Note: Descriptions are shown in the official language in which they were submitted.
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EN~ ~ N ~ Tt~ PROTEIN r~ T~'rIONAI,
MODIFICAT~ON IN TRI~NSGENIC ~R~:~NT~R
The present invention relates to transgenic non-human
transgenic multicellular org~rlj ~ having ce]ls of
5 altered capacity for posttranslational modification. The
invention relates to the transgenic organisms, methods
for producing the orgAn;~ ~ and substAn~c produced by
the organisms. One aspect of the invention particularly
relates to optimizing transgenic production of a
substance by altering the protein posttranslational
modification capacity of cells in a transgenic organism.
In another aspect, the invention relates particularly to
substances produced by transgenic animals having altered
capacities for posttranslational modification. In yet
another aspect, the invention relates particularly to
altering the constitution of a physiological compartment,
tissue organ or body fluid in a transgenic organism by
altering posttranslational protein modification in cells
of the organism. In this regard the invention also
:relates to cells, cellular products, tissues, organs and
Eluids modified by altering the posttranslational protein
modification of cells in the transgenic organism. In a
preferred aspect, the invention specifically relates to
:improved maturation of proteolytically processed proteins
by expressing a processing protease in cells of a
t:ransgenic organism. In this aspect, the invention
relates especially to expression of PACE/furin a~d,
i'urther, to the maturation of precursor proteins involved
in blood coagulation and clot dissolution.
BACKGROUND OF THE INVENTION
The expression of cloned or purposefully a]Ltered
ge~es in transgenic organisms has been seen to hold great
promise for the production of substances by transgenic
"bioreactors" and for the production of improved ~n;~ls
and plants, among other applications. Obstacl~es to
realizing the promise have been encountered, however. In
plarticular, it has not been possible to obtain highly
efficient production of properly modified use~ul
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substances in transgenic animals. The present invention
relates to methods and transgenic organisms that overcome
the problem and to products produced by the transgenic
organisms, inter alia.
Many DNAs have been cloned and expressed in cells in
culture to produce heterologous proteins, peptides and
other substances. Several genes also have been
introduced into plants or animals to produce heterologous
proteins, peptides and other substances. Although it has
been possible, in general, to engender expression and
production of proteins and other substances in cells,
~n;~l s and plants by expressing cloned genes, the
production levels that have been obtained often have been
low and the posttranslational processing of proteins
produced this way generally has been incomplete or
inefficient. These difficulties particularly have
limited, or precluded, the use of animal and plant
bioreactors to produce proteins with the proper
posttranslational modifications. Low production levels
and low specific activities in cultured cells have been
attributed to the expression of limiting amounts of
particular enzymes necessary for the production of the
expressed protein in its properly modified form.
Attempts have been made to increase production of
properly modified proteins in cells in culture by
expressing a cloned gene to increase the amount of a
limiting enzyme activity in the cells. For instance,
expression of a transfected yeast Rex2 cDNA in baby
hamster kidney ("BHK") cells that expressed human Protein
C (~HPC~) from an amplified Protein C gene increased the
conversion of Protein C from the single-chain zymogen
form to the mature two-chain form (Foster et al.,
Biochemistry 30: 367-372 (1991).
In another example, PACE/furin expressed at high
levels by a transfected DNA in chinese hamster ovary
cells ("CHO") apparently increased proper cleavage of the
propeptide of the co-expressed Factor IX precursor
(Wasley et al., J. Biol. Chem. 268: 8458-8465 (1993) ) .
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But, the processing activity apparently engendered
by expression of the PACE/furin gene in these experiments
was difficult to discern and varied. In addition,
apparent increases in protease activity seemed tc~ cause
cell toxicity, cytopathic effect and alterations in
markers of cellular differentiation. Indeed, it h~s been
s~ggested that PACE/furin and similar processing enzymes
may be deleterious or lethal to cells when they are
inappropriately expressed, even in culture, as no1_ed for
lo instance by Schalken et al ., J. Clin . Invest. 80 :~ 1545-
1549 (1987), Ayoubi et al., J. Biol . Chem. 269 : 9298-9303
(1994) and Decroly et al., J. Biol. Chem. 269: 12240-
1~247 (1994). The potential for improving the cellular
production of substances in this way is overshadowed by
tlle adverse affects observed in culture.
In light of such results, this approach to improving
production of proteins, polypeptides and other substallces
in transgenic orgAn;~ c has not been favorably
considered. For one, expression of enzymes that: alter
posttranslational modification in cells thus far has ~een
carried out only in abnormal cultured cells. These cells
generally exhibit aberrant growth, which allows t:hem to
propagate indefinitely in artificial media. Largely,
such cells are derived from tumors or are the outcome of
transduction with immortalizing viruses. Different:iation
and growth factors particularly are altered in such
cells. Thus, the response of these cells to altered
posttranslational modification capacity does not indicate
the expected response of cells in a healthy organism.
Indeed, it has been thought that such cells are far
more tolerant than an intact organism of adverse effects
stemming from altered expression of enzymes afiecting
posttranslational modifications. Given the role of
posttranslational modifications in controlling enzyme
cascades and in modulating the activity of factors that
control growth, mitosis and differentiation, including
processes that generally occur only in intact organisms,
altering posttranslational modification capacities poses
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a greater risk of being severely deleterious to an intact
organism, even when it would not adversely impact a cell
grown in culture.
A developing organism is especially sensitive to
inappropriate expression, particularly expression that
causes cytopathic effect. For example, loss of a single
cell at a critical stage can hopelessly incapacitate or
abort a developing embryo.
In addition, an organism can be adversely affected
not only by intracellular effects but also by
physiological communication of the activity from
expressing cells to other parts of the organism. The
complex physiological processes involved in transport and
metabolism of circulating proteins, moreover, often has
the potential to amplify a tolerable intracellular effect
into an effect that is intolerably damaging to the
organism as a whole.
For instance, it has been noted that expression of
some enzymes that carry out posttranslational modifi-
cations can activate toxins. Altering a posttrans-
lational modification that activates a toxin thus,
potentially could amplify ordinarily tolerable toxin
levels, such as those produced by a mild infection, to
levels seriously harmful or lethal to an organism (Chiron
et al., J. Biol. Chem. 269: 18167-18176 (1994)).
A variety of enzymes involved in posttranslational
modification of proteins, moreover, are expressed
differently in the cells of different tissues in an
organism. In addition, expression of such proteins in
cells often varies greatly over the course of embryonic
development. This is the case, for instance, of the
subtilisin-like PACEs, such as PACE/furin, which appear
to carry out proteolytic steps in the maturation of some
proteins. In fact, it has been suggested that
posttranslational modifications, such as the proteolytic
cleavages mediated by these processing proteases, not
only participate in, but also have a regulatory role in
the process of cell differentiation (Zheng et al., ~.
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Neuroscience, 14: 4656-4673 (1994). Inappropriate
e~pression of these enzymes in cells of a developing
embryo thus could have broadly deleterious or le-thal
effects.
Concern about deleterious effects in vivo have made
it seem unlikely that posttranslational modificat.ions of
proteins can be much altered in cells of transgenic
organisms. Thus, the idea of changing the posl:trans-
lational modification properties of cells has not seemed
lo to be a promising way to improve the abil.ity of
transgenic organisms to produce useful substances. Given
the potential of such animals for the production of
useful substances, it therefore remains an important goal
to devise methods that overcome these obstacles to
15 developing efficient bioreactors.
ABBREVIATIONS AND D~ 1N l'l'lONS
The following abbreviations and terms are used in
this application in accordance with their me~; ng as
understood by those of skill in the arts to whiLch the
20 present invention pertains. The following definitions
are illustrative and should not be construed to limit the
scope of the invention, however.
Abbreviations
. B - OH: ~ - hydroxyl group
BHK: baby hamster kidney cells
CH0: (1) chinese hamster ovary cell~s; (2)
glycoside or sugar moiety, a common
protein posttranslational modification
ELISA: enzyme-linked immunosorbant assay
HC:158-420 heavy chain of human Protein C, containing
amino acids 158-420
HPC: human Protein C
HRP: horseradish peroxidase
Gla or GLA: gamma-carboxy glutamic aci~l, a
posttranslational modification of
proteins, notable among the vitamin K-
dependent proteins involved in blood
clotting, clot dissolution, bone, lung.
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GPI: glycosylphosphatidylinositol
KR: Lysl56-Argls7dipeptide released during maturation
of human Protein C
LC:1-155: light chain of human Protein C, cont~in;ng
amino acids 1 through 155
MAb: monoclonal antibody
PACE: paired basic amino acid cleaving enzyme
PACEM: mutated PACE
PAGE: polyacrylamide gel electrophoresis
PC: prohormone convertase(s)
PCR: polymerase chain reaction
PRO: 24-amino acid propeptide of protein C
SDS: sodium dodecyl sulfate
S-S: disulfide bond
SP: signal peptide
t-PA: tissue plasminogen activator
WAP: whey acidic protein
rHFIX: recombinant human Factor IX
rHPC: recombinant human Protein C
Illustrative Definitions
MATURATION: The process by which a polypeptide
encoded by an mRNA or a region of an mRNA is altered
during and after addition of successive amino acid
constituents on the ribosome. Ultimately maturation of
the polypeptide produces proteins in their physiological
forms. Thus, for instance proteolytic events that
convert precursor forms to processed forms constitutes
maturation. Glycosyl groups also are added during
maturation of a protein. Generally, in fact, any
posttranslational modification of a protein, as defined
herein, affects maturation.
FURIN: One of the prohormone convertases (PCs)
or paired basic amino acid cleaving enzymes, referred to
herein as PACE/furin.
MULTICELLULAR ORGANISMS: all multicellular animals
and plants whose cells become differentiated to form
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tissues; among animals, the metazoa, which incluc,les all
animals except protozoa.
POSTTRA~SI,ATIONAL MODIFICATIONS: Covalent
alterations to the 20 naturally occurring amino acids
that make up the polypeptide chain(s) of a protein in its
unmodified form. Posttranslational modifications, occur
in the protein backbone and the side c-h~;nc of the
constituent amino acids. Generally, posttranslational
modifications are made by formation or breakage of a
covalent bond via an enzyme-catalyzed reaction. Some
modifications occur before protein synthesis is co~lpl~te,
while others take place after the newly synthesized
protein is released from the ribosome. A great variety
of posttranslational modifications have ~een
characterized in a diversity of proteins. AlL such
modifications useful in the present inventia,n are
encompassed by -the use herein of the term
"posttranslational modification."
