Note: Descriptions are shown in the official language in which they were submitted.
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EXPRESSION OF GONADOTROPINS IN DICTYOSTELIUM
The invention relates to gonadotropins or mutants thereof expressed in
Dictyostelium, pharmaceutical compositions containing the same, a method for
the
s preparation of the gonadotropins as well as a method for the selection of
gonadotropin
mutants with superagonistic or antagonistic properties.
The gonadotropins form a family of structurally related glycoprotein hormones.
Typical members include chorionic gonadotropin (CG), follicle stimulating
hormone
(FSH), luteinizing hormone (LH) and thyroid stimulating hormone (TSH). FSH, LH
io and TSH are present in most vertebrate species and are synthesized and
secreted by
the pituitary. CG has so far been found only in primates, including humans,
and in
horses and is synthesized by placental tissue.
The hormones are heterodimeric proteins of around 30 kD formed by a non-
covalent association of a con-unon a-subunit and a hormone specific ~3-
subunit.
is Within a species, the a-subunit is essentially identical for each member of
the
gonadotropin family; it is also highly conserved from species to species. The
(3-
subunits are different for each member, i.e. CG, FSH, TSH and LH, but show
considerable homology in structure. Furthenmore, also the (3 subunits are
highly
conserved from species to species. In humans, the a subunit consists of 92
amino acid
zo residues, whilst the (3 subunit varies in size for each member: 111
residues in hFSH,
121 residues in hLH, 118 residues in hTSH and 145 residues in hCG (Combarnous,
Y. (1992), Endocrine Reviews, ~, 670-691, Lustbader, J.W. et al. (1993),
Endocrine
Reviews, ~, 291-311). The (3 subunit of hCG is substantially larger than the
other ~i
subunits in that it contains approximately 34 additional amino acids at the C-
terminus
z s referred to herein as the carboxy terminal peptide (CTP). This CTP bears
four serine
linked oligosaccharides.
The gonadotropins serve important functions in a variety of bodily functions
including metabolism, temperature regulation and the reproductive process. The
hypophyseal gonadotropin FSH for example plays a pivotal role in the
stimulation of
a o follicle development and maturation whereas LH induces ovulation (Sharp,
R.M.
(1990), Clin Endocrinol., ~, 787-807; Dorrington and Armstrong (1979), Recent
Prog. Horn. Res., ~5, 301-342). Currently, FSH is applied clinically, either
alone or
in combination with LH, for ovarian stimulation i.e. ovarian hyperstimulation
for in
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vitro fertilisation (IVF) and induction of in vivo ovulation in infertile
anovulatory
women (Insler, V.(1988), Int. J. Fertility, ~, 85-97, Navot and Rosenwaks
(1988), J.
Vitro Fert. Embryo Transfer, ~, 3-13), as well as for male hypogonadism. The
human
choriogonadotropin (hCG) is involved in the maintenance of pregnancy in the
early
s stages after conception and has also important therapeutic applications.
The two subunits of the heterodimer display many conserved infra-subunit
disulfide bonds: five disulfide bridges in the a-subunit and six disulfide
bridges in the
~3-subunit. The corresponding cysteine residues are fully conserved among all
io members of the gonadotropin family. The recently obtained X-ray structure
of hCG
shows that these disulfide bonds are involved in typical three-dimensional
patterns
called disulfide knots.
The gonadotropins possess three or four asparagine residues that can be N-
glycosylated and have an important impact on its conformation and biological
is activity. In addition the C-terminal peptide (CTP) of hCG can be O-
glycosylated at
four serine positions. The major role of the glycosylated CTP seems to be the
prolongation of the circulatory half life of hCG.
The biosynthesis of the glycoprotein hormones is a highly complex process. In
ao the last decade it has become clear that folding, assembly and secretion of
gonadotropins is assisted by a large set of chaperones and folding enzymes,
residing
in the Endoplasmic reticulum and the Golgi apparatus. Since both the a- and
the (3-
subunit contain a so-called cystine-knot, it can be anticipated that protein
disulfide
isomerase plays a key role in the facilitation of the folding process. In
addition, it has
25 been shown that the N-linked oligosaccharide side chains are required for
proper
folding, disulfide formation and secretion of hCG (Feng, W. et al (1995) J.
Biol.
Chem. ~Q, 11851-11859). The successful assembly of the two subunits into the
dimer is an absolute prerequisite for the biological activity of the dimer.
a o Elucidation of functional determinants of the heterodimeric glycoprotein
hormones
is often hampered by unwanted secondary effects of mutations on the assembly
of the
subunits. To facilitate structure/function analyses studies mutants have been
produced
in which the coding regions of the hCG (3-subunit and the common a-subunit are
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connected via peptide spacers or intersubunit disulfide bonds. It has been
shown that
these covalently linked a- and (3-subunits of hCG are able to fold into
biologically
active conformations.
The gonadotropins have been expressed in Chinese Hamster Ovary (CHO) cells,
s and their recombinant derivatives have biological activities comparable to
the native
hormones (Olijve, W. et al. (1996) Mol. Hum. Reprod. ~, 371-382). This
indicates
that these host cells contain all chaperones and folding enzymes necessary to
assemble the a- and ~3-subunits of hCG and to perform all post-translational
modifications necessary for full biological activity. Furthermore, the use of
CHO cells
io in combination with site-directed mutagenesis has proven to be a valuable
tool for
elucidating functional determinants in the glycoprotein hormones (Puett, D. et
al H.
