-Gly
Amino acids 3-17 of the human sequence are
precisely homologous to amino acids 6-20 of the canine
32K protein except for the serine at position 9.
The isolated low molecular weight hydrophobic
proteins show bands corresponding to l6.5 kd, 14 kd and
10 kd when subjected to polyacrylamide gel
electrophoresis under non-reducing conditions. Under
reducing conditions, a single broad band corresponding to
10-11 kd is obtained.
D.2. Isolation of Canine Lunq mRNA
Total RNA was isolated from an adult canine
lung by the method of Chirgwin, J.M., et al, Biochemistry
(1979) l8:5294-5299. The lung tissue was first
pulverized by grinding with a mortar and pestle in liquid
N2, and homogenized in a solution of 6 M guanidine
thiocyanate, 0.05 M Tris-HC1, pH 7.0, 0.1 M-(3-
mercaptoethanol, 0.5% Sarcosyl. This homogenate was made
2.0 M in CsCl and layered over a 5.7 M CsCl cushion in
0.0l M ethylenediaminetetraacetic acid (EDTA) and 0.05 M
Tris-HC1, pH 9Ø The RNA was pelleted through this
cushion by centrifugation at
1340580
_ 34.
115,000 x g for 16 hr, thereby separating it from the
cellular DNA ~~nd protein which do not sediment through
the higher density CsCl solution. The RNA was then
dissolved in 0.01 M Tris-HC1, pH 7.4, 0.005 M EDTA, 1.0%
sodium dodecylsulfate (SDS), extracted with a l:l
mixture of chloroform and phenol, and precipitated from
70% ethanol. 'fhe polyadenylated RNA (poly A+ RNA)
fraction was obtained by affinity chromatography throughw--
oligo (dT) cellulose as described by Aviv, H., and
Leder, P., Proc Natl Acad Sci (USA) (1972)
69:1840-1412.
D.3. Construction and Screening of Canine Lund
cDNA Library
Adult: canine lung poly A+ RNA prepared as in
~[D.2 was used to construct a cDNA library as described
in 1[C.4, 5 ug mRNA yielded about 25 ng of cDNA)
size-selected to greater than 300 base pairs. The
library contained about 200,000 independent
recombinants. Of these, 40,000 recombinants were plated
on nitrocellullose filters. These filters served as the
masters for subsequent replicas (in accordance with the
method of EIanahan, D., and Meselson, M., Gene (1980)
l0:63-75.
cDNA Encoding the 32K Protein
Three probes were constructed: a mixture of 24
x 14-mer sequences complementary to the amino acids 1-5
having the seduence
A
5 ' A'L'CGAGAACAACAC 3 '
~I~ A T T
-35- 134Q5gp
(probe a): 64 x 14-mers complementary to the amino acids
7-11 having I:he sequence
A A
' C;Ar'G'fT'fGCGTTGG 3 '
T C T C
5 G G
(probe b); and a single 15-mer
5' ATCGAGAACAACACC 3'
selected on t:he basis of mammalian codon preference
(probe c). lM:ach oligonucleotide mixture and the single
unique oligonucleotide were synthesized on a Biosearch
SAM I oligonucleotide synthesizer (Biosearch) Inc., San
Rafael, CA) t>y a modif ication of the standard
phosphotriest:er method using mesitylenesulfonyl chloride
in the presence of N-methylimidazole as condensing
reagents as ~tescribed by Efimov, V. A., et al, Nucleic
Acids Res (1982) 10:6875-6894) and purified by
polyactylamide gel electrophoresis.
For hybridization. xix replica filters were
prepared from each master filter, so that each colony
could be screened in duplicate with each of three
oligonucleotide probes. Colonies recovered after
replication off the master filters were placed on agar
plates containing 170 ug/ml chloramphenicol for 18
hr. The colonies were then prepared for hybridization
according to the method of Grunstein, M.) and Hogness,
D.. Proc Natl Acad Sci (1975) 72:3961-3972.
The filters were baked for 2 hr at 80~C under
vacuum and then washed for 4 hr at 68~C with shaking in
a large volume of 3X SSC (where 1X SSC is 0.15 M NaCl,
0.015 M sodium citrate. pH 7.5), 0.1% SDS. The filters
were prehybri.dized in 6 x SSC, 0.1% SDS, 1 mM EDTA) 5x
Denhardt's solution (0.1% Ficoll;~ 0.1% polyvinyl-
pyrrolidone. 0.1% bovine serum albumin) 0.05% sodium
--~.. 1340580
-36-
pyrophosphate and 50 ug/ml denatured salmon sperm DNA
at 42~C for a minimum of 2 hr.
Duplicate filters were then hybridized with 5 x
106 rpm of o.ne of each 32P-labeled oligonucleotide
probe (phosp;horyltated in accordance with Maniatis, T.)
et al, Molecular Cloning) (1982) Cold Spring Harbor
Laboratories, pp. 122-123) per filter in 10 ml
hybridizatio n solution containing identical ingredients
as the prehybridization solution. Filters with
oligonucleotide probes a, b, and c were hybridized at
37~C, 45~C, and 41~C) respectively. After 1 hr, the
thermostat was lowered to 28~C for probe a and 37~C for
probe b, after which the bath was allowed to
equilibrate. Filters with probe c were not hybridized
at a lower temperature. The filters were washed twice
in 6 x SSC, 0.1% SDS at room temperature for 15 min,
then washed in 6 x SSC, 0.1% SDS at 37~C. 45~C, and 41~C
for probes a" b, and c, respectively, for 2 min. The
final washing temperature was obtained form the
empirical formula of Suggs) S. V., et al, Developmental
Bioloay Usind Purified Genes (ed. D. D. Brown and C. F.