SUMMARY OF THE lN v~N~l~lON
Therefore, in accordance with one aspect of the
invention, there is provided a transgenic non-human
multicellular organism comprising cells having
incorporated expressibly therein a first polynucleotide
that encodes a first protein and a second polynucleotide
that encodes a second protein, wherein expression of the
second protein affects the post-translational
modification of the first protein in the cells.
In preferred embodiments of this aspect of the
invention the transgenic organism is a matazoan animal.
In other preferred embodiments it is a plant.
In certain preferred embodiments of this aspect of
the invention the polynucleotides and proteins are
expressed in specific cells of the organism. In some
preferred embodiments the first protein is secrete!d into
a bodily fluid of the organism. Also, in some preferred
embodiments the post-translational modification is
required for maturation of the first protein.
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In addition, in some preferred particular embodiments
the second protein affects a post-translational
modification selected from the group consisting of:
acetylation, ADP-ribosylation, acylation, ADP-
ribosylation, amidation, covalent attachment of a flavin,
covalent attachment of a heme, covalent attachment of a
nucleotide or a nucleotide derivative, covalent
attachment of a lipid or lipid derivative, covalent
attachment of phosphatidylinositol, cross-linking,
cyclization, disulfide bond formation, demethylation,
formation of covalent cross-links, formation of cystine,
formation of pyroglutamate, formylation, gamma-
carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation,
prenylation, racemization, selenoylation, sulfation,
transfer RNA-mediated addition of amino acids to
proteins, such as arginylation, and ubiquitination.
In a particularly preferred embodiment the
polynucleotides and proteins are expressed in specific
cells of the organism and the first protein is secreted
into a bodily fluid. Among preferred bodily fluids in
this aspect of the invention are blood, milk, urine and
saliva.
In another aspect of the invention in this regard in
preferred embodiments the first polynucleotide encodes a
precursor protein, the second polynucleotide encodes a
protease and the precursor protein is processed by the
protease, in cells of the organism. In this regard, in
certain preferred embodiments, the first protein is a
vitamin-K dependent protein or it is Factor VII, Factor
VIII, Factor IX, prothrombin, Factor X, Protein C, Factor
VII, Protein S, bone Gla protein, matrix Gla protein,
growth arrest specific protein 6, antithrombin III, t-PA,
erythropoietin, fibrinogen, immunoglobulin or albumin.
In further preferred embodiments of this aspect of
the invention the cells are secretory cells of mammary
glands and the first protein is secreted into milk of the
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g
organism. In this regard, in certain preferred
embodiments, the first polynucleotide encodes a precursor
protein, the second polynucleotide encodes a protease,
and the precursor protein is processed by the protease.
- 5Among preferred particular embodiments of thisiaspect
of the invention, the first protein is Factor VII, Factor
'~III, Factor IX, prolthrombin, Factor X, Protein C, Factor
'~II, Protein S, bone Gla protein, matrix Gla prDtein,
~rowth arrest specific protein 6, antithrombin III, t-PA,
loerythropoietin, fibrinogen, immunoglobulin or alblamin.
In particularly preferred embodiments in this regard
the protease is a paired basic amino acid cleaving
enzyme. In some especially preferred embodiments in this
:regard the first polynucleotide encodes Protein C. Among
15the most particularly preferred in this regard are
emboA; ents in which the protease is PACE/furin.
Among other preferred emho~; ?nts in this asp,ect of
the invention are those comprising a third polynucleotide
that encodes a third protein that affects protein gamma-
20carboxylation, wherein the first protein re~uires gamma-
carboxylation for maturation and the third protein
affects gamma-carboxylation of the first protein in the
cells of the organism. In this regard, embodiment in
which the first polynucleotide encodes Protein C and the
25protease is PACE/furin are especially preferred.
In another aspect, the invention provides a method
Eor producing a posttranslationally modified protein
comprising the steps of incorporating expressibly into a
transgenic non-human multicellular organism a first
30polynucleotide that encodes a first protein and a second
polynucleotide that encodes a second protein, expressing
the first and the second proteins in cells of the
organism whereby the second protein affects the post-
ltranslational modification of the first protein in the
35cells, and then isolating the posttranslationally
modified first protein from the organism.
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Preferred embodiments of this aspect of the invention
include those in which the polynucleotides are expressed
in specific cells in the organism.
In addition, preferred embodiments include those in
5 which the first protein is secreted into a bodily fluid
of the organism. Especially preferred fluids are blood,
milk, urine and saliva.
Also preferred are embodiments in which the first
polynucleotide encodes a precursor protein, the second
polynucleotide encodes a protease and the precursor
protein is processed by the protease in the cells.
Among the highly preferred embodiments of this aspect
of the invention are those in which the cells are
secretory cells of mammary glands and the first protein
is secreted into milk of the organism. In this regard,
embodiments in which the first polynucleotide encodes a
precursor protein, the second polynucleotide encodes a
protease, and the precursor protein is processed by the
protease in the cells are especially preferred. In some
highly preferred embodiments of this type, the first
protein is Factor VII, Factor VIII, Factor IX,
prothrombin, Factor X, Protein C, Factor VII, Protein S,
bone Gla protein, matrix Gla protein, growth arrest
specific protein 6, antithrombin III, t-PA,
erythropoietin, fibrinogen, immunoglobulin or albumin.
In additional, in particularly preferred embodiments of
this aspect of the invention the protease is a paired
basic amino acid cleaving enzyme. Very highly preferred
in this regard are embodiments in which the first
polynucleotide encodes Protein C. Also highly preferred
are embodiments in which the protease is PACE/furin.
Particularly highly preferred are embodiments in which
the first polynucleotide encodes Protein C and the
protease is PACE/furin.
Additional preferred embodiments of this aspect of
the invention include those in which a third
polynucleotide encodes a third protein which affects
protein gamma-carboxylation, wherein the first protein
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requires gamma-carboxylation for maturation and the third
]protein affects gamma-carboxylation of the first protein
in cells of the organism. Especially preferred in this
regard are those embodiments in which the first
]polynucleotide encodes Protein C and the protease is
PACE/furin.
In yet another aspect, the previd~ss a
]posttranslationally modified protein made by a process
Icomprising the steps of incorporating expressibly in~o a
transgenic non-human multicellular organism a first
polynucleotide that encodes a first protein and a second
polynucleotide that encodes a second protein, wherein
expression of the second protein affects the post-
translational modification of the first protein in cells
of the organism and then isolating the modified protein
from the organism.
Among preferred embodiments of this aspect of
invention are those in which the polynucleotides are
expressed in specific cells in the organism, those in
which the first protein is secreted into a bodily fluid
of the organism, particularly those in which the f]uid is
selected from the group consisting of blood, milk, urine
or saliva. Also preferred in this aspect of the
invention are embodiments in which the first
polynucleotide encodes a precursor protein, the second
polynucleotide enclode a protease and the precursor
protein is processed by the protease in the cells.
Especially preferred in this aspect of the invention
are those embodiments in which the cells are secretory
cells of mammary glands and the first protein is se;creted
into milk of the organism. In this regard, embodiments
in which the first polynucleotide encodes a precursor
protein, the second polynucleotide encodes a pra~tease,
and the precursor protein is processed by the protease in
the cells are highly preferred. Particular preferred are
those embodiments in which the first protein is Factor
VII, Factor VIII, Factor IX, prothrombin, Fac1-or X,
Protein C, Factor VII, Protein S, bone Gla protein,
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matrix Gla protein, growth arrest specific protein 6,
antithrombin III, t-PA, erythropoietin, fibrinogen,
immunoglobulin or albumin. Perferred proteases in this
aspect of the invention include the paired basic amino
5 acid cleaving enzymes. Especially highly preferred are
embodiments in which the first polynucleotide encodes
Protein C. Particularly highly preferred in this regard
are embodiments in which the protease is PACE/furin.
Other preferred embodiments of this aspect of the
invention include those in which a third polynucleotide
encodes a third protein which affects protein gamma-
carboxylation, the first protein requires gamma-
carboxylation for maturation and the third protein
affects gamma-carboxylation of the first protein in cells
of the organism. In this regard, especially preferred
are embodiments wherein the first polynucleotide encodes
Protein C and the protease is PACE/furin.
In yet another aspect of the invention there is
provided a product of a transgenic non-human
20 multicellular organism, wherein the organism is
characterized by having incorporated expressibly therein
a first polynucleotide that encodes a first protein and
a second polynucleotide that encodes a second protein,
wherein expression of the second protein changes the
25 post-translational modification of the first protein in
cells of the organism and thereby changes the natural
composition of a product made from the organism.
In certain preferred embodiments of this aspect of
the invention the polynucleotides are expressed in
specific cells in the organism. Also among preferred
embodiments in which the second protein or the first and
the second proteins are secreted into a bodily fluid of
the organism, particularly blood, milk, urine or saliva.
Especially highly preferred are embodiments wherein the
product is milk or is derived from milk, the cells are
secretory cells of ~r~ry glands and the first, the
second or both the first and the second proteins are
secreted into and alter the milk of the organism.
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~bodiments wherein the first polynucleotide encodes a
precursor protein,~the second polynucleotide encodes a
protease, and the precursor protein is processed ]~y the
pr~tease in the cells also are highly preferred.
Additionaly highly preferred embo~ nts are those in
which the second protein is a protein that a;efects
protein phosphoylation or a protease precursor protein,
cmd the phosphorylation protein or the protease is
secreted into and alters the composition of the m:Llk.
In certain particularly preferred embodiments of this
aspect of the invention the first protein is Factor VII,
Factor VIII, Factor IX, prothrombin, Factor X, Protein C,
]?actor VII, Protein S, bone Gla protein, matrix ~la
protein, growth arrest specific protein 6, antithrombin
:[II, t-PA, erythropoietin, fibrinogen, immunoglobulin or
~1 lbumin .
In certain especially preferred embodiments the
protease is a paired basic amino acid cleaving enzyme.
In further preferred embodiments the first
polynucleotide encodes Protein C.
Particularly preferred embodiments of this aspect of
the invention are those in which the protease is
~P~CE/furin.
Other objects, features and advantages of the present
invention will become apparent from the following
detailed description. It should be understood, however,
that the detailed description and the specific examples,
while indicating preferred embodiments of the invention,
are given by way of illustration only, since various
I-hanges and modifications within the spirit and scope of
the invention will h~Come apparent to those skilled in
the art from this detailed description.