(1994) in Glycoprotein Hormones (Lustbader, J.W., Puett, D. & Ruddon, R.W.,
Eds.)
pp. 59-82, Springer-Verlag, New-York) and for designing potential new
therapeutic
analogs of these hormones ( Fares, F.A. et al. ( 1992), Proc Natl Acad Sci U S
A ,$~,
is 4304-4308).
Unfortunately, the use of mammalian cells such as CHO cells for the expression
of
human gonadotropins suffers from some limitations. Thus; it is not possible to
generate
the high number of transformants necessary for studies that involve random
mutagenesis
zo of protein domains. Random mutagenesis of selected domains in proteins has
been
shown to be a valuable tool for identifying the structural determinants for
receptor
binding and bioactivity. In addition, the use of CHO cells is expensive and
labour
intensive. Thus, there is a need for the expression of gonadotropins in a more
robust
expression host.
zs Host cells derived from lower organisms might meet the above mentioned
requirements, but it is expected that the complex folding of the gonadotropins
hampers proper expression and secretion of such complex recombinant proteins.
Now, unexpectedly it has been found that Dictyostelium is capable of producing
the highly complex glycoprotein hormones.
The soil amoebae Dictyostelium discoideum is an organism that provides an
attractive alternative for heterologous expression of the human glycoprotein
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hormones. While it can be grown and transformed with the same ease as the
yeast
Saccharornyces, it has some of the complex features that resemble mammalian
cells,
such as glycosylation and chemotaxis. Furthermore, it has recently been shown
that
Dictyostelium provides a useful system for random mutagenesis approaches.
s Nevertheless, there have been found differences between the glycosylation of
proteins produced in Dictyostelium compared to material produced in CHO cells.
It is
known that glycosylation plays an important role in hormone function, and
whereas
most glycosylation is performed by Dictyostelium, galactose, N-
acetylgalactosamine or
sialic acids are not attached to the oligosaccharide side chains {Slade, M. et
al. (1997)
io Biotech. Genet. Eng. Rev., fig, 1-35). Though the post-translational
modification is
not identical to that in higher eukaryotes, gonadotropins produced in
Dictyostelium
also were found to be biologically active. The protein is found to be capable
of
binding to its receptor and to stimulate cAMP production in cells expressing
the
human LH/CG receptor.
15 The heterogeneity of the expressed glycoproteins is greatly reduced leading
to
much more homogeneous preparations of isohormones. Therefore, gonadotropins
produced in Dictyosteliun: are chemically well-defined relative to the more
complex
CHO-produced gonadotropins. This is a major advantage for the maintenance and
analytical validation of the batch-to-batch consistency. Since the
glycosylation is a
ao very important determinant for the in vivo half live of gonadotropins,
production of
gonadotropins in Dictyostelium provides a tool to produce non-mutated
gonadotropins
with well-defined in vivo half lives. In addition, combination of the
expression in
Dictyostelium with protein engineering facilitates tailor made gonadotropins
for
several clinical applications.
is Because of the complex inter and intra molecular folding of the two
subunits, the
large number of disulfide bridges, disulfide knots and post-translational
modifications, it is remarkable that active gonadotropins can be expressed in
Dictyostelium and that properly folded molecules can be prepared according to
the
invention. Proper disulfide bond formation is a critical event in the folding
and
ao maturation of functional gonadotropins. Especially the disulfide bond
formation in the
(3 subunit is critical: all disulfide bonds are required for efficient
combination and
folding. Detailed studies of the folding of hCG revealed that the folding of
the
molecule does not proceed by a simple sequential pathway, but proceeds
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independently in different domains of the molecule. It was therefore thought
that cells
of lower organisms were not capable of secreting properly folded
gonadotropins.
The present invention provides for gonadotropins expressed in Dictyostelium.
Use of Dictyostelium discoideaun has the advantage that is a well-studied
s organism. The vegatative amoebae are easy and inexpensive to grow either in
axenic
culture or on Gram-negative bacteria. Furthermore, several transformation
vectors
have been described which are capable of directing expression of foreign
proteins.
The gonadotropins according to the invention can be dimeric i.e. composed of
two non-covalently bound subunits. Preferably, the gonadotropin is hCG or FSH.
The
io gonadotropins, however, can comprise modifications generally known in the
art.
In one such preferred modification of the gonadotropins according to the
invention, the C-terminus of the amino acid sequence of one of the subunits is
linked,
optionally through a linker moiety, to the N-terminus of the amino acid
sequence of
the other subunit. Preferably the linker moiety is a complete or partial CTP
unit or
is variant thereof, or a repeated oligopeptide e.g. a 5 times repeated Ser-Gly
peptide.
Another modification of the gonadotropins according to the invention can be an
extension of the a andlor ~3 subunit at their respective N- or C-terminus with
a
complete or partial CTP unit or a variant thereof. The extension may comprise
the
respective CTP units in single or multiple forms. Alternatively, a complete
CTP unit
ao or partial CTP unit or multiple forms thereof can be inserted in the N- or
C-terminus
of said subunits. Again another modification is the introduction of one or
more non-
native disulfide bridges.
Furthermore, the gonadotropins according to the invention may be either
glycosylated or partially glycosylated. Partially glycosylated gonadotropins
according
25 to the invention can be obtained by site-directed mufagenesis whereby one
or more of
the glycosyiation recognition sites in the gonadotropins are removed.
