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Description  |
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BRIEF DESCRIPTION OF THE INVENTION
Large amounts of various modified forms of Pseudomonas exotoxin are
produced. At least one of the modified forms (pJH8) of the exotoxin
exhibits low toxicity to human or mouse cells by itself but retains its
enzymatic activity and makes a very active cell specific immunotoxin with
very low nonspecific cytotoxicity. All the constructs with low cytotoxic
activity will be useful as vaccines to produce the antibodies to treat
pseudomonas sepsis. In addition, the protein encoded by domain I alone
could be administered directly to patients to treat pseudomonas sepsis
because that domain would block toxin binding to cells. Clones containing
domain II and particularly clone pJH12 can be fused to other toxins which
have low activity (such as ricin A chain or pokeweed antiviral protein) to
increase their cell-killing activity without increasing their nonspecific
binding to cells.
BACKGROUND OF THE INVENTION
Toxins are extremely potent cell-killing agents that are responsible for
many human diseases. Because of their high activity, these agents have
been attached to monoclonal antibodies in order to form cytotoxic agents
(immunotoxins) which specifically bind to target cells. These immunotoxins
are, therefore, most useful in cancer therapy.
Pseudomonas exotoxin A (PE) is an extremely active monomeric protein
(molecular weight 66Kd), secreted by Pseudomonas aeruqinosa, which
inhibits protein synthesis in eukaryotic cells through the inactivation of
elongation factor 2 (EF-2) by catalyzing its ADP-ribosylation (catalyzing
the transfer of the ADP ribosyl moiety of oxidized NAD onto EF-2).
The intoxication process is believed to proceed by the following steps:
First, PE binds to cells through a specific receptor on the cell surface.
Next, the PE-receptor complex is internalized into the cell. Finally, PE
is transferred to the cytosol where it enzymatically inhibits protein
synthesis. The transfer process is believed to occur from an acidic
compartment, since cellular intoxication is prevented by weak bases such
as NH.sub.4 +, which raises the pH in acidic vesicles. Upon exposure to
acidic conditions, the hydrophobic domain of PE enters into the membrane,
resulting in the formation of a channel through which the enzymatic
domain, in extended form, pass into the cytosol.
PE-containing immunotoxins are constructed by first reacting native PE with
iminothiolane. This reaction serves both to introduce two new sulfhydryl
groups used for coupling an antibody to the toxin, and to inactivate the
binding of PE to its own receptor. This approach relies on the chemical
inactivation of PE-binding sites in order to minimize undesirable side
effects due to the binding of PE to cells with PE receptors. While this
approach has been reasonably successful in producing a specific cell
killing reagent in tissue culture and in tumor-bearing mice, it has not
been possible to administer more than 2 ug of PE immunotoxin to a 20 gram
mouse or 1 mg to a 3 kilogram monkey or 4 mg to an adult human, due to the
toxic side effects of the immunotoxin. It is therefore desirable to be
able to administer larger amounts of immunotoxins to achieve greater
killing of tumor cells. The present invention fulfills this desire by
providing an immunotoxin with high potency and low toxicity. Furthermore,
to overcome the above-noted reliance on chemical inactivation of the PE
binding sites, the present invention incorporates recombinant DNA
techniques to clone the complete toxin gene (or segments of it) in order
to express at high levels the full length toxin molecule (or portions of
it) containing different functional domains, including one which lacks the
cell binding domain. See Example 1 for a comparative examination of these
clones.
The three-dimensional structure of PE has been determined by x-ray
crystallography. As shown in FIG. 2, the PE molecule contains three
structurally distinct domains: Domain I contains amino acid residues 1 to
252 (Domain Ia), and 365 to 404 (Domain Ib); domain II contains amino acid
residues 253 to 364; and domain III contains amino acid residues 405 to
613.