BRIEF DESCRIPTION OF THE FIGURES
FIGURE 1 is a schematic representation of the
posttranslational modifications observed in human Protein
C ("HPC"), showing ~hat HPC initially is synthesized in
hepatocytes as a single-chain 461 amino acid polypeptide,
that following synthesis an 18-residue signal peptide
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("SP") is removed, N-linked glycosides ("CHO") are added
to Asn~, Asn~8, Asn3'3 and Asn329, ~-carboxyl groups are
added to nine amino-terminal proximal glutamic acid
residues ("GLA") by vitamin K dependent gamma-
carboxylase, the N-linked CHOs are further processed in
the Golgi, a ~-hydroxyl group ("~-OH") is added to Asp
and the 443 residue polypeptide is cleaved by a protease,
releasing a 24-amino acid propeptide ("PRO") and a
dipeptide Lys'56-Arg'57 ("KR"), and the correctly modified
and processed HPC is secreted into the circulating plasma
as a two chain protein composed of a light chain ("LC:1-
155") and a heavy chain ('IHC:158-420'') linked by a
disulfide bond.
FIGURE 2 is a schematic representation of
illustrative mWAP/HPC and mWAP/PACE constructs of the
examples. Exons are depicted by solid boxes,
untranslated mWAP exon sequences by closed boxes, 5'- and
3' flanking and intron sequences by lines. The sequence
of the junction between the mWAP promoter and PACE cDNA
is GGTACCaCACCATG. The 3' junction between the PACE
cDNA and the mWAP gene has the sequence
TTTATCTGggGGATCCC. The mWAP sequence is in bold letters,
the linker sequence is in small letters and the PACE
se~uence is in italics. Both constructs contain identical
5l-flanking mWAP sequences from position -4098 to
position +25.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the expression of
cloned genes to alter protein posttranslational
modification and improve thereby the production of
substances in transgenic non-human multicellular
organisms.
In one particular aspect, the invention provides,
surprisingly, that the posttranslational modification
capacity of cells in non-human multicellular transgenic
organisms can be altered without deleterious effect on
the organisms.
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Furthermore, the invention provides thal: the
expression of enzymes that carry out posttranslational
modifications can be used to alter not only
]posttranslational modification of proteins in cells of
~the organism, but also to alter, for instance!, the
composition of the cells, tissues, organs~ and
]physiological fluids of the organism and, in addition,
~th.e composition of products derived from the organisms.
In a particular aspect, the invention provides that
~expression of proteases can be used to alter the
composition of milk and, thereby, of whey and other-milk-
,~omponents. Particularly in this regard, the invention
~can be used to alter the composition and properties of
l~airy products. Thus, the invention can be used not only
to enhance production of substances in transgenic
bioreactors, but also to increase the usefulness of
products produced by the organism, such as dairy mi.lk and
milk-products such as that made from pigs, sheep and
goats.
Also, the invention provides, surprisingly,. that
:membrane proteins when expressed transgenically in
mammary epithelial cells, are secreted into mil:k, are
enzymatically active and can alter the composition of the
milk in situ.
In a particular embodiment of the invention, D~A that
encodes PACE/furin can be expressed in secretory cells of
~rr~ry glands of transgenic animals using a whey acid
protein gene promoter ("WAP"). The PACE/furin ex~ressed
in the cells is secreted into milk; although, ordinarily
it is a membrane-bound protein. Further, the secreted
- PACE/furin apparently reduces the amount of whey acid
protein in the milk.
In addition, the invention provides co-expres;ion of
PACE/furin and human Protein C in secretory cells of the
~ ry glands of transgenic animals that not only bri.ngs
about the aforementioned alterations of cells and mi.lk,
but also increases the amount of properly proteolyt.ically
processed Protein C in the milk.
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In accordance with the invention, therefore, a geneis introduced into a non-human multicellular organism to
alter protein posttranslational modification in cells of
the organism. In a preferred embodiment, the gene
expresses a protein that alters the composition of a
protein, tissue or fluid in the organism. In the latter
case, a particularly preferred embodiment of the
invention involves a gene that expresses an enzyme in the
mammary qlands of a mammal, secretes the enzyme into milk
and alters the composition of the milk.
In another preferred embodiment, two genes are
introduced into an organism. The first gene encodes a
first protein that undergoes posttranslational
modification. The second gene encodes an enzyme that
carries out a posttranslational modification of the first
protein. In this embodiment, expression of the gene
encoding the modifying enzyme alters maturation in the
organism of the first protein encoded by the first gene.
In another preferred embodiment of the invention,
production of a substance in a transgenic organism is
improved by identifying a slow step of posttranslational
modification that limits production of the protein in its
desired form; and then augmenting the capacity of cells
in the organism that produce the substance to carry out
the modification so that it no longer is the limiting
step. The same process may be reiterated two or more
times to further optimize production until production is
optimized.
In yet another preferred embodiment, the
posttranslational modification is altered in specific
cells or tissue in an organism. Also, the
posttranslational modification may be inducibly altered
in the cells in response to endogenous stimuli, such as
hormones, or environmental variables, such a feed
components.
In addition, in certain preferred embodiments,
altered posttranslational modiciation affects maturation
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of protein in cells that secrete the protein intc~ a
bodily fluid, such as milk, urine, blood or saliva.
Posttranslational modifications, proteins for
modification, methods and organisms of the invention aLre
illustrated in the following generalized discussion. It
~rill be appreciated that the discussion illustrates the
scope of the invention, but does not, indeed cannot,
cLescribe each and every possible embodiment. Thu~;, the
c'liscussion is descriptive not limitative.
~:LX.USTRaq~IVE POSTTl2~t-Sr-~TIONAL MODIFICATIONS
The posttranslational modification capacity of cells
in a transgenic organism can be altered by expression of
a, transgene encoding an enzyme that modifies a protein.
~any posttranslational modifications have been desc:ri~ed
a~nd characterized. They have been the subject of' many
reviews, such as the reviews by F. Wold,
E'osttranslational Protein Modifications: Perspectiv~es and
Prospects, pp. 1-12 in POSTTRANSLATIONAL CO~TALENT
~[ODIFICATION OF PROTEINS, B.C. Johnson, Ed., Acaclemic,
N~ew York (1983); Seifter et al. Analysis for protein
modifications and nonprotein cofactors, Meth. En~,ymol.
182: 626-646 (1990); Rattan et al., Protein synthesis,
posttranslational modifications and aging, in Ann. NoY~
A,cad. sci . 663: 48-62 (1992); Han et al., Post-
translational chemical modification(sJ of proteins" Illt.J. Biochem. 24(1): 19-28 (1992); Han et al., Po~t-
translational chemic~l modifications of proteins - III,
In~. J. Biochem. 25 t7): 957-970 (1994) and Han et al.,
In~-. J. Biochem. 24(9): 1349-1363 (1994).
The invention can be carried out for a variety of
~ posttranslational modifications, in principle including
any modification. Among the posttranslat:ional
modifications useful in the invention in this respec-t are
acetylation, acylation, ADP-ribosylation, amida,tion,
covalent attachment of a flavin, covalent attachment of
a heme, covalent attachment of a nucleotide or a
nucleotide derivative, covalent attachment of a lipid or
lipid derivative, covalent attachment of
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phosphatidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of
covalent cross-links, formation of cystine, formation of
pyroglutamate, formylation, gamma-carboxylation,
glycosylation, GPI anchor formation, hydroxylation,
iodination, methylation, myristoylation, oxidation,
proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer RNA-
mediated addition of amino acids to proteins such as
arginylation, and ubiquitination, to name just some of
the known posttranslational modifications of proteins
that may be altered in accordance with the present
inventlon .
The alteration of such posttranslational
modifications of a protein in cells of a transgenic
animal may be accomplished by changing the activity of
proteins and enzymes that affect the occurrence of or
efficiency with which a specific modification occurs, in
selected transgenic cells or tissues in which the given
protein is to be expressed.
Enzymes that catalyze many posttranslational
modifications have been identified, purified and
characterized, including enzymes that directly catalyze
the modification, those that are involved in the
metabolism and catabolism of the modification and those
that affect the activity of a cell for the modification
of a given protein. Genes that encode such enzymes can
and, indeed, have been cloned, and expressed in cells of
transgenic animals as described elsewhere herein.
The following list illustrates some of the enzymes
that may be used in accordance with the present
invention. Illustrative cDNAs and genomic clones
encoding such enzymes also are set out below. It will be
appreciated, however, that any given modification of a
protein generally occurs as a result of the activities of
a variety of enzymes and other proteins and substances in
a cell. Thus, a modification of a protein in a cell of
a transgenic animal may be affected, in accordance with
.
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1:he present invention, not only by transgenic expr,ession
of enzymes directly responsible for posttranslational
~odi~ication, such as those illustrated below, but also
by expression of proteins that act indirectly to affect
- 5 ~;uch modifying enzymes, and thereby alter
]?osttranslational modification of another protein
expressed in the cells.
acetylation/dea~tylation
Posttranslational modification of proteins in cells
]~y attaching or removing an acetyl group often is carried
out by acetyltrans~erases and deacetylases; e.g.,
sp~ermidine/spermine Nl-acetyltransferase encoded by the
l"SSAT" gene, and arylamine 0-acetyltransferase, encoded
]by the NATl and NAT2 genes.
~cylation
Acylation of proteins posttranslationally in cells
often is carried out by acylesterases; e.g., rat
proteolipid protein fatty acylesterase, rat myelin
associated nonspecific esterase, P.chrysogenum acyl
coA:6-aminopanicillanic acid acyltransferase, encoded by
the cloned penDE gene, and maize G3P acyltransferase,
which has been cloned as a cDNA.
ADP-ribosylation
Illustrative of the enzymes that effectuate or alter
posttranslationalADP-ribosylation are mono (ADP-ribosyl)
transferases A, C and D, poly (ADP-ribose) polymerase,
which is encoded by the PARP gene, dinitrogen reductase
activating glycohydrolase, human ADP-ribosylating factor,
encoded by the ARF2 gene, and cholera toxin subunit A,
encoded by the cholera toxin subunit A gene.
amidation
Among the enzymes that affect posttranslational
amidation of proteins in cells is peptidylglycine ~-
amidating monooxygenase, encoded by the PAM gene, which
has been cloned.
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cros~-linking
Enzymes involved in cross-linking reactions that
occur in proteins posttranslationally in cells include
cross-linking peroxidase and extensin.
disulfide bond for~ation
Enzymes of disulfide bond formation include, among
others, disulfide oxidoreductases or isomerases; e.g.,
human protein disulfide isomerase, which has been cloned
as a cDNA, rat protein disulfide isomerase, which also
has been cloned as a cDNA, E. coli PDI-like protein,
encoded by the dipZ gene, A. castellanii PDI-like
protein, which also has been cloned.