Alternatively,
the glycosylation pattern of the gonadotropins according to the invention can
be
modified by the introduction of additional glycosylation recognition sites
and,
optionally, the removal of one or more glycosylation recognition sites,
resulting in a
3o modified glycosylation of said gonadotropins. A glycosylation recognition
site as
used herein consists of the amino acid sequence Asn-X-SerfThr, wherein X can
be
any amino acid.
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As used herein, the a and (3 subunits of CG, FSH, LH and TSH as well as the
heterodimeric fOnllS have in general their conventional definitions and refer
to the
proteins having the amino acid sequences known in the art per se, or allelic
variants
thereof, regardless of the glycosylation pattern displayed.
s "Native" forms of these proteins are those proteins which have the amino
acid
sequences as isolated from the relevant vertebrate tissue, and have these
known
sequences per se, or their allelic variants thereof.
These "variants" are those proteins which have deliberate alterations in amino
acid sequences relative to the native proteins. The alterations may include
single or
io multiple deletions, insertions, substitutions and combinations thereof, and
can be
produced by, for example, site specific mutagenesis.
As used herein, the "CTP unit" refers to the amino acid sequence found at the
carboxy terminus of the ~3 subunit of hCG which extends from amino acid 112-
118 to
residue 145 at the C-terminus or to a portion thereof. A "complete" CTP unit
contains
i5 28-34 amino acids, depending on the N-terminus of the CTP. A "partial" CTP
unit is
an amino acid sequence which occurs between positions 112-118 to 145
inclusive, but
which has at least one amino acid deleted from the shortest possible complete
CTP
unit (amino acid 118-145). "Multiple" CTP units are understood to encompass
tandem arrays of the complete CTP unit or partial CTP unit or combinations of
both.
zo Methods to constnict the gonadotropin genes according to the invention are
well
known in the art (Sambrook et al., Molecular Cloning: a Laboratory Manual,
Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, latest edition).
Techniques for
site directed mutagenesis, ligation of additional sequences, PCR, and
construction of
suitable expression systems are all, by now, well known in the art. Portions
or all of
25 the DNA encoding the desired protein can be constructed synthetically using
standard
solid phase techniques, preferably to include restriction sites for ease of
ligation.
Suitable control elements for transcription and translation of the included
coding
sequence can be provided to the DNA coding sequences.
The invention also provides for a method for the expression of gonadotropins
or
3o mutants thereof in Dictyostelium. Said method according to the invention
comprises
the steps of
- transforming a strain of Dictyostelium with a recombinant plasmid vector
comprising a DNA sequence encoding the gonadotropin genes or mutated genes
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under control of Dictyostelium regulatory sequences
- culturing the recombinant strain under conditions to allow expression of the
DNA sequence and
- isolating the expressed protein.
s Preferably the protein to be expressed is a single-chain protein, i.e. the
subunits
are covalently connected through a spacer molecule. More preferably the
gonadotropin is single-chain hCG.
Another aspect of the invention is to provide a method to easily screen for
mutated gonadotropins. Said method comprises
io - random mutagenesis of gonadotropin genes
- insertion of the mutated genes in a Dictyostelium plasmid vector
- transforming a strain of Dictyostelium with said recombinant plasmid vector
- culturing clones under conditions to allow expression of the DNA sequence
- determining the receptor binding / signal transduction ratio and
is - isolating clones with a ratio deviating from the ratio determined for
wild type
gonadotropins.
Preferably, the mutated gonadotropins show a receptor binding which equals the
binding of the native protein to its receptor. More preferably, the affinity
of the
mutated protein to its receptor is higher than its native counterpart. The
signal
ao transduction has to be at least two-fold higher or lower as compared to the
native
protein. Preferably, the difference in signal transduction amounts a factor
10.
Proteins exhibiting a high ratio are useful as antagonists whereas proteins
with a
low ratio can be used as super agonists.
Methods to determine receptor binding as well as in vitro and in vivo assays
to
zs determine biological activity of gonadotropins are well known.
Random mutagenesis need not to be performed on the complete gonadotropin
gene but instead can also be carried out on a single subunit gene or a well-
defined
region such as e.g. the determinant loop.
In order to introduce random point mutations in the gonadotropin gene(s),
3o amplification of the regions) of interest with Taq DNA polymerise can be
employed.
Since Taq DNA polymerise lacks a 3'~5' exonucleolytic editing activity, this
enzyme is an error-prone DNA polymerise, with a measured error rate of 10-5 to
10'~
error per nucleotide synthesized. Therefore, use of PCR with Taq DNA
polymerise
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_g_
under essentially standard reaction conditions can be used to introduce
mutations
(Zhou, Y. et al. ( 1991 ), Nucl. Acids Res. ~, S2). However, the frequency of
mutations using these conditions is adequate for mutagenizing relatively large
sequences, but not for small DNA fragments (< 500 bp). The infidelity of Taq
DNA
s polymerase can be increased by addition of Mn2+ and the use of relatively
high
concentrations of dNTP and Mg'-+ (Leungh, D.W. et al. (1989), Technique ~, 1-
1S).
An alternative method for the adjustment of the mutation frequency, which also
offers
the opportunity of influencing the types of mutation, is the use of dITP in
combination with limiting amounts of one of the four dNTPs (Spee, J.H. et al.