Plasmids have been constructed which express various portions of the PE
molecule, providing the ability to correlate different structural domains
with various functional activities and to determine (1) which portions of
the molecule are responsible for cell recognition (binding), (structural
domain I, amino acid 1-252); (2) which portion is required for enzymatic
activity (ADP ribosylating activity, domain III plus a portion of domain
Ib) (amino acid 385-613); (3) which portion is responsible for
translocation across cell membrane (domain II). The evidence that
structural domain Ia is involved in cell recognition is that proteins
produced by plasmids without domain Ia but containing domain II, Ib and
III, are not cytotoxic by themselves and do not show competitive
inhibition of the cytotoxicity of intact toxins, whereas a plasmid
encoding domain Ia, but missing other domains, blocked PE cytotoxicity of
sensitive cells. PE from plasmids with a deletion of the first half of
structural domain II exhibit both PE blocking activity and
ADP-ribosylation activity, but these molecules lost all cell killing
activity. Plasmids which encode only structural domain III produce large
amounts of the protein, but lack detectable enzymatic activity
(ADP-ribosylation). However, plasmids which encode all of structural
domain III plus adjacent amino acids from structural domain Ib express
large amounts of the protein and their ADP-ribosylation activity is high.
Based on the three dimensional structure of PE, plasmids have been
constructed which express different portions of PE. The protein pattern of
the cells expressing the different constructions was analyzed by SDS gel
electrophoresis and the ADP ribosylating activity of the recombinant
toxins was measured. Of these plasmids (described in Example 1) plasmid
pJH8, containing amino acids 253 to 613 (Domains Ib, II, and III), and
plasmid pJH17, containing amino acids 385 to 613 (Domain III, plus 20
adjacent amino acids from Domain Ib), are able to encode large amounts of
modified PE exhibiting low toxicity to human cells, but remaining
enzymatically very active.
Taken together, the plasmid patterns indicate that structural domain Ia is
a receptor binding domain, that the first half of structural domain II is
required for translocation of the toxin from a host cell's endocytic
vesicle to the cytosol, and that structural domain III alone is not
sufficient to express full ADP-ribosylation activity.
When administered to animals, PE characteristically produces death due to
liver failure. Immunotoxins made with PE also attack the liver and, when
given in large amounts, produce death due to liver toxicity. The
experiments shown in Table III indicate that Domain Ia is responsible for
cell binding and indicate that a PE molecule in which Domain Ia is deleted
is less toxic to mice than native PE. The data shown in Example 4 support
this conclusion, indicating that modified PE is at least 200-fold less
toxic to mice than native PE. Example 5 shows that when the protein made
by pJH8 is purified and coupled to an antibody to the human transferring
receptor, an immunotoxin is created that is approximately as active as an
immunotoxin containing native PE but is 100-fold less toxic on nontarget
cells (mouse cells).
One aspect of the present invention, therefore, is that PE molecules with a
deletion of Domain Ia are effective immunotoxins with diminished side
effects including diminished liver toxicity.
DESCRIPTION OF THE FIGURES
FIG. 1 shows the construction of the plasmids of the present invention used
for the expression of different domains of PE.
FIG. 2 is a simplified map of the different sized PE molecules produced as
a part of the present invention.
FIGS. 3A and 3B show the effect of PE.sub.45 -HB21 and PE-HB21 on protein
synthesis in human KB cells. PE.sub.45 is the toxin protein lacking Domain
I encoded by plasmid pJH8. Cells were incubated for 24 hours with the
appropriate immunotoxin and then incubated with .sup.3 H-leucine for 1
hour. Incorporation of (.sup.3 H)-leucine into protein was measured. In
panel 3B Swiss 3T3 cells were incubated for 24 hours with either native
pseudomonas toxin (PE), native pseudomonas toxin coupled to HB21,
PE.sub.45 alone, or PE.sub.45 coupled to HB21. Cells were exposed to these
agents for 24 hours and then protein synthesis measured for 1 hour as
above.
SPECIFIC DISCLOSURE OF THE INVENTION
In the preferred embodiment, the present invention is the production of a
modified form of Pseudomonas Exotoxin (PE) wherein the modification
results in an active immunotoxin. The modified toxin and the immunotoxin
made from it have markedly diminished non-specific toxicity on human and
mouse cells in tissue culture and with greatly diminished toxicity in mice
in vivo.