Fox), Academic Yress, NY, pp. 683-693; that is. Td -
4(G+C) + 2(A~-T). The hybridized filters were then dried
and autoradiographed on Kodak" XAR film with DupontTf"t
Cronex intensifying screens until complete exposures
were obtained.
A colony was considered positive if it
hybridized ire duplicate with all three oligonucleotide
probes or with both probes a and b. Of several
potential positive clones, one hybridized much more
intensely with probes a and b as compared to the
others. Sequencing of this clone demonstrated that it
encoded a portion of the sequence of canine 32K ASP. It
--
a340580
-37-
was designated DS-1 and used to obtain the entire 32K
canine ASP.
The purified DNA insert of 375 base pairs was
excised from pDS-1 by restriction with PstI and prepared
using small miniprep methods (Maniatis, et al, supra at
p. 366) and was isolated on agarose gels. The intact
DNA insert was then subcloned into bacteriophage M13
(Messing, J., and Vieira, J.) Gene (1982) 19:259-268)
and sequenced using the dideoxy method of Sanger, F., et
al, Proc Natl Acad Sci (USA) (1977) 74:5463-5469. The
sequence encoded the N-terminal portion of the
approximately 300 amino acid protein, i.e., the 32
residue N-terminal amino acid sequence determined from
the purified canine ASP of 1(D.1, and 101 additional
downstream amino acids. It also contained 50 base pairs
of the 5' unt.ranslated region.
The mRNA pool was assessed to determine the
presence of sequences of sufficient length to encode the
entire canine ASP sequence by Northern blot. Poly A+
RNA of 1(D.2 was subjected to Northern blot using nick
translated DS-1 insert DNA after fractionation by
electrophoresis on a 1.4% agarose gel containing
methylmercuric hydroxide by the method of Bailey, J. M.
and Davidson, N., Anal Biochem (1976) 70:75-85. mRNA
hybridizing to probe was 1800-2000 nucleotides in
length, clearly larger than the approximately 700
nucleotides needed for the coding sequence.
The DS-1 insert probe was therefore used to
rescreen one duplicate set of original filters, which
had been treated at 100~C for 10 min to remove residual
oligonucleotide probe. Filters were prehybridized in
0.75 M NaCl, 0.075 M Na citrate, 50% formamide, 0.5%
SDS, 0.02% bovine serum albumin, 0.02% Ficoll - 400,000,
0.02% polyvinyl pyrollidone, 0.1% sodium pyrophosphate,
..:
1340580
_38-
50 ug.ml yeasty tRNA and 50 ug/ml denatured sheared
salmon sperm I)NA) at 42~C for 18 hr. 5 x 105 cpm of
32P-labeled boiled DS-1 cDNA was added per ml fresh
hybridization buffer and the filters were incubated in
this buffer at 42~C for 16 hr. They were then washed in
0.03 M NaCl and 0.003 M sodium citrate and 0.1% SDS two
times each for 30 min at 50~C, and exposed for
autoradiography overnight. Two additional clones, DS-4
and DS-31, were identified, which, together with DS-1,
comprise roughly 1700 base pairs (Figure 1).'
DS-4 and DS-31 were also excised using PstI,
subcloned into the Pstl site of M13mp9) and sequenced by
dideoxy sequencing according to the procedure of Sanger,
F. (supra). The entire sequence contains two internal
PstI sites. Confirmation of correct sequencing was
obtained by d:ideoxy sequencing of fragments obtained
from deduced internal restriction sites, as shown in
Figure 1. The entire nucleotide sequence including the
amino acid sequence of ASP deduced from the 256 codon
open reading :frame is shown in Figure 1.
cDNA Encoding Canine 10K ASP
Two ~~ligomeric probes were synthesized
corresponding to the N-terminal sequence of the 16.5 kd
protein using mammalian codon preference tables for
codon choice. Probe 1198 was a 36-mer of the sequence
5'-GGTCACAGCCAGGCCCTTGGGGATCATGGCCTGGAT-3'; probe 1199
was a 45-mer of the sequence
5'-CTTGATCAGGGTTCTGCACAGCCAGCAGTAGGGCAGGGGGATGGG-3'.
Both were labelled with 32P by kinasing.
For hybridization, filters were baked at 80~C
for two hours under vacuum and then washed for 4 hr at
68~C with shaking in a large volume of 3 x SSC
containing 0.1% SDS. The filters were prehybridized for
~''~ 13e580
-39-
several hours at 42~C in 6 x SSc. 5 x Denhardt's, 20%
formamide, O.:l% SDS, and 100 ug/ml sheared) denatured
salmon sperm I)NA. Duplicate filters were hybridized in
the above buffer containing either 13 ng/ml probe 1l98
or 16 ng/ml probe 1199 at an intial temperature of 68~C,
and then at 42~C overnight. The filters were washed
twice for 15 min at room temperature in 6 x SSC, 0.1%
SDS, 0.05% sodium pyrophosphate, then for 5 min at 65~C
in the same buffer, and then dried and autoradiographed.