( 1993),
io Nucl. Acids Res. ?~, 777-778). For mutagenesis of short target DNA(< SO
bp), the use
of degenerate oligonucleotides seems to be the method of choice (Kirchhoff, F.
and
Desrosiers, R.C. (1996), Meth. Molec. Biol. ~, 323-333). Usually, the
procedures
have four steps. In step one, the region of interest is amplif ed by
(modified) PCR. In
step two, the amplified DNA is digested with a pair of restriction
endonucleases that
is cut at each end of the DNA sequence of interest. In step three, the DNA
fragment
containing the DNA sequence of interest is ligated with restriction
endonuclease
digested vector DNA. In step four, the resulting recombinant DNA molecules are
introduced into the cells by transfonnation or electroporation.
ao DNA vectors encoding any of the gonadotropins according to the invention
are
also within the scope of the invention. DNA vectors according to the invention
can be
obtained by operatively linking the DNA encoding the native gonadotropins or
variants thereof to DNA comprising Dictyostelium regulatory sequences.
Optionally,
these vectors also might contain regions which contain origins of replication
and/or
as polypeptide-encoding sequences facilitating extrachromosomal replication.
Such
vectors have the advantage that they can replicate extrachromosomally in the
Dictyostelium host cell.
As explained, the variant gonadotropins according to the invention can be
3 o agonists or antagonists, depending on the mutation site. The mutation site
may lead to
subtle changes in the conformation of the molecule. If the mutation site e.g.
is
selected in parts of the protein that are associated with receptor binding
and/or signal
transduction, the excreted protein according to the invention may lead to a
partial or
complete loss of signal transduction activity. Such altered gonadotropins,
wherein the
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receptor binding properties are retained, can be used as antagonists. Also
gonadotropins with improved binding and signal transduction activities may be
selected.
The agonist gonadotropins according to the invention can be used for the same
s clinical purposes as the native gonadotropins. In addition the proteins can
be used as
diagnostic tools to detect the presence or absence of antibodies with respect
to the
native proteins in biological samples. They are also useful as control
reagents in assay
kits for assessing the levels of gonadotropin hormones in various samples.
Antagonists can be used e.g. in the treatment of gonadotropin dependent
tumors;
i o LH/hCG antagonists to prevent LH surges during controlled ovarian
hyperstimulation
and FSH antagonists for male contraception.
Suitable pharmaceutical compositions according to the invention comprise one
or
more of the gonadotropins according to the invention and a pharmaceutical
acceptable
carrier.
is Pharmaceutical acceptable carriers are well known to those skilled in the
art and
include, for example, sterile satin, lactose, sucrose, calcium phosphate,
gelatin,
dextrin, agar, pectin, peanut oil, olive oil, sesame oil and water.
Furthermore the pharmaceutical composition according to the invention may
comprise one or more stabilizers such as, for example, carbohydrates including
2o sorbitol, mannitol, starch, sucrosedextrin and glucose, proteins such as
albumin -or
casein, and buffers like alkaline phosphates.
Suitable administration routes are intramuscular injections, subcutaneous
injections, intravenous injections or intraperitoneal injections, oral and
intranasal
administration.
The following examples are illustrative for the invention and should in no way
be
interpreted as limiting the scope of the invention.
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LEGENDS TO THE FIGURES
Figure 1: Plasmids and cloning strategy. (lA) Maps of plasmids MBl2n and
MBl2n/PsA. Both plasmids consists of four main fragments, containing E.coli
s maintenance sequences (bluescript), blasticidine resistance gene under Dd
promoter
control (BSR), a cloning cassette with Dd promoter (actl5) and terminator
(2H3), and
sequences for extrachromosomal maintenance in Dictyostelium (G4/D5, GS/D6).
MBl2n/PsA contains a PsA linker sequence (Table 1) inserted into the unique
BgIII
site of MBl2n. (1B) Cloning diagram of JV158. The oligonucleotides used are
shown
io by the horizontal arrows. The xxx shown in oligonucleotide b20aarev
indicate that
sequence substitutions have been made to optimize the sequence for
Dictyostelium
codon preference. ( 1 C) Cloning diagram of JV l OPSA. All oligonucleotide
sequences
are given in Table 1.
is Figure 2: Binding activity of wildtype hLH, hCG and single chain hCG (sc
hCG) produced in Chinese Hamster Ovary cells (CHO) or Dictyostelium
discoideun: (Dd) to membranes of CHO cells stably expressing the human
LHICG receptor. The membranes were incubated with '25I-labeled hCG in the
absence or presence of varying concentrations unlabeled wildtype hLH, hCG or
the
ao single chain hCG's. Displacement curves are presented as the percentage of
maximal
binding at each dose of unlabeled hormone.
Figure 3:In vitro biological activity of wildtype hLH, hCG and single chain
hCG (sc hCG) produced in Chinese Hamster Ovary cells (CHO) or Dictyostelium
25 discoideurn (Dd). Extracellular cAMP was measured by specific ltIA after
stimulation of CHO cells stably expressing the human LH/CG receptor.
Figure 4: Ire vitro biological activity of wildtype hCG produced in Chinese
Hamster Ovary cells (CHO) or Dictyostelium discoideum (Dd). Luciferase
ao production was measured after 4 h incubation at 37 °C and
stimulation of CHO cells
stably expressing the human LH/CG receptor and a reporter construct.
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Figure 5: In vitro biological activity of wildtype FSH produced in Chinese
Hamster Ovary cells (CHO) or Dictyosteliunt discoideum (Dd). Luciferase
production was measured after 4 h incubation at 37 °C and stimulation
of CHO cells
stably expressing the human FSH receptor and a reporter construct.