The formation of disulfide bonds in proteins within
cells has been the subject of many reviews, such as
Bardwell et al., Cell 74: 769-771 (1993).
gamma-carboxylation
Enzymes which effectuate posttranslational gamma-
carboxylation of proteins in cells include vitamin k-
dependent carboxylases, for instance human and bovine
glutamyl-carboxylase, which have been cloned as cDNAs.
Posttranslational gamma-carboxylation also is affected by
enzymes of vitamin K synthesis such as vitamin K
epoxidases, vitamin K epoxide reductases, NADH- and
dithiol-dependent vitamin K reductases. Gamma-
carboxylation of proteins in cells has been the subjectof much research and of numerous reviews, such as Furie
et al ., Blood 75 (9) 1753-1762 (1990).
glyco~ylation
The many enzymes that affect the glycosylation of
proteins in cells include glycosyltransferases such as
oligosaccaryl-, N-acetylglucoseaminyl-, fucosyl-,
galactosyl- and sialyl-transferases, glucosidases and
mannosidases, of which several cDNAs and genes cloned,
including, for instance the gene for human ~-
fucosyltransferase. Post-translational glycosylation of
proteins in cells has been the subject of many reviews,
including, for instance, Goochee et al ., BIO/Technol ogy
9: 1348-1354 (1991).
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GPI anchor form8tion
E n z y m e s i n v o 1 v e d i n f o r m i n g
~31ycosylphosphatidylinositol (GPI) anchors include GPI
r,ynthases, such as those encoded by the GPI synthase
class A, F, H cDNAs, which have been cloned.
hydroxylation
Enzymes that affect posttranslational hydroxylation
of proteins in cells include prolyl 4-hydroxylase,
including human brain tryptophan hydroxylase, whi~h has
iDeen cloned as cDNA, rabbit brain tryptophan hydroxylase,
which also has been cloned as cDNA, human cholest~rol 7
alpha-hydroxylase, which has been cloned as a cDNA, human
l~yrosine hydroxylase, the gene for which has been cloned,
human phenylalanine hydroxylase, the gene for which also
has been cloned, and lysyl hydroxylase.
iodination
Iodination of proteins in cells occurs primarily at
tyrosine, and the enzymes that affect this
posttranslationalmodification include thyroidperoxidase
encoded by the thyroid peroxidase ("TP0") gene, which has
]Deen cloned.
lipid modification
A variety of lipidation and other lipid-related
modifications occur posttranslationally in proteins in
cells that may be used in accordance with the present
invention. Some of these modifications are discussed
individually elsewhere herein. Lipid modifications have
been much studied and are described in a variety of
reviews such as Chow et al., Structure and biol~gical
effects of lipid modifications on proteins, Current
~pinion Cell Biol. 4: 629-63 (1992).
methylation and demethylation
Methylation commonly seen in proteins includes
methylation of carboxyl-, N- and 0- groups, carried out
Eor instance, by protein methyltransferases, such as
catechol 0-methyltransferases, illustrated by the N-
~nethyltransferase encoded by the P. aeruginosa gene pilD.
Methylation also has been studied in detail and has been
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the subject of a variety of reviews, including, for
instance, Clarke, S., Protein Methylation, Current
Opinion Cell Biol. 5: 977-983 (1993). A review of
methylation at carboxyl-terminal cysteine residues in
particular is provided by Clarke, S., Ann. Rev. Biochem.
61: 355-386 (1992).
oxidation
Posttranslational oxidation of proteins in cells is
affected by, among others, lipid peroxidases, for example
the lipid peroxidases of fatty acid oxidation.
proteolysis
A diverse array of proteases are known to carry out
proteolytic events of protein maturation in cells.
Specific proteolytic processing steps often are
associated with transport across membranes and out of
cell compartments, including excretion out of the cell.
In addition, proteolytic cleavage often is required to
activate proteins which initially are synthesized as
inactive precursor polypeptides. The use of such enzymes
is preferred in the invention. Particularly preferred
are proteases that carry out cleavages of secretion or
activation.
Enzymes involved in proteolytic posttranslational
modification of proteins include, to name just an
illustrative few, trypsin, chymotrypsin, E.coli signal
peptidase, the metallo-carboxypeptidases such as
carboxypeptidase B, the dipeptidyl aminopeptidases such
as cysteine-, serine or aspartate proteases, including
the human dipeptidyl peptidase IV encoded by the DPP4
gene and rat mitochondrial processing peptidase.
Proteolytic processing of proteins has been the
object of prolonged and careful study and it has been
reviewed extensively. One review, focusing on
proteolytic processing particularly as it relates to
physiological processes is Neurath, H, Proteolytic
processing and physiological regulation, TIBS 14: 268-271
(1989).
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phosphorylatio~L
Phosphorylation is a an important aspect of protein
aLctivity in cells and it is effectuated by a diversity of
enzymes, including phosphorylases, kinases such as
protein kinases A and C, including human TGF-~ receptor
type II kinase which has been cloned as a cDNA, human
]protein phosphatase 1 (PP1) G-subunit, which al:,o has
]been cloned as a ~DNA, A. thal iana kinase-associated
]protein phosphatase ("KAPP") encoded by a cloned cDNA, S.
typhimurium phosphatase encoded by the cobC gene.
prenylation
Prenylation of proteins posttranslationally in cells
is carried out by, among others, prenyltransferases,
including farnesyl-transferases and geranylgeranyl-
transferases, illustrated by human heme
~:farnesyltransferase which has been cloned as a cDNA,
]human CAAX farnesyltransferase which also has been cloned
as a cDNA, yeast farnesyl-transferases encoded by the
RLM1 and RAM2 gen~s, E. coli octaprenyl diphosphate
synthase encoded by the ispB gene, beta subunit of P.
sativum farnesyl-transferase which has been cloned as a
cDNA, geranylgerany] pyrophosphate synthase of C. annum,
1which also has been cloned as a cDNA. Protein
]prenylation is reviewed by, for instance, among others,
IClarke S, Ann. Rev. Biochem. 61: 355-386 (1992), which
focuses on isoprenylation of carboxyl-terminal cysteine
residues.
sulfatio~L
Posttranslational sulfation of proteins in cells
involves, for example, tyrosyl protein sulfotransferase
("TPST") and sulfate sulfatases, illustrated by human
placental estrogen aryl sulfotransferase, and human brain
estrogen aryl sulfotransferase, both of which have been
cloned, and rat hepatic aryl sulfotransferase IV, whLich
has been cloned. Sulfation of proteins is illustraLted by
thLe sulfation of Factor VIII. Posttranslational
sulfation of tyrosine in proteins is reviewed by Niehrs
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et al., Chemico-Biological Interactions, 93: 257-271
(1994).
ubiguitination
Isopeptide formation by the covalent attachment of
ubiquitin is carried out by ubiquitin-conjugating
isopeptidases, many of which are called E2s and are
illustrated by at least ten identified yeast UBC genes
and over 20 identified Arabidopsis UBC genes. Ubiquitin
activating enzymes also can be used in accordance with an
aspect of the invention to affect posttranslational
ubiquitination of proteins. Among these types of enzymes
are the E1 enzymes that activate the ~-carboxyl group of
ubiquitin prior to isopeptide bond formation.
Illustrative of enzymes of this type are those of the E1-
encoding UBA1 gene of yeast and those of the three genesidentified in Arabidopsis as E1-encoding genes. Also
useful in this regard are enzymes of deubiquitination and
substrate recognition factors involved in
unbiquitination. The metabolism and role of ubiquitin
and the ubiquitin protein degradation pathway are
reviewed in M. Hochstrasser, Current Opinion in Cell
Biology 4: 1024-1031 (1992).
Posttranslational modifications often are specific
to an amino acid, as noted in some places in the
foregoing illustrative discussion. Thus, the
posttranslational modification of specific amino acids in
a protein can be selectively altered in cells of a
transgenic organism by manipulating, in accordance with
the present invention, the efficiency with which an amino
acid-specific posttranslational modification is carried
out in cells or tissues of an organism. The fo,llowing
list of known amino acid-specific posttranslational
modifications of proteins illustrate this aspect of the
invention. It will be appreciated that point mutations
may be introduced into a given protein to avoid or
engender one or more of these modifications at a specific
amino acid residue in a protein.
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amino terminus: formylation, acetylation,
pyroglutamate formation, N-tel ; n~ 1 arginylation,
~ myristoylation.
arginine: N-methylation, ADP-ribosylaLtion,
- 5 phosphorylation.
asparaginQ: glycosylation, ~-hydroxylation,
cleamidation, ADP-ribosylation.
~ spartic acid: ~-hydroxylation, ~-carboxyliltion,
phosphorylation, methylation, racemiziation
lo l'isomerization).
carboxy terminus: phosphatidylinor,itol
clerivatization, glycine-amidation.
cysteine: cystine formation, selenocysteine
formation, heme linkage, myristoylation, prenylation,
~DP-ribosylation, heme addition, palmitoylation,
oxidation.
glutamic acid: ~-carboxyglutamate formation, C-
methylation, ADP-ribosylation.
glutamine: deamidation, cross-linking, ~Loylut:amate
formation.
histidine: methylation, diphthamide formation,
phosphorylation, flavin addition.
lysine: N-acetylation, N-methylation, oxidation,
hydroxylation, cross-linking, biotinylation,
ubiquitination, hypusine formation.
methionine: selenomethionine formation.
phenyl~l~n;ne: hydroxylation.
proline: hydroxylation, N-terminal methylation.
serine: phosphorylation, glycosylation, acetylaLtion,
phosphopantetheine addition.
threonine: phosphorylation, glycosylation.
tyrosine: iodination, phosphorylation, o-sulfation,
flavin linkage, nucleotide linkage.
Any of the foregoing modifications may be altered in
transgenic animals in accordance with the preslently
disclosed invention. Any of the foregoing enzymes, and
genes that encode them, may be used in accordance with
the invention herein disclosed to alter posttranslational
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modification of proteins expressed in transgenic
org~n-~c.
ILLU8TRATIVE PRO~lN~ T~AT MAY BE PRODUCED MORE
EFFI~~ ~Y BY ALTERING POSTT~ T~IONAL MODIFICATION OF
CELLS IN A TRANSGENIC ORGANISM
The invention is applicable generally to any protein
produced in cells or tissues of a transgenic organism.