Genomic DNA is obtained by completely digesting a strain of Pseudomonas
aeruginosa with EcoRI and PstI. Although any strain of P. aeruginosa is
suitable for use in this invention, the preferred strain is PA103, a
strain which produces a large amount of active toxin. DNA fragments
ranging in size from 2.6 to 2.9 kb are isolated from the genomic DNA
library, and ligated with a 2.7 kb DNA fragment from plasmid pUC13 cut
with EcoRI and PstI. Plasmid pUC13 is commercially available from
Boehringer Mannheim. A suitable host, preferably an E. coli strain, is
then transformed with the ligated products (the preferred strain of E.
coli is HB101, commercially available from Bethesda Research
Laboratories). The 2.7 kb DNA fragment derived from ptoxETA, a plasmid
which contains the PE structural gene, is used as a probe and plasmid DNA
from several positive colonies were prepared and characterized by Southern
blotting. The different sizes of recombinant PE are then expressed in a
suitable host. A host which carries the bacteriophage T7 RNA polymerase
gene in lysogenic and inducible form [BL21(DE.sub.3)]is preferred, and is
obtainable from Dr. F. W. Studier at Brookhaven National Laboratories.
Also preferred are plasmids which carry the bacteriophage late T7 promoter
and AMP.sup.r and Shine-Dalgarno box--pAR2156, also obtainable from Dr.
Studier.
As is shown in the Examples, the preferred plasmids are pJH8 and pJH17.
pJH8 contains a DNA fragment of 5.1 kb and is capable of expressing
Domains II, Ib, and III of PE. This plasmid is produced by partially
cutting plasmid pJH4 with AvaI. The linearized DNA fragment is then
completely cut with HindIII in order to obtain a 5.1 kb DNA fragment which
has AvaI and HindIII sites at its ends. This fragment is then incubated
with S1 nuclease to remove its cohesive ends, followed by ligation with T4
ligase to form plasmid pJH8.
Plasmid pJH17 contains a DNA fragment of 4.8 kb and is capable of
expressing Domain III with the adjacent 20 amino acids of PE. This plasmid
is produced by completely cutting plasmid pJH4 with HindIII and ApaI. The
4.8 kb DNA fragment is then isolated and incubated with Klenow DNA
polymerase I and dNTP to fill the cohesive ends, followed by ligation with
T4 ligase.
Expression of Recombinant Toxins in BL21(DE.sub.3).
BL21(DE.sub.3) containing plasmids for expression of different sizes of PE
is cultured in LB broth with 50 ug Ampicillin/ml at 37.degree. C. When
absorbance at 650 nm reaches 0.3, IPTG (isopropyl
beta-D-thio-galactopyranoside) is added to the culture at a final
concentration of 1 mM. Cells are harvested 90 minutes later and analyzed
for the amount of recombinant toxins by SDS-PAGE, immunoblotting,
ADP-ribosylation, and cell killing experiments.
SDS-PAGE and Immunoblotting
Cells pellets are dissolved in Lemmli buffer. Samples are boiled for 5
minutes prior to application to a 0.1% SDS, 10% acrylamide slab gel. For
immunoblotting, samples after electrophoresis are transferred to a
nitrocellulose paper, followed by reaction with antibody to PE, then a
second antibody (goat anti-rabbit) and staining. The antibody to PE is
obtained by hyperimmunizing rabbits with glutaraldehyde (0.2%) reacted PE
(250 ug of PE per injection). An IgG fraction was prepared for
immunoblotting.