Figure 6: Li vitro biological activity of wildtype hCG from CHO cells and
selected hCG mutants produced by Dictyostelium discoideum (Dd). Luciferase
production was measured after 4 h incubation at 37 °C and stimulation
of CHO cells
stably expressing the human LH/CG receptor and a reporter construct.
io
EXAMPLES
is Design of the expression constructs
A gonadotropin mutant consisting of a completely intact a-subunit connected
via
a peptide decamer containing five Ser-Gly repeats to the (3-chain only lacking
its C-
terminal peptide, was selected for expression in Dictyostelium.
Two constructs were generated, which differ in their leader sequences. The
ao first one contains the natural leader sequence of the (3-subunit of hCG. To
limit
possible problems in mRNA translation due to the presence of a considerable
amount
(about 40% in the (3-subunit of hCG) of infrequently used codons, the first 30
bases of
the coding sequence were altered conform to Dictyostelium preferred codon
usage.
For the construction of the second construct it was considered that the
proteins
as involved in the secretion route of mammalian cells, via the ER and Golgi,
may not be
conserved between different species. To facilitate transport in Dictyostelium,
the
leader peptide of the human ~i-subunit was exchanged with a leader peptide of
a
Dictyostelium glycoprotein, Prespore protein A (PsA), which is transported
over the
plasma membrane. This leader had been used previously to express secreted
ao heterologous proteins (Dittrich ,W. et al. (1994) Bio/Technology ~, 614-
618).
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The expression plasmids were called JV 158 (adapted codons with (3-subunit
leader peptide) and JV10PSA (with the PsA leader peptide) and were derived
from
MB 12n.
MB 12n is an 8.25 kb extra-chromosomally maintained Dictyostelium discoideum
s (Dd) plasmid, containing an unique restriction site between a Dd promoter
and
terminator. MB 12n consists of 2.9 kb (a CIaI -HincII partial digest fragment)
from
p155d1 (Hughes, J.E. et al. (1994) Mol. Cell. Biol. ],~, 6117-6124) containing
the Dd
origin of replication and two Dd genes required for replication (G41D5 and
GS/D6),
2.95 kb from pBluescript (Strategene) for propagation in E. coli, 1.35 kb
containing
is the blasticidine resistance gene between the Dd Actine 15 promoter and the
Dd Actin
8 terminator (from pBsr2, Sutoh, K. (1993), Plasmid ~Q, 150-154), and a 1.05
kb
cloning cassette containing the Dd Actin 15 promoter, an unique BgIII
restriction site
and the Dd 2H3 terminator (from BS 18.2H3, Kumagai A. et al (1989) Cell ~, 265-
275). The BgIII site in the blasticidine resistance gene has been removed by
is mutagenesis. Figure lA illustrates the relative position of these
components in
MB 12n. Plasmid MB 12n/PsA contains a linker sequence encoding the 19 amino
acid
PsA leader peptide (Early, E.A. et al (1988) Mol. Cell. Biol. $ 3458-3466;
Dittrich , et
al (1994) Bio/Technology ~, 614-618).cloned in the unique BgIII site. The
plasmid
has an unique NdeI site in the 3' part of the PsA sequence and an unique BgIII
site 5'
ao of the 2H3 terminator, to facilitate 'in frame' directional cloning. The
BgIII site
adjacent to the Dd Actinl5 promoter was eliminated during cloning (see Table
1).
Using primers b20aarev and alphater (Table 1) a single chain hCG was
amplified by PCR from a plasmid containing hCG mutant 1 [(3-(1-111)-(Ser-Gly)5-
a-
(1-92), (Heikoop, J. C. et al. (1997) Eur. J. Biochem. ~S4 , 656-662). The
resulting
25 fragment was cloned in MBl2n after digestion with BgIII. Primer b20aarev is
designed to optimize the first 10 amino acids of the (3 chain for codon usage
in
Dictyostelium. The same template was amplified with primers bmature (Table 1)
and
alphater to produce a product that, after digestion with NdeI and BgIII, was
cloned
into MB 12n/PsA. The resulting plasmids were labeled JSV 158 and JV l OPSA,
3 o respectively (see Figure 1 B and 1 C). PCR was performed with the Expand
High
Fidelity (Boehringer Mannheim) system, using the following cycle parameters:
denaturation for 3 minutes at 94 °C, then 25 cycles with 30 seconds at
94 °C, 30
seconds at 37 °C and 60 seconds at 72 °C. PCR products were
separated by gel
electrophoresis, excised and purified with Qiaex II (Qiagen) before
restriction
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digestion and cloning. All DNA sequences were analyzed and confirmed by
dideoxy
sequencing.
Table 1: DNA sequences for oligonucleotides and PsA linker. The PsA sequence
s also shows the peptide sequence of the PsA leader, as well as the unique
restriction
sites BgIII and NdeI.
Oligo ID: Sequence:
io PsA: (BgIII) NdeI BglII
agatcatgaa attccaacat acatttattg cattattatc actattaaca~caaatg c_agatct
M K F Q H T F I A L L S L L T Y A N A
b20aarev: gcagatctat ggagatgttc caaggtctcc tccttttatt actcctcagc atgggtggta
catgggcatc caag
is alphater: ccagatctaa acatttaaga tttgtg
bmature: gctatcgaca tatgcaaatg catccaagga gccgcttcg
20 Ex~g~:
Expression of single chain hCG in Dictyostelium
Dictyostelium strain AX3 was grown to a density of 2x106 cells per ml in
axenic
medium before electroporation. The electroporation conditions were basically
as
described (Mann S.K.O. et al (1994) in: Cell Biology: a Laboratory Handbook,
J.E.