Of 40.000 clones screened, 8 hybridized to both-~--w
probes) and ware subjected to restriction analysis. Two
overlapping c:Lones which when combined span 1520
nucleotides were sequenced, with the results shown in
Figure 2. The arrow indicates the beginning of the
mature 16.5 k~i protein.
D.4. Isolation of the human 32K ASP Gene
A human genomic library cloned into bacterio-
phage Charon :Z8 (Rimm. U. L., et al, Gene (1980)
l2:301-310) was obtained from Ur. T. Maniatis, Harvard
University. Approximately 1.5 x 106 phage were grown
on E. coli K803, and plaque lysates were transferred to
nitrocellulose filters as described by Benton, W. D., et
al, Science (1977) 196:180-182. The filters were probed
with DS-1 cDNA which had been kinased by the nick-
translation method of Rigby, P. W. J., et al, J Mol Biol
(1977) 113:23'7-251. Filters were prewashed in
hybridization buffet (0.75 M NaCl, 0.75 M sodium
nitrate, 40% formamide, 0.05% SDS) 0.02% bovine serum
albumin, O.02'% Ficoll -400.000, 0.02% polyvinyl
pyrollidone) 0.1% sodium pyrophosphate, 50 ug/ml yeast
tRNA, 50 ug/ml denatured sheared salmon sperm DNA) at
42~C for 1 hr. 5 x 105 cpm probe was added per ml
fresh hybridization buffer and the filters were
~1~~058t~
-40-
incubated in this buffet at 37~C fot 16 hr. They were
then washed in 0.45 M NaCl and 0.045 M sodium citrate
and 0.1% SDS two times at 50~C, and exposed for
autoradiography overnight. Six potential clones
containing sequences hybridizing to DS-1 cDNA were
purified. The most strongly hybridizing clone, gHS-15,
was characterized.
A 700 by EcoRI fragment from gHS-15 hybridized
with the DS-1 probe and was chosen for sequence
analysis. Thi:~ EcoRI fragment was purified, inserted
into M13mp9, s~aquenced and found to be extensively
homologous with the corresponding canine sequence.
The entire human coding region was contained
within two contiguous BamHI fragments: a 5' 1.2 kb and
a 3' 3.5 kb fragment. Both BamHI fragments were
individually subcloned into the BamHI site of M13mp8 and
sequenced. Ad<jitional fragments were similarly
sequenced according to the strategy shown in Figure 3.
The sequence information was analyzed using various
ZO Intelligenetics (Palo Alto, CA) computer programs in
accordance with the instructions of the manufacturer.
The regions containing the signal peptide, precursor
sequence and mature apoprotein were identified by
comparison to the canine ASP cDNA. From the sequence
analysis, the ~>' terminus of the gene is encoded within
the 1.2 kb Bamtll fragment and the 3' terminus within the
3.5 kb BamHI fragment. The gene is interrupted by three
introns at positions 1218 bp) 1651 by and 2482 bp, with
position 1 being the first by of the 1.2 kb BamHI
fragment. The entire sequence, including the amino acid
sequence of human ASP protein deduced is shown in Figure
3.
134o~so
-41-
D.S,. Expression of Human 32K ASP
The phage isolate gHS-15 identified in 1[D.5 as
harboring an insert of approximately 16 kb containing
the entire human ASP gene was transferred into CHO cells
which had been grown in McCoy's 5A medium with 10% fetal
bovine serum by co-transformation with pSV2:NE0
(Southern, P.., et al, J Mol Appl Genet (198Z)
1:327-341), a plasmid containing a functional gene
conferring resistance to the neomycin analog G148, which
is toxic to ~aammalian cells. In the transformation, 15
ug of the '~.:~1HS-15 and 2 ug of pSV2:NE0 were
applied to a 100 mm dish of CElO cells in a calcium
phosphate/DNA coprecipitate according to the method of
Wigler, M., pt al. Cell (1979) 16:777-785, with
inclusion of a 2 min "shock" with 15% glycerol 4 hr
after exposure to the DNA. The cells were transferred
to medium containing 1 ug/ml G418, and yielded about
50 stable transformants per 100 mm dish.
Stable transformants were cultured prior to
labeling in media supplemented with 0.25 mM ascorbic """,
acid. Two pools of stable transformants and one pool of
untreated CHO cells were grown for 1 hr in medium
containing 1/10 of normal methionine concentration and
then labeled with 35S-methionine for 8-16 hours, and
the 35S-met labeled total secreted proteins were
analyzed by SDS-polyacrylamide gel electrophoresis. The
results ate shown in Figure 4. Lane 1 shows the normal
CHO secreted proteins. Lanes 2 and 3 display ~.:gHS-15
secreted proteins: both of which have an additional
30-36 kd protein corresponding to an expressed ASP
protein. To further document the identity of the 30-36
kd protein one can immunoprecipitate the total secreted
protein samples with canine ASP antibodies. The vector
~.:gHS-15 was deposited with the American Type Culture
134~~8~J
-42-
Collection on 7 December 1984 and has accession no. ATCC
40146.