As noted above, the invention may be used to alter the
efficiency of naturally occurring posttranslational
modifications of a protein expressed in cells of a
transgenic organism. It also may be utilized to
manipulate posttranslational modification of a site
introduced into a protein by point mutagenesis, or by
more dramatic alteration of amino acid sequence of a
protein.
Preferred embodiments of the invention in this regard
relate to enzymes that perform posttranslational
modifications of serum proteins, particularly the serum
factors that play a role in hemostasis, blood clotting
and the dissolution of blood clots. Especially preferred
in this regard are the vitamin K-dependent proteins.
Among the proteins of particular interest in
accordance with this aspect of the invention are Factor
VII, Factor VIII, Factor IX, Factor X, Factor XIII,
fibrinogen, prothrombin, plasminogen activators, such as
t-PA, plasminogen, Protein S and Protein C, bone Gla
protein, matrix Gla protein, growth arrest specific
protein 6, antithrombin III, erythropoietin,
immunoglobulins and albumin.
Particularly preferred alterations in the
posttranslational modification of these proteins in
transgenic organisms in accordance with the invention in
this regard include enzymes for proteolytic processing,
~-carboxylation, ~-hydroxylation, sulfation and
glycosylation.
Especially preferred in this regard are enzymes for
proteolytic processing, glycosylation, sulfation and
~-carboxylation. In some aspects of the invention,
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-- 27 --
proteolytic processing particularly especially is
preferred.
In this regard, enzymes of the PACE fami:Ly are
preferred, especially PACE/furin. PACE/furin is
- 5 described, for instance, by Roebroek (1986) cited abc~ve.
PACE/furin has been shown to process pro-von Willebrand
factor, pro-nerve growth factor, proalbumin, and
complement protein pro-C3, as described in Wise et al .,
Pr-oc. Nat'l Acad. sci., U.S.A. 87: 9378-9382 (199O), Van
de Ven et al., Nol. Biol. Rep. 14: 265-275 (199O),
Br~hn~h~n et al., J. Cell Biol. 111: 2851 - 2859 (199O),
Brennan et al ., J. Biol. Chem . 266 : 21504-21508 (1991)
and Misumi et al., Biochem. Biophys. Res. Comm. 171:
3~64 - 3568 (199O). Moreover, PACE/furin, and PACE4 as
well, likely also process a variety of other proteins,
including growth factors~ receptors, viral glycoproteins
and coagulation factors, as indicated by their
~disseminated expression in many cell-types. Thus,
PACE/furin may be expressed in cells of transgenic
organisms to improve processing and maturation of these
proteins, among others.
Notably, P1 and P4 arginine residues have been
identified as amino acids important for efficient
propeptide cleavage by PACE. For instance, see Derian et
al., J. Biol . Chem. 264 : 6615-6618 (1989).
Guidanceregardingexpressionconstructsspecifically
in this regard is provided by Wasley (1993) and others,
which report on genes encoding PAC enzymes expressed in
cultured cells. The DNAs and cloning methods used to
make PACE expression constructs for use in cultureaLcells
~ can be adapted to the engineering of vectors for
~expression in transgenic organisms. It will be
~esirable, of course, to use promoters and other
regulatory signals that will target expression of the
PACE transgene to particular cells in an organism as
described elsewhere herein.
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ILLUSTRATIVE A~ OF C~N~ ~UCTS FOR TRANSGENICEXPRESSION USING R~u~ MRINANT DNA ~ ~NlQUE8
DNAs for producing transgenic organisms can be
obtained by conventional methods of recombinant DNA
cloning. A general discussion of well known t~chni ques
for making suitable DNAs in this regard is provided by
Maniatis et al., MOLECULAR CLONING, A LABORATORY MANUAL
(Cold Spring Harbor Laboratory, 1982) and Sambrook et
al., MOLECULAR CLONING, A LABORATORY MANUAL, Second
Edition, Vol. 1-3 (Cold Spring Harbor Laboratory, 1989).
Examples of DNA constructs that have been introduced into
transgenic animals for systemic or tissue-specific
expression are provided in GENETIC ENGINEERING OF
ANIMALS, A. Puhler, Ed., VCH Verlagsgesellschaft,
Weinheim, New York (1993).
DNA coding for a given protein can be fused, in
proper reading frame, with appropriate regulatory
signals, as described in greater detail below, to produce
a genetic construct which then may be amplified, for
example, by propagation in a bacterial vector or by PCR,
for subsequent introduction into a host organism,
according to conventional practice.
Generally, the genes will be linked operatively to
the cis-acting signals necessary for expression in a
desired manner in an organism. Particularly preferred in
this regard are promoters and other cis-acting regulatory
elements that provide efficient expression in a
particular cell-type. In the following discussion, the
term promoter is used broadly and extends to cis-acting
elements such as enhancers that may not always be
considered in a strict technical sense, promoters.
The cis-acting regulatory regions useful in the
invention include the promoter used to drive expression
of the gene. Particularly useful in the invention are
those promoters that are active specifically in given
cell-types. In this regard, preferred promoters are
active specifically in cells that secrete substances into
bodily fluids. Especially useful in this regard are
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cells that can secrete substances into milk, urine, blood
and saliva. Notably, therefore, cells of mammary gland,
for instance, urinary tract, liver and salivary gland are
especially useful.
- 5 Very particularly useful promoters in this regar~ in
some aspects of the invention are active in mammary
tissue. Particularly useful are promoters that are
specifically active in mammary tissue, i.e., are more
active in mammary tissue than in other tissues under
lo physiological conditions where milk is synthesized. Most
preferred in this regard are pr~moters that are both
specific to and efficient in mammary tissue By
"efficient" is meant that the promoters are strong
promoters in ~rr~ry tissue and support the synth~sis of
large amounts of protein for secretion into milk.
Among such promoters, the casein, the lactalbumin and
the lactoglobulin promoters are preferred, including, but
not limited to the ~ and ~-casein promoters cand the
~-lactalbumin and ~-lactoglobulin promoters. Pre!ferred
among the promoters are those from rodents (e.g., mouse
and rat), rabbits, pigs, sheep, goat, cow and horse,
especially the rat ~-casein promoter, the sheep ~-
lactoglobulin promoter and the rat and goat ~-lactalbumin
promoters.
The most preferred promoters are those that regulate
a whey acidic protein (WAP) gene, and the most preferred
WAP promoter is the murine WAP promoter. A most highly
preferred promoter is the 4.2 kb Sau3A - Rpnl promoter
fragment of the mouse whey acidic protein promoter. It
has been found that this fragment is highly effective in
directing the production of high levels of a protein in
the milk of a transgenic animal.
Among the sequences that regulate transcription that
are useful in the invention, in addition to the promoter
sequences discussed above, are enhancers (which may be
considered part of the promoter), splice signals,
transcription termination signals and polyadenylation
signals, among others. Particularly useful regulatory
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- 30 -
sequences increase the efficiency of mammary cell
specific expression of proteins in transgenic An;~-l5.
Especially useful in this regard are the other
transcription regulatory sequences of genes expressed at
high levels in mammary cells, such as the ~ - and ~-
casein genes and the ~-lactalbumin and ~-lactoglobulin
genes mentioned above. Preferred sources for regulatory
sequences in this regard are rodents (mice and rats),
rabbits, pigs, sheep, goat, cow and horse. Exemplary of
preferred regulatory sequences are those associated with
the rat ~-casein gene, the rat and goat ~-lactalbumin
genes and the sheep ~-lactoglobulin gene, respectively.
Among the regulatory sequences most preferred for use
in the present invention are those that encode hormone-
induced milk proteins that are expressed only during
pregnancy and lactation, such as those associated with
whey acidic protein genes. Particularly preferred in
this context are regulatory sequences of the murine whey
acidic protein gene.
Among the sequences that regulate translation, in
addition to the signal sequences discussed above, are
ribosome binding sites and sequences that augment the
stability of RNA. Especially useful are the translation
regulatory sequences of genes expressed at high levels in
mammary cells. For instance, the regulatory sequences of
the ~ - and ~-casein genes and the ~-lactalbumin and
~-lactoglobulin genes are preferred, especially those
from rodents (mice and rats), rabbits, pigs sheep, goat
cow and horse. Even more particularly preferred are the
regulatory sequences of rat ~-casein and the sheep ~-
lactoglobulin genes.
In another aspect, inducible promoters are preferred,
particularly those that can be induced by environmental
variables, such as food components. Notable in this
regard are metallothionien promoters, which may be
induced in animals by incorporating an appropriate metal
inducer in feed. Metallothionien promoters have been
used to express osteoglycin, epithelin, and bovine
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-- 31 --
oncostatin M in transgenic ~ni ~l ~, for instance, and
~[alik et al ., Molec. Cell . Biol . 15: 2349-2358 ~1995)
provides a review of promoters that can be used :Eor
t:issue-specific or inducible expression or both. Also
preferred are milk gene promoters which may be indulced by
]actogenic or steroid hormones.
Such promoters, useful in the invention for t:issue-
~:pecific expression or inducible expression or bo-th,
i.nclude albumin promoters for liver-specific expression,
ctl-antitrypsin promoters for liver-specific expression,
~:eratin-14 promoters for expression in epithelial cells
an~ basal cells, RI~ promoters for expression especially
i.n neurons, the insulin-1 promoters for expressi.on in
pancreatic ~-cells, the LcK promoters for express:ion in
t:h~mocytes, the metal-induced metallothionien promoters
and the hormone-induced milk protein gene promoters for
expression in the mammary gland.
It will be appreciated that there may be additional
r.egulatory elements that aid the production of transgenic
organisms that express high levels of a protein. Some of
t:hese signals may be transcriptional regulators, or
signals associated with transport out of the cell. Other
signals may play a role in efficient chromosomal
integration or stability of the integrated DNA.
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- 32 -
ILLU8TRATIVE ORGANISMS IN W~ICH POST~N~T~TIONA~
MODIFICATION MAY BE ALTERED
Non-human multicellular organisms suitable for
practicing the invention include plants and animals.
Particularly preferred are mammals, other than humans,
for producing substances in milk. All lactating animals,
that is, all mammals, are suitable for use according to
the present invention. Preferred mammals include mice,
rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs,
sheep, goats, cows and horses. Livestock and research
~n; ~1 ~ particularly are preferred. Among livestock,
cows, goats, sheep and pigs are preferred, especially
sheep and pigs. Among research animals are the foregoing
and dogs, cats, hamsters, rabbits, rats and mice. Among
these, hamsters, rats and mice are particularly
preferred. Rats and mice are especially preferred in
this regard.