25 Celis edt, Academic Press, Vol 1, pp 412-452), using 1 p,g plasmid DNA for
electroporation of 10' cells.
Plasmids JV158 and JV10PSA were transformed to Dictyostelium, as well as a
MB 12n control plasmid. After electroporation, the cells from one cuvette were
seeded
in a 10 cm plate. 12 hours after electroporation blasticidine was added to a
final
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concentration of 5 pg/ml. 24 hours after electroporation the medium,
containing dead
cells, was aspirated, the cells were resuspended by pipetting in medium with
blasticidine, and the cells were distributed over 24 wells in a 96 well
microtiter plate.
The 24 wells were each serially diluted 10, 100 and 1000 fold. The medium
s containing blasticidine was replaced every 3 days. Positive wells were
identified 5-9
days after seeding, and transformation efficiency was estimated from the
dilution
series. Typically, between 1 x 104 and 6 x 104 colonies per pg DNA were
obtained.
Single wells were then selected for further experiments. A single well
contains 200 p,l
of medium, sufficient for a DELFIA~ hLH assay which has a 100% cross-
reactivity
io with hCG. The assay is based on the direct sandwich technique, in which
monoclonal
antibodies directed against a specific antigenic site on the ~3-subunit are
immobilized.
After binding of intact (single chain) hCG to the solid phase antibody,
europium
labelled antibodies directed against a specific antigenic site on the a-
subunit are
bound and quantified. The assay is performed as described by the manufacturer
is (Wallac Oy, Turku, Finland).
The results cleariy show that immunological active single chain hCG is
produced
from both expression constructs and not from the control plasmid. Moreover,
the
amount of single chain hCG produced from JV158 (267 mU/ml) seems to be
considerably higher than the amount produced from JV10PSA (4.9 mU/ml),
ao suggesting that in this case the human (3-hCG leader peptide is more
effective then the
Dictyostelium leader peptide from PsA.
The kinetics of single chain hCG production were studied for JV158 transformed
cells. Different densities of transformed cells were seeded on plates in
axenic media,
25 grown to confluence and maintained for several days, without any medium
change.
An aliquot of medium was taken every 24 hrs and analyzed for the presence of
single
chain hCG. The expression-level reaches a maximum 4 to 5 days after cells
reach
confluence (data not shown). Since the cells start to detach from the plate at
about 6-7
days after reaching confluence, medium was harvested at day 5 after reaching
3 o confluence.
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Example 3:
Activity of single chain hCG expressed in Dictyostelium
The ability of single chain hCG froiri Dictyostelium to bind to the human
LH/CG
receptor was determined by a competitive binding assay with heterodimer hCG.
CHO
s cells expressing the human LH/CG receptor (Jia, X.-C. et al. (1991) Mol.
Endocrinol.
~, 759-7G8) were simultaneously incubated with 'ZSI-hCG and purified material
from
the cell culture supernatant of Dictyostelium.
The receptor-binding activity of purified single chain hCG was quantified with
a
radioligand receptor displacement assay on membrane fractions isolated from
io exponentially growing cells. In a total volume of 0.5 ml buffer {final
composition 10
mM Tris-HCI, 5 mM MgCl2, 0.1 % bovine serum albumin, pH 7.4) a fixed amount of
membrane protein was incubated with 'ZSI-hCG (20,000 epm, approximately 12 pM)
and increasing amounts of competitor protein for 18 hours at ambient
temperature.
'ZSI_labelled hCG (NEX-lOG) was obtained from Du Pont de Nemours. Specific
is binding was 10-12% of the total radioactivity added. After incubation bound
and free
hormone were separated by centrifugation. Highly purified, recombinant
gonadotropins were used as standards.
For purification of hCG from Dictyostelium cell culture medium was harvested
from large (22 x 22cm) culture plates. Purification was performed with the aid
of a
zo progran unable FPLC system (Pharmacia, Roosendaal, The Netherlands) using
the
control and chromatography supervision system UNICORN (Pharmacia, Roosendaal,
The Netherlands). Purification of single chain hCG was accomplished using a
combination of hydrophobic interaction and immuno chromatography with LH/CG (3-
subunit specific monoclonal antibodies. For this purpose, 150 mls of medium
were
zs produced with a single chain hCG content of 0.372 unit/ml as determined by
DELFIA~. Using this medium, approximately 3 units of purified single chain hCG
(~5% yield) were obtained. All procedures were carried out 4°C.
Coincubation with varying concentrations of wildtype hCG, wildtype hLH or
single
chains hCG displaced 'ZSI-hCG binding in a dose-dependent manner (Fig. 2). The
3o displacement curves indicate the material produced by Dictyostelium is
indeed able to
bind to the human LH/CG receptor. The affinity for the receptor is comparable
to the
affinity of single chain hCG produced by CHO cells, which has been shown to be
considerably less effective in displacing 'ZSI-hCG binding than the
heterodimer of
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hCG.~'Thus, there seems to be no significant difference in binding capacity
between
single chain hCG produced in Dictyostelium compared to CHO cells.