D.6. Preparation of a Human cDNA Clones for
the 32K and 10K Proteins
Human 32K ASP
Human lung was obtained from two fetuses, one
22 weeks, the other 24 weeks of age. 7 g of lung tissue
was first pulverized by grinding with a mortar and
pestel in liquid N2, and total poly A+ RNA prepared
as set forth in ~(D.2 (supra).
A cDNA library was prepared from the mRNA as
set forth in ~(C.4. Five ug of lung poly A+ RNA
yielded about .25 ng of cDNA) size-selected to greater
than 500 base pairs, and gave a library of 300,000
independent recombinants.
60,000 members of the human cDNA library were
screened with the canine DS-1 cDNA in the manner
described above for the screening of the genomic
library. The recombinant colonies were plated on
nitrocellulosa filters which served as masters for two
sets of replicas. The colony filters were then prepared
for hybridization according to the method of Grunstein,
M~. and Hognese~, D. (supra). The filters were baked for
2 hr at 80~C under vacuum and then washed for 4 hr at
68~C with shaking in a large volume of 3R SSC and 0.1%
SDS. Next the filters were prehybridized in 0.75 M
NaCl, 0.075 M sodium nitrate, 40% formamide, 0.5% SDS,
0.02% bovine serum albumin) 0.02% Ficoll - 400,000)
0.02% polyvinyl pyrollidone) 50 ug/ml yeast tRNA, 50
ug/ml denatured sheared salmon sperm DNA) at 37~C for
18 hr. One x 106 cpm of 32P-labeled DS-1 probe was
added per ml of fresh hybridization buffer then
1340~8t~
-43-
incubated for 16 hr at 37~C. The filters were then
washed in 0.45 M NaCl and 0.045 M sodium citrate and
O.01% SDS two times each for 30 min at 50~C, and exposed
for autoradiography overnight.
One positively hybridizing clone, HS-6, was
further analyzed by sequence determination: HS-6 harbors
a 1.2 kb insert which can be released from the vector
using PstI digestion) and which bears an internal EcoRI
site. Both Pstl-EcoRI fragments from the insert were
subcloned into the PstI-EcoRI site of M13mp8 and mpg,
and partial sequences obtained. The over 200 by
sequenced portion corresponds perfectly to the 3' end of
gHS-15. The nucleotide sequence of HS-6 is shown in
Figure 5.
As the HS-6 cDNA insert contained only the
3'-terminal region of the ASP mRNA. the remaining clones
were screened far adjacent surfactant sequences using
HS~-6 as probE~. No clones were found in the remainder of
the library.
To obtain the complete cDNA encoding human 32K
ASP, a randomly primed cDNA was prepared from adult
human lung and cloned in the bacteriophage vector gtl0
using EcoRI linkers by the procedure of Huynh. T., et
al, cDNA Cloning Techni4ues: A Practical Approach
(Glover) D., ed) IRL, Oxford. Adult lung is greatly
enriched in ASP transcripts as compared to fetal lung
tissue (our observations) and therefore affords a
greater frequency of obtaining a complete ASP cDNA.
Phage plaques were screened with a 32P
labelled insert from pHS-6 using 5x10 5 cpm/ml in 50%
formamide, 5 x SSC, 0.05% SDS) 5 x Denhardt's, tRNA and
salmon sperm DNA at 42~C for 16 hr. The filters were
washed twice at 50~C for 30 min each in 0.2 x SSC. 0.1%
SDS, dried and autoradiographed.
-44- 134D580
Two positively hybridizing clones, designated
pHS-2 and pHS-~5 were isolated. Each contained the
entire 3ZK ASP encoding sequence and most of the 5'
untranslated region. Each overlapped with HS-6, which
contained most. of the 3' untranslated region: the 3'
terminus of each clone corresponds to the EcoBI site
within the coding region.
Human 10K' ASP
The same cDNA library in lambda gtl0 was
screened on ni.trocellulosE filters as above using
1x106cpm of the canine clone pDlOk-1 described above
in 40~ formami.de, 5 x SSC. 0.05 SDS, 5 x Denhardt's, 50
ug/ml yeast tRNA and 50 ug/ml salmon sperm DNA for
16 hr at 37~C. The filters were washed twice at 50~C
for 30 min in Z x SSC, 0.1~ SDS, dried and
autoradiographed. Of 40,000 plaques, two were positive,'
and one, designated lambda HlOk-1 containing a 1.5 kb
insert was chosen for sequencing. Preliminary partial
sequencing results are shown in Figure 6. The under3.inec~
amino acid residues represent homologies with the canine
protein.