Assay of ADP-ribosylation Activity
Known procedures are used for assay of ADP-ribosylation activity. Briefly,
rabbit reticulocyte preparations or wheat germ extracts enriched with
elongation factor 2 (EF-2) are used as a source of EF-2. Assays (500 ul
total volume) contain about 10 pmole of EF-2, 37 pmole of .sup.14 C-NAD
(0.06 u Ci), 0.25 to 1.25 ug of PE and buffer (40 mM DTT, 1 mM EDTA, and
50 mM Tris, pH 8.1). Activity is measured as pmoles of NAD transferred to
EF-2 in 30 minutes. A standard curve of known concentrations of PE is
established and used to determine the activity of PE in extracts from E.
coli. After incubation for 30 minutes at 37.degree. C., 0.5 ml 12% TCA is
added to each assay mixture. The assay mixtures are then set in an ice
bath for 15 minutes, followed by centrifugation at 4.degree. C., 3,000 xg
for 10 minutes. The pellet is washed with 1 ml 6% TCA and centrifuged as
above. The pellet is then measured for .sub.14 C radioactivity in a liquid
scintillation counter as the index of the ADP-ribosylation activity.
Cell Cytotoxicity Test
Tests of the cytotoxic activity of the modified PE are performed in NIH 3T3
cell cultures and human KB cells. NIH 3T3 cells or KB cells are seeded 24
hours prior to the cytotoxicity test in a 24-well tissue culture plate at
a density of 2.times.10.sup.4 cells per well. After incubation for 48
hours with various concentrations of PE or protein extracts isolated from
BL21(DE.sub.3) with plasmids which express different sizes of PE, the
monolayers are stained with methylene blue to detect the surviving cells.
The results are shown in Table 1.
Inhibition of Protein Synthesis
Assays for the inhibition of protein synthesis by PE or PE with extracts
from BL21(DE.sub.3)/pJH8 are performed in Swiss 3T3 cell cultures. Swiss
3T3 cells are seeded one day before assay in 24-well tissue culture plates
at a density of 10.sup.5 cells per well. Cells are washed once by
replacing media with DMEM and 0.2% BSA before adding PE (100 ng/ml) or PE
(100 ng/ml) with extracts which contained an excess amount of different
sizes of PE (Table 2). After 15 minutes at 37.degree. C., the medium is
then removed and replaced with fresh DMEM and 0.2% BSA. Four hours later,
the rate of protein synthesis is assayed by adding [.sup.3 H] leucine to
the medium (to a final concentration of 2-4 uCi/ml) for 1 hour.
In the preferred embodiment of the invention, the structural domain of the
Pseudomonas exotoxin gene is inserted into a T7 expression vector
downstream of the ribosome binding site and its accompanying ATG
initiation codon (as shown in FIG. 1), as described above. To produce
recombinant proteins, cells are grown at 370.degree. C. to an A.sub.650
=0.3. IPTG is then added at 1 mM to induce T7 RNA polymerase and incubated
for 2 hours. A series of plasmids are produced, as shown in Example 1.
Next, a clone is constructed which encodes a PE molecule without a leader
sequence (pJH4) and in which a methionine is placed adjacent to the
alanine at the amino terminus of the processed form of native PE (Table
1). The protein produced by pJH4 is designated Met-PE. Large amounts of
Met-PE are produced upon induction by IPTG, representing 20% of total cell
protein. ADP ribosylating activity equivalent to 0.1 mg native PE is found
in the supernatant, and 0.2 mg in the pellet per mg of total cell protein.
The PE molecule produced by pJH4 differs from native PE molecules by the
presence of one extra methionine residue at the amino-terminus.
pJH8, produced from pJH4 as noted above, expresses a protein in which most
of Domain Ia is deleted (retaining only the added methionine and three
amino acids at the amino terminus. Immunoblotting shows that this protein
is 45 kd, and is expressed at a concentration of approximately 0.04 mg/mg
cell protein. High ADP ribosylating activity is exhibited when urea and
DTT is excluded from the reaction mixture. In contrast to native PE (where
the addition of urea and DTT activates ADP ribosylating activity), the
addition of urea and DTT to pJH8 reduces (by about 30%) ADP ribosylating
activity.
Because the ADP ribosylating activity of PE resides in the carboxyl end of
the molecule, plasmid pJH17, constructed from pJH4 (as noted above), was
produced. This plasmid contains Domain III and 20 amino acids from Domain
Ib. Extracts from this plasmid contain high levels of ADP ribosylating
activity--equivalent to 0.06 mg PE (Table 1). By immunoblotting, a 31 kd
protein is detected, present at a concentration of 0.03 mg/mg cell
protein.