ILL~STRATIVE ~N~T- II~ ~O~S FOR MARING TRAN8GENIC
0~ ~NT SMS
Genes may be introduced into an organism in
accordance with the invention using stAn~Ard, well-known
t~-hn; ques for the production of transgenic organisms.
These evolving techniques have been the subject of
numerous reviews, including, for instance, TRANSGENESIS
TECHNIQUES, Murphy et al., Eds., Human Press, Totowa, New
Jersey (1993) and G~N~llC ENGINEERING OF ANIMALS, A.
Puhler, Ed., VCH Verlagsgesellschaft, Weinheim, New York
(1993).
For instance, DNA can be introduced into totipotent
or pluripotent stem cells by microinjection, calcium
phosphate mediated precipitation, liposome fusion,
retroviral infection or by other means. Cells containing
the heterologous DNA then can be introduced into cell
embryos and incorporated therein to form transgenic
organisms.
In a preferred method, developing cells or embryos
can be infected with retroviral vectors and transgenic
animals can be formed from the infected embryos.
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-- 33 --
In a highly preferred method, DNAs are microinjected
into embryos, preferably at the single-cell stage, and
the embryos are developed into mature transgenic animals.
Double and other multiply-transgenic animals cal be
made by introducing two or more different DNAs into the
genomic DNA of a multi cellular organism using t~chn;ques
described above. The DNAs may contain the s,~me or
different promoters and other expression-controlling
signals. The cDNA or genomic DNAs encoding first and
second proteins may be in separate or in a single
construct. Furthermore, multiply-transgeinc organisms
also can be made breeding. For instance, two singly-
transgenic organisms can be crossed, using appropriate
generally well known breeding tec-h~;ques, to genexate
double-transgenic offspring having the transgenes of both
the parents. Successive breeding can be u;ed to
introduce additional transgenecs, as well.
CE~LS, TI8SUES, OR~N~, FL~IDS AND OTHER PHYSIOI,OG~CA~
COMP~h,r~ FOR E~PRESSING TRANSGENES I~STRATIVE OF
T~E ~Nv~r.,~ON
Generally, any cell or tissue of an organism may be
used in accordance with the present invention.
All-ty~pe specific expression that isolates altered
posttranslational modifications to a particular
physiological compartment is preferred in some aspects of
the invention. By compartment is meant a physiologically
and/or physically distinct aspect of an organism that
localizes the alteration and insulates the organism as a
whole from potentially undesirable systemic affects.
Preferred, in this regard, are cells and tissues that
secrete substances into bodily fluids. In this regard,
cells and tissues that secrete proteins into blood,
saliva, urine and milk are highly preferred. Those that
secrete proteins into urine or milk are very highly
;preferred. Among these, - ~ry epithelial cells that
secrete proteins into milk are especially preferred.
~Also preferred are cells that secrete proteins into
urine.
CA 02220109 1997-11-03
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-- 34 --
ILLIJSTRATIVE PRODUCTS OF THE lNv~ LoN
It will be appreciated that the invention can be used
to improve maturation of proteins in transgenic metazoic
organisms. By altering maturation of proteins in cells
of transgenic organisms the invention can be used to
affect the production of substances in the organism. The
production of substances can be directly affected by
altering posttranslational modification or it can be
indirectly affected. Thus, in the latter case, for
instance, the invention can be used to alter the activity
of an enzyme in a cell to influence the production of
non-proteinaceous, as well as proteinaceous, products of
cell metabolism and catabolism. For instance in this
regard, the invention can be useful to alter
phosphorylation of a protein posttranslationally and
thereby alter its activity in converting a substrate to
a product. In this way, altering posttranslational
modification in accordance with the invention can be used
to alter the production of any substance produced by
cells in a transgenic organism.
More particularly, the invention can be used to alter
protein maturation in a transgenic organism. For
instance in this regard, the invention can be used to
alter the posttranslational modification of milk
proteins. In a particular example of this aspect of the
invention, the phosphorylation and proteolytic processing
of caseins in milk can be altered by means of the
invention.
It will be appreciated that altered posttranslational
modification of this type can be used, in a further
aspect of the invention, to produce novel products in
transgenic organisms. For instance, by means of the
present invention the posttranslational modifications of
caseins, known to play an important role in determining
the qualities of milk and in making milk-derived
products, may be altered. Thus, the aforementioned
alteration of phosphorylation and proteolytic processing
of caseins in milk can be used to produce transgenic
CA 02220109 1997-11-03
W~ 96J34966 PCT~US~6~06~2
-- 35 --
~ that provide novel milk, milk protein and ~ilk
products.
In addition, the present invention also can be llsed
to produce the enzymes that carry out posttranslational
~ 5 modification, and ~o produce compositions, such clS ~ilk
and milk-derived products, that contain the enzymesO
It will be appreciated in this regard, therefore,
that the present invention can be applied generaLlly to
providing novel foods and f OOa products.
A~J ILLUSTRATION: PACE/F~IN--AU(.~ v ~aT~JRaTION OF HPC
IlJ TRANSGENIC MOUSE MII K
Vitamin K-dependent coagulation proteins repre~ent
one class of proteins that generally are synthesized as
precursors and then undergo a series of proteolytic
cleavages, among other events, that ultimately produce a
fully matured protein. Proteases are responsible, in
part, for maturation of precursor forms of coagula~ion
proteins. Proteolytic processing of precursor forms of
coagulation proteins are one type of posttranslational
modification that can altered in transgenic org~n' cr~ to
improve production of a substance in accordance with the
present invention. This aspect of the invention is
exemplified by the expression of PACE/furin in ~lAr~ry
epithelial cells to increase production of mature HPC in
milk of transgenic mice.
Efficient transgenic expression of vitamin K-
dependent coagulation proteins with complete
posttranslational modification and processing has
rem~;ne~ a difficult challenge, despite some promising
results. Similar obstacles have been observed for the
expression of several of these proteins in a number of
conventional cell lines.
For instance, it has been observed that HPC
transfected cells in culture do not completely process
the single chain of HPC. In fact, human liver cells
apparently do not completely process single-chain t:o two-
chain HPC in vivo. Observers have reported th,at the
amount of precursor processed to the mature two-chain
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form was about 85-95~ in the human liver, about 50% in
liver-derived HepG2 cells, 30% in transfected baby
hamster kidney cells and 80% in human kidney 293 cells.
Chinese hamster ovary (CHO) and C127 mouse fibroblast
cell lines not only did not process the protein to the
mature form at all, but even secreted rHPC with the
propeptide attached, as reported by Suttie, J. W.,
Thromb. Res. 44: 129-134 (1986) and Yan et al., Trends in
Biochemical sci. 14: 264-268 (1989).
rHPC secreted into the milk of transgenic animals was
reported to be a mixture of pro- and single chain rHPC
(Drohan et al., Transgenic Res. 3:355-364 (1994), and
Velander et al., PNAS, U.S.A. 89:12003-12007 (1992)).
It has been suggested that varying inefficiency of
the endoproteolytic maturation of rHPC in different cells
may be due to saturation of the endogenous processing
protease by rHPC production in the cells.
Expression of an appropriate protease might improve
the endoproteolytic processing of rHPC in cells of a
transgenic organism. Among the proteolytic processing
enzymes mentioned above are the subtilisin-like serine
proteases called prohormone convertases (PC) or paired
basic amino acid cleaving enzymes ("PACE"). These
proteins can be expressed in transgenic organisms in
accordance with the invention to augment the proteolytic
maturation of precursor forms of coagulation proteins.
As a first step in making transgenic mice with
improved processing of HPC in mammary glands, DNAs were
constructed in which cloned genes encoding HPC and
PACE/furin were placed under expression control that
directs expression of HPC and PACE/furin to ~ ry gland
epithelia cells.
A preferred promoter for this purpose is the 4.2 kb
Sau3A - Rpnl fragment of the mouse whey acidic protein
promoter. Other promoters and promoter fragments may be
used to express proteins in mammary epithelial cells and
other cells in like fashion.
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-- 37 --
In a preferred embodiment in this regard hu~an
c~enomic DNAs encoding Protein C are employed for
expression of HPC. Among the most highly preferred h~man
genomic DNAs is the fragment of the human Protein r gene
- 5 beginning 2l basepairs upstream of the Protein C start
codon and ending at the NheI site in the 3' end of the
]?rotein C gene, which is 9.4 kb long and contains
regulatory elements that engender high expression of
human Protein C in m~lk. It will also be appreciated that
1:he 9.4 kb Protein C fragment described is merely one
highly preferred DNA in this regard. Of course, many
other DNAs also may be employed in much the same ~ay.
For expression of PACE/furin, any of a vari~ty of
]?ACE/furin DNAs ~hat have been made and characterized can
be employed. DNAs that encode PACE/furin and are
suitable for use in this regard are described, for
instance, in van de~len et al., Molecular Biology R,eports
L4: 265-275 (l990) and Wasley et al., J.Biol.Chem. 268:
~3458 (1993).
A DNA containing a promoter is ligated to a DNA
encoding Protein C and to a DNA encoding PACE/furin. The
DNAs are arranged so that expression of the proteins is
driven by transcription from the promoter. The ligation
products can be inserted into an appropriate vector for
propagation. Finally, DNA for injection is purified and
ased to produce transgenic mice. All of the t~chn;ques
involved in these processes, which are described further
above~ and in references cited elsewhere herein, are ~ell
]known and routinely practiced by those of skill in the
art.
Pups developed from the injected embryos c:an be
tested for the presence of the transgenes using standard
t~c-hn;clues. For instance, the presence of transgenes in
the animals can be determined by Polymerase Chain
Reaction ("PCR") using primers specific for the injected
PACE/furin and HPC DNAs in genomic DNA obtained from a
small piece of tail tissue.
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Likewise, expression of Protein C by lactating micecan be assayed using standard techn;ques, such as western
blots, ELISAs, assays of procoagulant activity and the
like described in the literature pertaining to transgenic
expression of HPC, inter alia, as discussed elsewhere
herein.
In particular, the Protein C contained in milk can
be purified by known means without unduly affecting
activity. One suitable approach to purification in this
regard is immunoaffinity chromatography. Alternatively,
the expressed Protein C can be isolated from the milk by
other conventional means using methods described by, for
instance, Drohan et al., pages 501-507 in ADVANCES IN
BIOPROCESS ENGINEERING, Galindo et al . Eds., Kluwer
Academic, Netherlands (1994).
It is preferred that Protein C produced in milk
pursuant to the present invention should be isolated as
soon as possible after the milk is obtained from the
transgenic mammal, thereby mitigating any deleterious
effect(s) on the stability of the protein.