D.7. Construction of Expression Vectors
Vectors suitable for expression o~f the genomic
human 32K ASP encoding sequence in mammalian cells.
which are capable of processing intron-containing DNA
were constructed. Expression is controlled by the
metallothionein II (hMTII) control sequences, as
described by Karin. M.) et al) Nature (1982)
299:797-802. '
The host vector, pM'f is obtained by ligaCing
the promoter into pUCB as follows:
j
y
134058U
-45-
Plasmid 84H (Karin, M., et al (supra)) which
carries the hMTII gene was digested to completion with
BamHI) treated with exonuclease Bal-31 to remove
terminal nucleotides, and then digested with HindIII to
liberate an 840 by fragment containing nucleotides -765
to +70 of the hMTII gene (nucleotide +1 is the first
nucleotide tr,anscribed). The 840 by fragment was
isolated and ligated with HindiIl/HincII digested pUCB
(Vieira, J., ~et al, Gene (1982) 19:259-268) and the
ligation mixture transformed into E. coli MC1061. The
correct construction of pMT was confirmed by dideoxy
nucleotide se~~uencing.
In addition, a derivative of the pMT, pMT-Apo,
containing C-terminal regulatory signals was also
prepared. pM'r-Apo harbors a portion of the human liver
protein ApoAl gene (Shoulders) C. C.) et al. Nucleic
Acids Res (1983) 11:28Z7-2837) which contains the
3'-terminal regulatory signals. A PstI/PstI 2.2 kb
fragment of ApoAl gene (blunt ended) was cloned into
the SmaI site of the pMT polylinker region, and the
majority of the ApoAl gene removed by digestion with
BamHI, blunt ending with Klenow, digestion with StuI,
and teligation. The resulting vector contains roughly
500 by of the ApoAl gene from the 3' terminus as
confirmed by dideoxy-sequence analysis.
Five constructs of the human ASP gene and the
pMT and pMT-Apo expression vectors were prepared using
the 1.2 kb and 3.5 kb BamHI fragments of gHS-15. A11
constructs were isolated and confirmed by both
restriction analysis and dideoxy sequencing. These
constructs were prepared as follows:
1. the 1.2 kb and 3.5 kb BamHI fragments were
cloned into the BamHI site of pMT to give pMT:gHS;
134058g
-46-
2. the 1.2 kb BamHI fragment was truncated at
the 5' terminus by digestion with HinfI (position 950)
and filled in with Klenow. The truncated fragment was
cloned, along with the 3.5 kb fragment into the BamHI
site of pMT to give pMT: gHS(Hinf I ) ;
3. the fragments of 1f2 were cloned instead
into the BamHI site of pMT-Apo to give
pMT-Apo:gHS(Hinfl);
4. the 3.5 kb BamHI fragment was truncated at
the 3' terminus by digestion with EcoRI (position 3434')
and filled in with Klenow. This truncated fragment was
cloned, along with the truncated 1.2 kb fragment
truncated with HinfI as above into the BamHI site of
pMT-Apo to give pMT-Apo : gEIS (Hinf I /EcoRI ) ;
5. the 1.2 kb fragment was truncated at the
BstEII site at position 356 and the 3.5 kb fragment at
the BstEII site at position 4024. These fragments were
cloned into the BamHI site of pM'f-Apo to give
pMT-Apo:gHS(BstEIi).
The resulting pM'f:gHS constructs were
transferred into CHO cells as set forth in ~[D.6 except
that 10 4 M ZnCl2 was added with 35S-methionine to
induce the metallothionein promoter and label the
proteins produced.
After .B-16 hr the medium is analyzed for
35S-met labeled total secreted protein which
immunoprecipitates with antibodies to canine ASP.
Non-immune IgG are used as a control.
D.B. Optimization of Expression
Conditions of expression were optimized) and
additional expression vectors containing the SV40 viral
enhancer were used to increase the levels of expression
in CHO cells. 'Phree vectors were used:
134~~.~~.,_.
-47-
pMT-Apo:gHS(H:infI/EcoRI) described above and further
characterized below) and pASPc-SV(10) and pASPcg-SV(10)
which are constructed as described below.
Enhancer-containinu Vectors
To obtain host expression vectors containing
the SV40 enhancer in operable linkage to the MT-II
promoter an 11.00 by SV40 DNA fragment was inserted into
the HindIII site preceding the MT-II promoter sequences
in pMT. The SV40 DNA fragment spans the SV40 origin of
replication and includes nucleotide 5171 through
nucleotide 5243 (at the origin), the duplicated 72 by
repeat from nucleotide 107-250, and continues through
nucleotide 1046 on the side of the origin containing the
5' end of late viral mRNAs. This HindIII 1100 by
fragment is obtained from a HindIII digest of SV40 DNA
(Buchman, A.R.) et al, DNA Tumor Viruses) 2d ed (J.
Tooxe, ed.), Cold Spring Harbor Laboratory. New York
(1981), pp. 799-841), and cloned into pBR322 for
amplification. The cloning vector was cut with HindIII)
and the 1100 by SV40 DNA fragment isolated by gel
electrophoresis and ligated into HindIII-digested.