None of the plasmids produced by the above-noted process (and described in
the Examples), except for pJH1, pJH2, and pJH4, exhibit significant cell
killing ability, even though many of these exhibit high enzymatic activity
(Table 1). These plasmids are shown to prevent inhibition of protein
synthesis or cell killing by native PE by exposing cells for 15 minutes to
native PE at 0.1 ug/ml in the presence of 3-5 ug/ml of various modified
toxins. The data in Table III shows that plasmids expressing either Domain
Ia alone, or Domain I, half of Domain II and Domain III prevent PE from
inhibiting protein synthesis.
Animal Toxicity
Mice receiving native PE succumb due to liver destruction. The lethal dose
for a 20 gram mouse is 0.1-0.2 ug. The data in Table IV shows that PE with
a deletion of Domain Ia exhibits greatly diminished toxicity in mice. Mice
were injected I.P. with either native PE or lacking Domain Ia. All mice
receiving 1.0 ug of native PE and one-half receiving 0.2 ug PE were dead
at 48 hours. Two of three mice receiving 50 ug of PE Ia died; all
receiving 20 ug of PE Ia lived; and all mice receiving 5 ug PE Ia lived.
Thus, PE Ia is more than 100 times less toxic on a weight basis than
native PE.
Gene Fusions Using Recombinant Modified Pseudomonas Exotoxin
The gene for modified PE of this invention specifically contained in pJH8
was fused with DNA sequences encoding the human alpha transforming growth
factor (a-TGF) or human interleukin 2 (IL-2). See Examples 7 and 8 for the
specifics of these gene fusions. Modified PE is suitable for use in
fusions with any peptide hormone, growth factor, or other polypeptide cell
recognition protein for which a specific receptor exists on cells. A few
examples include: insulin, glucagon, endorphins, growth hormone,
melanocyte stimulating hormone, transferrin, bombesin, low density
lipoprotein, luteinizing hormone, and asialoglycoproteins.
EXAMPLES
Example 1
As shown in Table 1, the process of the present invention was used to
construct several plasmids containing fusions of the different sizes of PE
structural gene and the T7 late promoter. pJH2 contains the intact PE
structural gene with a segment coding for a modified leader sequence in
front. pJH4 contains the intact PE structural gene with the addition of a
methionine codon at the amino-terminus. pJH7 contains the gene encoding
structural Domain III of PE (amino acid 405-613). pJH8 contains the gene
encoding structural Domain II, Ib, and III of PE (amino acid 253-613.
pJH13 encodes PE with a deletion of the first half of structural domain II
encompassing amino acids 253-307. pJH14 encodes structural domain Ia of PE
(amino acids 1-252). pJH17 encodes structural domain III with an
additional adjacent sequence of 20 amino acids from Domain Ib (amino acids
385-613).
pJH1 was constructed by partially cutting the native PE (pE 0) with BamHI,
and the linear form of DNA was eluted and completely cut with EcoRI. The
2.0kb DNA fragment derived from pE 0, which has BamHI and EcoRI at the two
ends, was then inserted into pAR 2156, which had been completely cut with
BamHI and EcoRI.
pJH2 was constructed by partially cutting pJH1 with BamHI. The linear form
of DNA was isolated and completely cut with NdeI. The 610 kb DNA fragment
was saved to construct pJH2 by ligating it with synthetic oligonucleotide
duplex
##STR1##
pJH4 was constructed by partially cutting pJH2 with TaqI. The linearized
DNA (610 kb) was isolated and completely cut with NdeI. The largest DNA
fragment (5.9 kb) was separated and ligated with synthetic oligonucleotide
duplex
##STR2##
pJH7 was constructed by partially cutting pJH4 with AatII. The linearized
DNA fragment (5.9 kb) was then completely cut with HindIII. The 4.7 kb DNA
fragment which has AatII and HindIII sites at its ends after separation
was incubated with S1 nuclease to remove the cohesive ends, followed by
ligation with T4 ligase.
pJH8 was constructed as described in the Specific Disclosure.