The degree of proteolytic maturation of the expressed
Protein C can be assessed by separating the whey proteins
from whole milk, resolving the proteins by size by
SDS/PAGE and blotting the proteins onto a filter and
probing the filter with an antibody that recognizes all
forms of HPC to visualize rHPC in the milk.
rHPC in milk from nontransgenic mice, mice transgenic
for HPC, and mice transgenic for HPC and PACE/furin can
be compared side-by-side in blots to assess directly the
effect of PACE/furin expression on conversion of
recombinant preproHPC to proHPC, proHPC to mature rHPC,
and single-chain rHPC to two-chain rHPC. The observed
forms of rHPC can be sequenced to determine if
PACE/furin-mediated cleavage sites correspond to the
sites of natural processing. Activity assay also can be
applied to assess the fidelity of rHPC produced in
transgenic mice with natural HPC.
=
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-- 39 --
The foregoing i~ illustrated in yet more detail in
1:he specific examples below. It will be appreciated that
- 1he illustrations are not limitative and that the same
paradigm can be used to alter posttranslational
- 5 processing of any protein in accordance wit:h the
:invention.
Thus, for instance, other enzymes that can properly
cleave rHPC in cells in a transgenic animal can be used
:in the same way as PACE/furin, and other proteins can be
expressed and their processing augmented in transgenic
;ln; ~1~ in the same way that proteolytic maturat.ion of
]IPC is augmented above.
The present invention is further described by
reference to the following illustrative examples.
As a demonstration that the processing of complex
heterologous proteins in organs of transgenic anima.ls can
]De improved, a DNA encoding human PACE/furin has been
expressed in mammary gland cells of transgenic m.ice to
.increase the production of properly processed two-chain
]~Pc and to alter the composition of mouse milk and mouse
whey.
~EXAMPLE 1 Construc~ion of WAP/HPC and WAP/PACE DNAs and
eneration of HPC a~d HPC/PACE Double-Transgenic ~ice
Mice transgenic for an intact HPC gene and eith.er (i)
an intact PACE cDNA or (ii) a mutated PACE cDNA encoding
an enzymatically inactive protein (I'PACEM") were produced
using standard methods by co-injecting constructs
contA;n;ng the two cDNAs into mouse embryos. T h e
]posttranslational processing of human Protein C is shown
diagramatically in Figure l. Illustrative DNA constructs
of the present examples are depicted schematically in
:Figure 2.
To target expression of PACE and PACEM to mam~ary
glands, the cDNAs were placed under expression cont.rol of
a mouse WAP promoter. The promoter is well known a.nd has
:been used to direct expression and secretion of rHE~C into
:mi.lk in transgenic mammals, as described in, for
i~stance, Drohan e~ al., Transgenic Res. 3: 355~364
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- 40 -
(1994) and Paleyanda et al., Transgenic Res. 3: 335-343
(1994).
Intronless WAP/PACE cDNAs were used to express low
amounts of PACE. Several groups have documented the
inefficient expression of intronless transgenes in the
mammary gland (Whitelaw et al., Transgenic Res. 1: 3-13
(1991) and Hennighausen, L., Protein Expr. and Purif. 1:
3-8 (1990).
The WAP/HPC DNA construct comprised a 4.1 kb mouse
whey acidic protein (WAP) promoter (33) and a 9 kb HPC
gene with 0.4 kb 3' nontranslated sequences. It was
constructed from readily available DNAs using well-known
techniques, (29) as described in Drohan et al.,
Transgenic Res. 3: 355-364 (1994) and Hogan et al.,
MANIPULATING THE MOUSE EM8RYO, Cold Spring Harbor Press
(1986).
To make WAP/PACE and WAP/PACEM, a 4.1 kb Sau3A-KpnI
WAP promoter fragment, and BamHI-EcoRI fragment
containing 1.6 kb of 3' WAP nontranslated sequences were
cloned into pUC19; generating plasmid pHL215. A 2.47 kb
EcoRI-SalI fragment, comprising the 794-codon human PACE
coding sequence and 74 bases of 3'untranslated sequence,
was excised from a PACE cDNA clone (Wise et al., Proc.
Nat'l Acad. sci., USA 87: 9378-9382 (1990)) and treated
with Mung bean nuclease. PACE or mutated PACE Ser26l to
Ala ("PACEM"), fragments then were inserted into SmaI-
digested pHL215 plasmids in which PACE or PACEM cDNA is
under expression control of the WAP promoter, generating
plasmids pHL252 and pHL255, respectively.
WAP/PACE and WAP/PACEM DNAs for injection were
released from the plasmid by NotI-HindIII digestion, and
then purified for microinjection.
Transgenic mice were generated by coinjecting WAP/HPC
and WAP/PACE fragments in a 2:1 molar ratio, at a total
concentration of 2 ~g/ml. PCR was used to detect the
transgene DNAs in tail DNA from mice produced from the
injected embryos. Transgenic mice were identified by HPC
and PACE-specific PCR products in DNA from tail samples.
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-- 41 --
Primers that define a 502 bp region at the 5' end of the
HPC gene and a 216 bp region of the mouse WAP gene were
used as probes for the WAP/HPC DNA (Drohan et ~l.,
~ransgenic Res. 3: 355-364 (1994). Primers that define
~ 5 a 260 bp region at the 5' end of the PACE cDNA were used
as probes for PACE DNA (see Wise et al., Proc. Natil.
Acad. Sci., U.S.A. 87:9378-9382 (1990)).
Four founders carrying both HPC and PACE traI~sgenes
were detected amon~ thirteen mice screened. All Eour
HPC/PACE founders ~ransmitted the transgenes to their
offspring. The male, C1.2, showed high trans~ene
trAn! ;~-cion frequency. The three females, c2.~, c4.1
and C5.2, showed low transmission frecluencies and
appeared to be mosaic. Two founders carrying both the
HPC and PACEM transgenes and one founder carrying the
PACE transgene alone were detected among seven ~ice
screened. One double-transgenic founder, M2.3,
transmitted both PACEM and HPC transgenes to offspring.
Lines were established from the five founders that
exhibited transmission to progeny and mice from these
lines were employed in further studies.
EXAMPLE 2 Detection of PACE and WAP expression by
Northern blotting
Total RNA was prepared from tissues of transgenic
females of the F1 or F2 generations and from control mice
using standard techniques. RNA was isolated from fresh
or frozen tissues in a single step procedure using acid
guanidinium thiocyanate phenol-chloroform extrac1:ion
(available commercially, for instance, as ~WAzol,
Molecular Research Center, Inc. and descri~,ed in
Chomczynski et al ., Anal . BiocAem. 162: 156-159 (~987)).
The RNA was analyzed on Northern blots using 15 ~g of
total RNA for each sample. RNA from two Anir~ls or more
was analyzed in each experiment. The sample RN~s were
separated under denaturing conditions on formalclehyde-
1.2% agarose gels and then transferred by downward
alkaline blotting for 2.5 h onto nylon membranes
(available, for instance, as GENESCREEN PLUS DuPont ~EN)
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- 42 -
as described in Chomczynski, P., Anal. Biochem. 201: 134-
139 (1992) .
The membranes were screened with probes specific for
HPC, PACE and 18S rRNA. A 0.5 kb BamHI-NheI fragment
5from the HPC 3' region of HPC was used as a probe for
PACE mRNA. A 0.85 kb BamHI-SalI fragment from the PACE
cDNA was used as a probe for PACE mRNA. A 0.7S kb BamHI-
SphI fragment of the 18S ribosomal RNA (rRNA) gene in
pN29111 (ATCC No. 63178) was used as a probe for 18S
10rRNA. An intact 2. 5 kb human PACE cDNA fragment was used
as a probe for the endogenous murine PACE mRNA. The
probes were labeled with 32p by random primer
polymerization labelling.
Filters were prehybridized for 0.5 h at 68~C using an
15accelerated hybridizing solution (such as QUIK-HYB from
Stratagene). They then were hybridized for 2 h at 68 ~C
in a buffer containing 0.5 to 1.0 ng/ml denatured probes
and sonicated salmon sperm DNA.
After hybridization, the filters were rinsed twice
20in 2X SSC, 0.1% SDS for 15 minutes at room temperature,
then washed once in O.lX SSC, 0.1% SDS for 30 min at 60
~C and thereafter autoradiographed.
Northern hybridization of replicate blots with the
three probes revealed that both the HPC and PACE
25transgenes were expressed in the lactating mammary glands
of HPC/PACE mice.
The major species of HPC mRNA was approximately 1725-
1775 nucleotides (nt) in length, with a distinct minor
species approximately 200 nucleotides shorter. The
30shorter mRNA may have been produced by an alternative 3~
polyadenylation site (29). Several HPC precursor RNAs of
about 2250, 2400 and 4800 nucleotides also were detected.
Human PACE mRNA of the expected size, 2700
nucleotides, was detected by the PACE cDNA probe.
35Endogenous mouse PACE transcripts were not detected, even
upon prolonged exposure of autoradiograms. However, upon
hybridization under conditions of lower stringency,
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endogenous transcripts were detected at levels at leas
one order of magnitude lower than the transgene mRN~.
The amount of rHPC secreted into the milk of founder
and later generation mice was determined by a sandwic
- s ELISA, using HPC-~specific polyclonal antibodie:s, as
described in EXAMPLE 3 and Table 1 below.
E~AMP~E 3 Detection of maturQd r~PC in milk of
transgenic ~nimals
Mice were administered 0.3 ml (0.6 IU) of oxytocin
i.p. to facilitate collection of milk. Milk samples were
collected between days 7 and 15 of lactation. The milk
was diluted with 2 volumes of phosphate-buffered saline,
pH 7.4, contA;n;ng 50 mM EDTA, centrifuged twice at
:L5,000 rpm for 15 min at 4 ~C, and stored at -80 ~C before
use. EDTA was used to solubilize casein miceller" and
:improve recovery of micelle-associated rHPC.
The concentration of rHPC in the milk was measured
by sandwich ELISA (enzyme linked immunosorbent a,say).
';heep anti-HPC polyclonal antibody immobilized in
microtiter wells was used to capture rHPC in the diluted
milk samples (Drohan et al ., Transgenic Res . 3: 3.'~5-364
llg94)). A rabbit anti-HPC antibody was used to detect
c:aptured rHPC bound to the immobilized ant:ibody.