CIP-treated) pMT. The resulting vectors) designated
pMT-SV(9) and pMT-SV(10), contain the fragment in
opposite orieni:ations preceding the MT-II promoter. In
pMT-SV(9), the enhancer is about 1600 by from the 5'
mRNA start site; in the opposite orientation it is
approximately 980 by from the 5~ mRNA start site. Both
orientations are operable) but the orientation wherein
the enhancer sequences are proximal to the start site
provides higher levels of expression.
pASPc-SV 10_: The coding sequences for ASP were
inserted into the above-described modified form of the
,"
1340g0
-48-
host vector pMT-SV(10). First) the 500 by apoAI
fragment was inserted into pMt-5V(10) by isolating this
fragment, obtained by digestion of pMT-Apo (described
above) and ligating the isolate into EcoRI/BamHI
digested pMT-SV(10). The modified vector was digested
with BamHI, blunted) and ligated to the cDNA sequences
obtained from pHS-5 (White, R.T., et al, Nature (1985)
317:361-363) as a blunted EcoRI digest. The cDNA
fragment extends from the EcoRI linker joined to the 5'
untranslated region to the naturally occurring EcoRI
site in the 3' untranslated region (900 bp). The
relevant nucleotide sequences are shown in Figure 7,
where the starred amino acids represent differences in
the primary amino acid sequence from that of the protein
~gbtained from vpMT-Apo:gHS(HinfI/EcoRI). (The
differences result from base changes between human cDNA
and the genomic sequences.) Initiation of translation
is at nucleotide 56, as in the native sequence.
pASPccZ SV(10): An additional modification was
prepared by integrating pABPc-SV(10) and
pMT-Apo:gHS(HinfI/EcoRI) sequences. Plasmid
pASPc-SV(10) was digested with BamHI and EcoRI, and the
isolated larger fragment ligated to the 3' portion of~ ~~
the ASP gene obtained by BamHI/EcoRI(partial) digestion
of pMT-Apo:gHS(Hinfl/EcoRI). This represents the
portion of the human ASP gene beginning at nucleotide
1154 and extending to nucleotide 3432, this being
ligated to the ApoAI gene fragment as above. This
construct results in a protein identical to that
obtained from pMT-Apo:gHS(HinfI/EcoRI), but different at
amino acid positions 25, 30) and 34 from that obtained
from pASPc-SV(7.0). The nucleotide sequence of the
relevant insert: is shown in Figure 8.
...,
1340g0
-49-
pMT-Apo:dHS(HinfI/EcoRI
For the genomic DNA-containing vector,
pMT-Apo:gHS(HinfI/EcoRi), the coding sequences were
obtained as an HinfI/EcoRI fragment of the gene
extending from nucleotide 950 to nucleotide 3432,
containing exons 2, 3, and 4, and part of exon 5. See
also,.White, R.T.) et al, Nature (1985) 3l7:361-363.
This fragment was ligated to a 500 by fragment from the
3' end of the human ApoAI gene (Shoulders, C.C., Nucleic
Acids Res (19B3) 11:2827-2837) which contains the
polyadenylation signal and polyadenylation site as set
forth above. The entire ASP-encoding genomic insert is
shown ligated to the Mf-II promoter in Figure 9.
It was expected that this vector would produce
a protein 23 amino acids longer than the native
preprotein (which includes the signal sequence). The
construct lacks exon 1 and therefore translation
probably initiates at the ATG beginning at~nucleoti~e ~~~
987 of the genomic sequence complementary to native
preprotein mRNA, which nucleotide normally resides in
the first intron. In the production of native
preprotein) exon 1 is spliced to exon 2 at nucleotide
1022, deleting this start codon, and permitting
translation to initiate at nucleotide 1046. However,
the additional residues do not appear to interfere with
secretion, and the normal mature protein is secreted
from cells expressing this modified form of the gene.
Transformation Procedure
Each of the vectors described above was
transformed into CHO cells as follows: Chinese hamster
ovary (CHO)-K1 cells were grown on medium composed of a
l:l mixture of Coon's F12 medium and DME21 medium with
10% fetal calf serum. The competent cells were
134058U
-50-
co-transformed with the vector of interest and pSVZ:NEO
(Southern, P., et al. J Mol Appl Genet (198Z)
1:327-341). pSV2:NE0 contains a functional gene
conferring resistance to the neomycin analog G418. In a
typical transformation) 0.5 ug of pSV2-NEO and 5 ug _,....
or more of the expression vector DNA are applied to a
100 mm dish of cells. The calcium phosphate-DNA
co-precipitation according to the protocol of Wigler,
M., et al. Cell (1979) 16:777-785, was used with the
inclusion of a two minute "shock" with 15% glycerol in
PBS after four hours of exposure to the DNA.
Briefly, the cells are seeded at 1/10
confluence) grown overnight, washed 2x with PBS, and
placed in 0.5 ml Hepes-buffered saline containing the
CaP04~DNA co-pr.ecipitate for 15 min and then fed
with 10 ml medium. 'the medium is removed by aspiration
and replaced with 15% glycerol in PBS for 1.5-3 min.
The shocked cells are washed and fed with culture
medium. Until induction of MT-II-controlled expression,
the medium contains F12/DMEM21 1:1 with 10% FBS. A day
later, the cellLs are subjected to 1 mg/ml G418 to
provide a pool of G418-resistant colonies. Successful
transformants, also having a stable inheritance of the
desired plasmid, ate then plated at low density for
purification of-_ clonal isolates.
Assa for Production Levels of ASP
The transformants are assayed for production of
the desired protein, first as pools, and then as
isolated clones in mufti-well plates. The plate assay
levels are somewhat dependent on the well size - e.g.
results from 24 well plates are not directly comparable
with those from 96 well plates. Clones which are found
by plate assay to be producing the protein at a
1340580
..