The bioactivity of single chain hCG from Dictyostelium was analyzed by
s examination of its ability to stimulate cAMP production in CHO cells
expressing the
human LH/CG receptor. Cells were incubated for 4 hours with increasine
concentrations of hormone in the presence of 0.1 mM 3-isobutyl-1-
methylxanthin.
The extracellular CAMP was determined by RIA (Immunotech).
The results demonstrate that the single chain hCG produced by Dictyostelium is
io indeed able to activate the human LH/CG receptor which results in the
production of
cAMP. Only a small difference in potency was observed between single chain hCG
produced in Dictyostelium compared to the material produced in CHO cells (ICso
value approximately five-fold lower.
is Exam,pte
Expression and bioactivity of heterodimeric hCG in Dictyostelium
We aimed for the expression of heterodimeric hCG in Dictyostelium. For the
expression of the a- and (3-subunits of hCG, two plasmids were generated.
Their
structure and overall organisation is essentially identical to that of MBl2n
which has
so been described in example 1. In order to facilitate expression of two
independent
plasmids in Dd we replaced the blasticidine resistance cassette in MBI2n with
the 2.4
kb KpnI-XbaI fragment from p155d1 (Hughes, J.E. et al. (1994) Mol. Cell.
Biol.. ~,
6117-6124) containing a neomycin cassette, creating MB l2neo. For the
construction of
the expression vector for the a-subunit, its natural cDNA sequence was
produced by
25 PCR using primers which introduce a BgIII restriction site both at the 5'
and 3' end of
the fragment, so that it could be cloned in MB l2neo. For the conshuction of
the
expression vector for the (3-subunit of hCG, MBl2n was modified to contain
another
unique restriction site (SphI) 3' in the BgIII cloning site [see example 1].
The hCG (3-
subunit cDNA was amplified using a 5' primer resulting in the alteration of
the first 30
s o bases of the coding sequence conform the Dictyostelium preferred codon
usage [see
example 1 ]. The primers also introduced appropriate restriction sites at both
the S'
(BgIII) and 3' (SphI) end of the fragment to facilitate directional cloning.
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The two expression plasmids were transformed simultaneously to Dictyostelium.
After transformation, cells were plated to clonal dilution. Clonal
transformants were
then selected and further grown for analysis. The amount of hCG secreted by
Dictyostelium was determined as described by the manufacturer using a DELFIA~
hLH
s assay (Wallac Oy, Turku, Finland), which has a 100% cross-reactivity with
hCG. The
results clearly show that immunologically active hCG is indeed produced by
Dictyostelium
Although the presence of heterodimeric hCG was demonstrated in the medium
by means of epitope detection, additional experiments were necessary to
establish
io whether hCG produced by Dictyostelium is biologically active.
Quantification was
based on immuno-reactivity. The bioactivity of heterodimeric hCG from
Dictyostelium was analysed by examination of its ability to activate the human
LH/CG receptor in a luciferase reporter assay. The results demonstrate that
the
heterodimeric hCG produced by Dictyostelium is indeed able to activate the
human
is LH/CG receptor (Figure 4). Moreover, its bioactivity is comparable
(ICS° value
approximately two-fold higher) to the bioactivity of wildtype hCG produced by
CHO
cells.
zo Expressio~z and bioactivity of heterodimeric FSH in Dictyostelium
To investigate if Dictyostelium is also capable to produce other complex
glycoprotein hormones, we studied the production of FSH in this organism. In
line with
the strategy for hCG (example 4), two expression plasmids were generated. For
the
construction of the expression vector for the (3-subunit of FSH, the cDNA was
amplified
z5 using a 5' primer resulting in the alteration of the first 27 bases of the
coding sequence
conform the Dictyostelium preferred codon usage and cloning was performed as
described for the (3-subunit of hCG. After transformation of Dictyostelium,
cells were
plated to clonal dilution. Clonal transformants were then selected and fiuther
grown for
analysis.
3 o The amount of FSH secreted by Dictyostelium was determined by a sandwich
immunoassay [SS-artikel]. The results demonstrate that immunological active
FSH is
produced by Dictyostelium. Moreover, heterodimeric FSH produced by
Dictyostelium
is also able to activate the human FSH receptor (Figure 5).
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Example 66:
Random mutagenesis of a selected region of hCG in Dictyostelium
Since we showed that Dictyostelium is capable of producing biologically active
s gonadotropins, we aimed for the development of a random mutagenesis
approach. For
this purpose, we selected two amino acids in the determinant loop of hCG which
have
been demonstrated to be involved in receptor binding and signal transduction.
Specific base substitutions were introduced by site-specific mutagenesis and
combining PCR-fragments that overlap in sequences as described using standard
PCR
io conditions. The primers were designed to alter aminoacids 94 and 95 of the
(3-subunit of
hCG. The first two nucleotides of both codons were altered fully random (A,C,T
or G),
while the third base was restricted to G or T to minimise to percentage of
stopcodons
introduced. The PCR fragments were separated and subcloned in pCR~ 2.1
(Invitrogen,
Leek, The Netherlands). The primers were chosen in such a way that the
subcloned
is PCR-fragments contained a BgIII site at the 5' end and a SphI site at the
3' end. After
PCR and cloning, the pool of pCR~ 2.1 constructs was transformed to E.coli.
Subsequently, DNA was isolated from a pool of 200 transformants and after
restriction
digestion the BgIII/SphI mutated fragments were subcloned in MBl2n containing
the
BgIII and SphI site. After plating E.coli transfonnants of MB 12 plasmids
containing the
zo random mutmt fragments, 400 colonies were pooled and DNA was prepared.