E[orseradish peroxidase (HRP) conjugated to a goat anti-
rabbit IgG antibody then was bound to the immobili2ed
rabbit anti-HPC antibody. Bound Peroxidase was det:ected
by activity assay using a calorimetric subst:ra~e,
3,3',5,5'-tetramethylbenzidine (TMB). Subs;trate
utilization was measured by the change in absorbance at
650 nm during 10 minutes. Purified plasma-derived HPC
s diluted in control milk served as a standard.
Results from these experiments are shown in Table 1,
below.
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TABLE 1 rHPC in milk of RPC/PACE transgenic mice
Milk was collected between days 7 and 15 of lactation and
the concentration of rHPC in defatted milk was assayed by
ELISA, as described herein. Six to eight different milk
samples were analyzed from each HPC/PACE line. Each
sample was analyzed in duplicate. (Except "*" indicates
a value from ELISA of a single milk sample.)
Fo GENERATION F~3 GENERATIONS
MOUSE LINE rHPC (mq/ml) rHPC (mq/ml)
Cl.2 - 0.4Sl - 0.896
C 2.2 0.020* 0.308 - 1.626
C 4.1 0.004 - 0.075 0.908 - 1.352
C 5.2 0.001 - 0.004 0.734 - 1.154
M 2.3 - 0.169 - 0.331
6.4 0.512 - 0.706 0.190 - 0.532
The level of rHPC detected in the milk of the founder
generation HPC/PACE mice was significantly lower than in
the milk of mice from later generations, which ranged
from 0.45 to 1.63 mg/ml. This may reflect mosaicism of
the founder mice. Lower amounts of rHPC were detected in
the milk of HPC mice from the 6.4 line and in HPC/PACEM
mice.
Coexpression of rHPC and PACE in the mammary gland
did not result in deleterious effects on the health of
the females over successive lactations. Similar to
normal nontransgenic micé from HPC/PACE lines C2.2, C5.2,
as well as HPC/PACEM transgenic mice were able to raise
litters of 10-15 pups, indicating that PACE did not
affect nursing capabilities even at expression levels an
order of magnitude higher than the endogenous gene.
Animals from HPC/PACE lines C1.2 and C4.1 reared only 2-6
of their pups, although their health remained unaffected.
EX~MPLB 4 Western blot analysis of HPC in transgenic
milk show~ that maturation is augmented by co-expression
of PACE/furin
Milk proteins were separated under reducing
conditions on 10% SDS-polyacrylamide gels alongside
prestained molecular weight markers. Following
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-- 45 --
electrophoresis, the gels were silver stainled or
transferred to nitrocellulose membranes (such as HYBOND-
ECL from Amersham).
The blots were reacted serially with (i) the 8861
5 anti-HPC monoclonal antibody that recognizes an epitope
on the activation peptide of the heavy chain or a sheep
anti-HPC polyclonal antibody, and then with (ii~ HRP-
conjugated secondary antibodies. After binding t;he HRP
~_onjugates and washing the filters, the activity of bound
l~P was determined using enhanced chemiluminescence.
'~c~nn; ng densitometry of the developed Western blots was
~mployed to quantitate the proteins.
The analysis showed that rHPC from the milk ~f HPC
or HPC/PACEM transgenic mice consisted of approximately
40-60% rHPC single chain form, which is more than the 5-
~L5~ present in human plasma. In sharp contrast:, the
amount of single chain form in the milk of HPC/PACE
clouble-transgenic mi~e expressing PACE averaged less than
~i~, indicating efficient conversion of the precursor to
t:he mature two-chain form by the transgenic expression of
t:he heterologous PACE gene.
~:XAMPLE 5 Purification and amino acid analysis o~. r~PC
f'r~m milk of transge~ic mice shows that co-expression of
}'ACE/furin increases correct maturation
Pooled whole milk, 1.5-2 ml, from several F2 and F3
animals of the C5.2 line was thawed, diluted with 20 ml
of 50 mM Tris, 0.15 M NaCl, 2 mM EDTA, 2 mM benzamidille,
pH 7.2 and centrifuged at 30,000 g for 15 min at 4~C.
I'he spun aqueous phase was filtered through a 0.45 ~m
m~embrane (for instance, MTTT~-HA from Millipore). The
filtrate was loaded at a linear flow rate of 17 cm/hr
onto a 1.5 cm X 2.7 cm column consisting of 8861 MAb
im~obilized on Sepharose CL-4B resin (from Pharmacia)
equilibrated with 50 mM Tris, 0.15 M NaCl, 2 mM E~TA, 2
mM benzamidine, pH 7.2. The loaded column was washed
with 5 mM ammonium acetate, pH 5.0 and then bound r~IPC
was eluted in 0.5 M ammonium acetate, pH 3.0, ;rr~~;~tely
neutralized with 3 M Tris and stored at -80 ~C.
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The recovery of rHPC from milk was more than 80%.
Electrophoretic analysis of rHPC purified from the milk
of HPC/PACE mice as described above, revealed that rHPC
polypeptides migrated slightly faster than plasma-derived
HPC. The difference probably was due to differences in
glycosylation (Drohan et al., Transgenic Res. 3: 355-364
(1994)).
Some single-chain rHPC was removed from rHPC obtained
from HPC mice by the previously described purification
process, but the amount of single-chain rHPC in rHPC
purified from HPC/PACE mice was substantially lower
(Drohan et al., Transgenic Res. 3: 355-364 (1994).
Furthermore, no other rHPC-specific lower molecular
weight bands were observed upon nonreducing and reducing
SDS-PAGE.
Amino acid sequence analysis of rHPC from the milk of
HPC/PACE mice revealed two amino terminal sequences,
beginning at positions 1 and 158 of HPC, as shown in
Table 2, below.
Alal has previously been identified as the site of
removal of the HPC propeptide (Foster et al., Proc. Nat'l
Acad. sci., USA 82: 4673-4677 (1985)). Aspls8 is the site
of cleavage of the internal Lysl5fi-Argl57 dipeptide during
conversion of the HPC zymogen to mature, two-chain HPC.
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-- 47 --
TABLE 2 Amino-Terminal sequence of Transgenic r~PC
Purified rHPC was subjected to automated Edman
degradation and the sequence compared to that of rHPC
from HPC transgenic mice. Numbering indicates the! first
,amino acid of the propeptide (-24), of the light chain
~ (+l) and of the heavy chain of mature HPC (+158). (~)
represents non-detected residues corresponding to ~-
,carboxyglutamic acid residues present in the HPC
,sequence.
CHL~IN COMPOSITION PER CENT
]~PC from Human Plasma
]Light A N S F L ~ ~ L R H S S L ~ R ~ C 100
chain +l
IIeavy D T E D Q E D Q V D P R L I D G K 10 0
chain +158
rHPC f rom HPC Transc~enic Mice
l?ro- T P A P L D S V F S S S :20-30
~eptide -24
I.ight A N S F L E E L R H S S L E R E C 70-30
Chain +1
Heavy D T E D Q E D Q V D P R L I D G K 100
c:hain +158
rH~C from HPC/PACE Tran~genic Mice
I.ight A N S F L E E L R H S S L E R E 100
c:hain +1
H!eavy D T E D Q E D Q V D P R L I D G K 100
chain +158
The results show that processing ofboth 1:he
30 propeptide and the single chain of the Protein C
precursor occurs at the appropriate sites in mammary
cells in HPC/PACE mice. In contrast, 20-30% of the rHPC
secreted into milk contains the propeptide in HPC
transgenic mice (Drohan et al., Transgenic Res. 3: 355-
364 (1994)).
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-- 48 --
Furthermore, the good yields of Glu residues at
positions 6, 7, 14, 16, 19, 20, 2S, 26 and 29 in the
light chain indirectly indicate that ~-carboxylation of
glutamic acid residues is inefficient at these expression
levels, consistent with earlier observations (Drohan et
al ., Transgenic Res. 3: 355-364 (1994) ) .
EX~MPLE 6 Expression of PACE/furin decreases whey acid
protein content of milk of transgenic animals
Western blot analysis of mouse whey acid protein
("WAP") in milk proteins from control non-transgenic
mice, mice transgenic for HPC, mice doubly transgenic for
HPC and PACEM and mice doubly transgenic for HPC and PACE
showed that HPC/PACE expression decreased the WAP content
of milk.
Milk proteins were analyzed by western blots
essentially as described above. Briefly, defatted milk
proteins were separated by electrophoresis through 14%
SDS-PAGE gels. The separated proteins were transferred
onto filters by standard western blotting procedures and
then WAP was visualized on the filters using an anti-WAP
antibody. Whey proteins from non-transgenic (i.e.,
normal mice), HPC/PACE transgenic mice, HPC transgenic
mice, and HPC/PACEM transgenic mice were analyzed side-
by-side. The amount of WAP protein in milk from non-
transgenic, HPC and HPC/PACEM mice was roughly the same
in all cases, while the amount of WAP protein HPC/PACE
transgenic mice decreased by about 40-60%.
EXAMPLE 7 PA~E/furin is secreted into milk of transgenic
animals
The presence of PACE/furin was analyzed in milk of
transgenic mice using the western blotting t~chniques
described herein above. Whey proteins from the milk of
control and transgenic mice and separated by PAGE in 8%
SDS-polyacrylamide gels. The gels were blotted onto
filters and PACE was visualized using a PACE/furin-
specific antibody. A band of approximately 50 kD that
appeared in all of the samples apparently was due to
artefactual non-specific binding of the secondary
antibody. No furin-specific band was seen in milk from
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non-transgenic control mice, HPC or HPC/PACEM transgenic
mice. A PACE/furin-specific band of approximately 80 kDa
was detected in milk proteins from HPC/PACE transgenic
mice.
S EXAMP~E 8 PACE/furi~ is ~n ~ctive protease in milk of
transgenic animal~
PACE/furin activity was determined in mil]k from
control and HPC/PACE transgenic animals by monitoring
conversion of single-chain rHPC to two-chain rHPC, as
follows.
Whole milk from mice was incubated at 37~C for 0, 1
and 3 hours, defatted by centrifugation and proteins from
each sample were resolved by SDS-PAGB in 8-16% gels. In
addition, milk from HPC/PACE mice was mixed with milk
from HPC mice in a 1:10 ratio to detect activity of
secreted PACE/furin in the milk of the HPC/PACE mice.
The gels were blotted onto filters and HPC was visualized
by western blotting techniques using an HPC-specific
antibody, as described herein above.
Samples cont~i~;ng milk from HPC/PACE mice contained
less single-chain HPC than milk from the other mice.
~ctivity of the secreted PACE/furin was indicated by the
decrease in precursor.