-51-
satisfactory level can then be grown in production runs
in roller bottles. Typically, the levels of production
are higher when the scale up is done. However, there is
not an absolute correlation between performance in the
plate assay and in roller bottles - i.e. cultures which
are the best producers in the plate assay are not
necessarily the best after scale-up. For this reason,
typically 100-200 or more individual clones are assayed
by various screening methods on plates and 5-10 of the
highest producers are assayed under production
conditions (roller bottle).
Plate Assays
Pools of cells transformed with the various ASP
encoding plasmids were grown in multi-well plates and
then exposed to 5 x 10 5 to 1 x 10 4 zinc ion con-
centration to induce production of ASP. ASP assays were
conducted using Western blot employing immunoprecipation
with rabbit anti-human ASP polyclonal antiserum followed
by 125I protein A and autoradiogtaphy.
In more detail) semiconfluent monolayers of
individual cell lines growing in McCoy's 5A medium with
10% FBS were washed with phosphate-buffered saline (PBS)
and refed with McCoy's containing 10% FBS, 1 x 10 4
zinc chloride, and 0.25 mM sodium ascorbate. (Ascorbate
may be helpful in mediating the hydroxylation of proline
residues.) Twenty-four hours post induction, the cells
were washed with PBS and refed with serum-free McCoy's
containing the zinc chloride and ascorbate. After 12
hours, the conditioned media were harvested, made 20 mM
in Tris, pH 8, and filtered through nitrocellulose in a
BRL dot-blot apparatus. The nitrocellulose filter was
blocked in 50 mM Tris, pH 7.5, 150 mM NaCl (Tris/salt)
containing 5% nonfat dry milk, and then incubated with
.~-.. 1344 ~~0
-52-
1:5000 dilution of rabbit anti-human ASP polyclonal
antiserum in the blocking solution, washed several times
in the above Tris/salt, and incubated with 25 uCi of
125I protein A in blocking solution, washed, and
autoradiographed.
Most pools transformed with the ASP encoding
vectors did not produce ASP detectable in this assay.
However, a positive, ASP-secreting cell line, designated
A-38) was selected from pMT-Apo:gHS(HinfIlEcoRI)
transformants. In addition, certain pools from cells
transformed with pASPc-SV(10), designated ASP-I, or with
pASPcg-SV(10), designated ASP-F and ASP-G, produced
levels of ASP comparable to those produced by the cell
line designated D-4 described below (~2-5ug/ml).
Characterization of ASP Protein
The A-38 cells (supra) were grown to 25%
confluence in McCoy s 5A medium containing 10% FBS and
then induced with l0 4 M zinc chloride in McCoy s
containing 10% FBS and 0.25 mM sodium ascorbate. (Half
of the cells were also treated with 10 6 M
dexamethasone.) Twenty-four hours later, the cells were
washed with PBS and refed with RPMI medium containing
10% dialyzed FBS, 1 x 10 4 M zinc chloride, 0.25 mM
sodium ascorbate, and 0.5 mCilml 35S-methionine.
Eighteen hours later, the cell supernatant was
made 1 mM phenylmethylsulfonylfluoride and
immunoprecipit.ated with rabbit anti-canine ASP antiserum
using protein A as carrier. Half of the precipitated
protein was boiled in SUS-PAGE sample buffer) and the
other half eluted into 0.75% TritonTMX-100, 0.075% SDS,
0.75% 2-mercaptoethanol, 30 mM EUTA, 75 mM sodium
phosphate, pH 1 and incubated for 1 hr at 37~ with 0.5
units of endoglycosidase-F (endo-F). Endo-F treated and
A
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untreated protein fractions were subjected to SDS-PAGE
with the results shown in Figure 10. The Endo-F treated
fraction showed a 30 kd protein (lane F) as compared to
38 kd protein for the untreated (lane E). (Lane M
contains size markers, lanes A and B supernatants from
untransformed CHO cells, and lanes C and D supernatants
from A-38 cells untreated and treated with
dexamethasone) respectively.)
Supertransfection to Prepare D-4
An additional cell line, designated D-4, was
obtained by supertransfection of A-38 with a mixture of
pMT-Apo:gHS(Hi.nfI/EcoRI) (20 ug) and pSV2:GPT (1
ug). Semiconfluent monolayers of A-38 growing in
F12/DMEM21 with 10% FBS were co-transfected, as
described above. After 48 hours the cells were split
1:5 into F12/DMEM21 containing 10% FBS and HAT selection
drugs. After 17 days of HAT selection, the pool of
surviving resistant clones was screened for individual
clones producing high levels of ASP by the i~mmunofilter. .,....
screen method of McCracken) A.A.) et al Biotechnigues
(March/April l.984) 82-87. Briefly, the cells were
seeded onto plates at 100 cells per 100 mm dish in
F12/DMEM21, lO% FBS. After 5 days (when colonies
contain 50-200 cells each), the cells were washed with
PBS, refed with serum-free F12/DMEM21, and overlayed
with a sterile teflon mesh. On top of the mesh was
placed a nitrocellulose filter which was left in place
for 8 hr. The nitrocellulose was removed and treated as
an immunoblot, first with rabbit anti-canine ASP
polyclonal antiserum, then 125I protein A) followed by
autoradiography. Of approximately 2000 colonies
screened, two gave a detectable signal and one,
designated D-4) was shown to express the ASP gene at
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10-20 times the level of A-38, or at an amount
corresponding to an estimated 2-5 ug/ml ASP.