Dictyostelium was transformed simultaneously with the expression vector for
the a-subunit by electroporation. Selection with blasticidine (10 p,g/ml) was
introduced
hours after electroporation. The next day, cells were clonally diluted in 96
well plates
using 4 fold dilutions and neomycin selection (10 pg/ml) was iniated. Medium
was
zs replaced every 3-4 days, maintaining selective conditions. Positive wells
were identified
11-14 days after electroporation, and the transformation efEciency was
estimated from
the dilution series. Typically, about 500 transformants were obtained by
electroporation
of 10' cells with 1 p,g of both the hCG a and hCG (3 vectors. Single wells
were then
selected for furkher experiments. A single well contains 200 pl of medium.
Larger
a o amounts of media for purification were harvested from large {22 x 22cm)
culture plates.
The supernatants of 85 Dictyostelium clones were analysed for the presence of
immuno- and bioactivity. As controls, several wildtype hCG producing clones
and non-
transformed Dictyostelium clones were present on the 96-well plates.
Concentrations of
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wildtype and mutant hCG were measured using a DELFIA~ hLH assay (Wallac Oy,
Turku, Finland), which has a 100% cross-reactivity with hCG. Subsequently, the
in
vitro biological activity on the human LH/CG receptor was determined. Half of
the 85
mutmts analysed, show B/I ratios varying from 0.35 to 1.05. Taking variations
of 2
s single analyses into account, the activities of these mutants are considered
to be
comparable to wildtype hCG. Approximately 40% of the mutants show decreased
B/I
ratio's. This could be anticipated, since the mutated aminoacids 94 and 95
have been
shown to be involved in receptor binding and activation. The fact that 11
clones do not
show any hCG production is probably due to interference of the altered
aminoacids with
io appropriate folding of the mutated (3-polypeptide and/or association with
the a-subunit
or the fact that not all clones contain both an a- and a (3-subunit expression
construct.
Twenty clones with varying B/I ratios were selected for detailed analysis.
Selected clones were further grown on 6-well plates until confluence and cell
culture supernatants were collected after 4 additional days. Four of the
twenty clones
is did not produce any detectable hCG. This is in agreement with the results
for these
clones in the initial screen. Among the other clones, the amount of hCG
produced varied
considerably (116-2118 mU/ml). The cell culture supernatants were analysed for
the
presence of in vitro biological activity of hCG at a range of concentrations.
All mutants
which showed B/I ratios in the range of wildtype hCG in the initial screen,
also
ao displayed a wildtype in vitro biological activity when analysed in more
detail. The four
supernatants that did not show any immunological activity, were also not able
to
activate the LH/CG receptor. Therefore, we conclude that these clones are not
producing
any hCG. Furthermore, all mutants with decreased B/I ratios in the initial
screen indeed
show a clear increase in ICS. Their B/I ratios vary from a 2 fold to >100 fold
lower than
2s wildtype hCG produced by Dictyostelium. The dose/response curves for eight
of the
mutants are shown in Figure 6.
To correlate the observed biological activities with the mutations present in
the ~i-
subunit, the mutated expression vectors were sequenced. We isolated total DNA
from
selected Dictyostelium clones and transformed it to E.coli. For each
Dictyostelium
ao clone a number of transformants was analysed by colony-PCR for the presence
of the
a-subunit plasmid or the (3-subunit plasmid. Subsequently, DNA was isolated
from
several transformants containing the (3-subunit plasmid and sequence analysis
was
performed on the mutated region using an automated sequencer (Pharmacia).
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Thus, the majority of Dictyostelium clones contains only one type of mutation
in the hCG(3 expression vector. These clones produce a single type of mutant.
In
addition, we conclude that the majority of Dictyostelium clones analysed
contain
unrelated sequences on position 94 and 95 of the ~i-subunit, suggesting that
the
s mutagenesis has been random indeed.
Mass spectrometric analysis of rec hCG from Dictyostelium discoideum
Recombinant hCG, produced by Dictyostelium was isolated from culture
io supernatant by subsequently hydrophobic interaction chromatography and
immunochromatography with MoAb 119A that reacts with an epitope common to LH
and hCG. The antibody was immobilised to NHS-Sepharose. The acid-eluted,
purified
rec hCG was dialysed against 50% acetonitrile and lyophilised. The protein was
dissolved in 0.1 % TFA and mass spec analysis was carried out with MALDI-TOF
i5 using superDHB as matrix. Pure rec hCG produced by CHO cells and isolated
with
the same purification procedure was used as a reference.
Both a- and (3- subunits of the Dictyostelium material were of lower molecular
weight than the corresponding subunits from CHO cells , the median values of
the
peaks being 11578 and 17351 D for the Dictyostelium subunits and 14013 and
23284
ao D for the CHO polypeptides. This observation indicates that less and/or
shorter
carbohydrate chains are present on the Dicyostelium-produced hormone.
Moreover,
the peak width of the corresponding subunits differed considerably i.e. 11110-
12452
D ( 1342 D) for the Dictyostelium a- vs 12959-15591 D (2632 D) for the CHO a-
chain and 17006-18104 D (1098 D) for the Dictyostelium (3 vs 20674-25208 D
(4534
25 D) for the CHO ø-chain. This indicates that the glycosylation of the
Dictyostelium
product is far less complex.