Characterization
The secreted ASP from the D-4 cell line was
isolated from the serum-free medium by affinity
chromatography and sequenced at the N-terminus on a
gas-phase microsequencer. Determination of a 16 amino
acid sequence showed complete homology with the
N-terminal portion of the protein isolated from lung
lavage; 70% oi: the total contained an N-terminal Glu
residue; the remaining 30% was clipped so as to contain
an N-terminal Val (position 2 relative to Glu). This is
the same composition as the isolated lavage protein.
Hydroxyprolines were present at positions 10, 13, and
16, indicating the ability of the cells to exhibit
post-translat:ional processing.
In addition, the protein secreted by D-4 along
with the secreted protein fraction from pool ASP-I
(supra) and from pool ASP-G (supra) was compared to
human proteinosis lung lavage protein using Western
blot. Serum-.free medium from induced cells was TCA
precipitated, treated (or not) with Endo-F and subjected
to SDS-PAGE in 12.5% gels. The gel was electroblotted
and dot-incubated with rabbit antihuman ASP polyclonal
antiserum followed by 125I protein A. The results are
shown in Figure 11.
Lanes A and F contain 1 ug alveolar
proteinosis protein before and after Endo-F digestion;
lanes B, C, and D represent media from D-4, ASP-I pool)
and ASP-G pool respectively untreated with Endo-F; lanes
G, H, and I represent proteins from these supernatants ""_"
treated with Endo-F. It is evident that Endo-F
,..
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treatment reduces the apparent molecular weight of a11
proteins, and results in mote discrete bands.
Production Runs
The superttansfected cell line containing
multiple copies of pMT-Apo:gHS(HinfI/EcoRI) (cell line
D-4) was used in a production level run in roller
bottles. An 850-cm square roller bottle was seeded with
a 10 cm dish containing 2 x 106 cells in 10% FCS, 15
mM Hepes, pen/strep, and glutamine. After the cells
reached confluence (2-3 days), they were washed 2 x with
PBS and replaced with 250 ml of F12/DMEM21, 10 mM Hepes
without FCS. The following day the cells were refed
with 250 ml of F12/DMEM21, 10 mM Hepes, 5 x 10 5 zinc
chloride, 10 6 M dexamethasone, and 0.25 mM
ascorbate. The cells were harvested every 2 days, spun
for ten minutes at 1000 rpm, and frozen at -20~C.
Production was 1-5 ug/ml/day, assayed by dot-blot
Western using polyclonal anti-canine ASP antiseta at
1:5000 dilution) as described above. Production drops
after about 14-17 days.
D.9. Activity of the ASP Components
The .ability of the isolated ASP components to
enhance the formation of lipid film at an air/aqueous
interface was assessed in vitro using the method
described by iHagwood, S., et al, Biochemistry (1985)
24:184-190. :Briefly, a preparation of phospholipid
vesicles with the appropriate ratio of test proteins is
added carefully in a small volume to the bottom of a
teflon dish containing aqueous buffer, a magnetic
stirrer, and a platinum plate suspended at the surface
of the buffer and attached to a strain gauge. Changes
in surface tension registered on the strain gauge are
- ~..
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recorded as a function of time upon starting the
stirrer.
10K proteins were added to the phospholipid by
mixing a chloroform solution containing them with a 2:1
v/v chloroform:methanol solution of the lipid. The
solvents were evaporated, and the solids hydrated in
buffer to obtain vesicles. 32K proteins can be added in
aqueous solution directly to a suspension of the
vesicles, and association with and aggregation of the
vesicles can be detected by turbidity measurements.
As reported by Hawgood) et al (supra), 32K
canine ASP was capable of aggregating phospholipid
vesicles and of enhancing the formation of film when
included in the phospholipid vesicles, when the
phospholipids were those obtained from the canine lung
surfactant complex. The activity of the proteins of the
invention is assessed using the same procedures for
measuring aggregation and film formation enhancement as
set forth in Hawgood.
Both the phospholipid preparation from canine
lung prepared as described above (300 ug) and a
synthetic mixture of phospholipids were used. The
synthetic phospholipid contained 240 ug of
commercially available DPPC and 60 ug egg PG) and is
much more reluctant to form films than is the natural
lipid. However, the test phospholipid was chosen so as
to dramatize most effectively the activity of the
proteins.
The 32K protein and the mixture of 10K ASP were
isolated from canine lung as described above. While the
addition of 60 ug of the 32K protein was able to
enhance film formation by the "natural" phospholipid
obtained from lung almost to the level exhibited by the
complex per se, it only moderately enhanced film
t
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formation using synthetic lipid. Similar results were
obtained for addition of 13 ug of the 10K protein
alone. However) when 13 ug of the 10K preparation was
incubated with the synthetic phospholipid vesicles prior
to the addition of 60 ug of 32K protein, film
formation occurred at a rate and to a degree comparable
to that of the natural complex per se. These results
are shown in Figure 12.
to
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