pJH13 was constructed by partially cutting pJH4 with AvaI. The linearized
DNA fragment was then completely cut with EcoRI. The 4.7 kb DNA which has
AvaI and EcoRI cut at both ends was incubated with S1 to remove cohesive
ends (DNA fragment 1). pJH4 was partially cut with SalI. The linearized
DNA fragment was then completely cut with EcoRI. The 1.0 kb DNA fragment
which has SalI and EcoRI sites at the ends was incubated with Klenow DNA
polymerase I and dNTP to fill the cohesive ends (DNA fragment 2). DNA
fragment 1 (4.7 kb) and DNA fragment 2 (1.0 kb) were ligated at 4.degree.
overnight.
pJH14 was constructed by partially cutting pJH4 with AvaI. The linearized
DNA fragment was then completely cut with EcoRI. The 4.7 kb DNA which has
AvaI and EcoRI sites at its ends was incubated with S1 to remove the
cohesive end, followed by ligation with T4 ligase.
pJH17 was constructed as described in the Specific Disclosure.
Example 2
The amount and activity of the recombinant toxins (produced by the plasmids
described in Example 1) were measured by SDS-PAGE, ADP-ribosylation, and
cell killing ability. The results are tabulated in Table 1.
EXAMPLE 3
Determinations were made of the sizes of the protein and the domains
present in various constructions of the present invention. The results are
shown in Table 2.
Example 4
The effect of various deletions on cell protein synthesis was determined,
in the presence and absence of native PE. The results are shown in Table
3. Structural Domain Ia (pJH14) and structural domain I, half of II, and
III (pJH13) were extracted from the pellet of the sonicated cells with 8M
urea. 10 ul of each extract equivalent to 3-5 ug of recombinant proteins
was used in each assay. Structural II, Ib, and III (pJH8) were present in
the supernatant of the sonicated BL21(DE.sub.3)/pJH8 cells. 10 ul of
extract equivalent to 2 ug of recombinant toxin was used. Cells were
treated as indicated in Table 3 for 15 minutes with and without native PE
at 0.1 ug/ml followed by 1 ml DMEM washing; incubated for 4 hours in DMEM
and 0.2% BSA, then incubated with .sup.3 H-leucine for 1 hour.
Example 5
As shown in Table 4,the dose causing death in mice was determined by
injecting Balb/c mice I.P. with various amounts of PE contained in 1.0 ml
of sterile saline and 10 mgs/ml sterile human albumin. The animals were
monitored daily for two weeks. All deaths occurred at 48 hours.
Example 6
As shown in FIGS. 3A and 3B an immunotoxin composed of the 45 kD protein
produced by pJH8 conjugated by a disulfide bond to an antibody to the
human transferrin receptor (PE.sub.45 -HB21) kills human cells expressing
the transferrin receptor (ID.sub.50 3 ng/ml). It has little or no effect
on mouse cells which do not express the human transferrin receptor at 1000
ng/ml, whereas native PE conjugated by a sulfide bond to HB21
nonspecifically kills mouse cells at an ID.sub.50 of 29 ng/ml. The data in
FIG. 3 indicate the non-specific toxicity of PE.sub.45 -HB21 is 100-fold
less than PE-HB21.
Example 7
Construction of alpha Transforming Growth Factor-PE fusion gene
pJH8 was treated with Tth lllI and SphI to construct a smaller plasmid pVC8
which has fewer AvaII sites. pVC8 was partially cut with AvaII and ligated
to a synthetic oligonucleotide (30 bp) which contains a STuI site, a Tth
lllI site, and a stop codon in order to create pVC31. pVC31 was cut with
Tth lllI, and filled in with a Klenow fragment, a fragment of DNA
polymerase, and ligated to a blunt ended clone containing the alpha-TGF
gene (pVC 33). The alpha-TGF DNA, p-hTGF-10-925 [Derynck etal, Cell,
38:287-297 (1984)] was cut with EcoRI and BglI to give a 322 bp fragment
which was isolated and cut with Fnu 4HI and treated with T4 polymerase to
give a 152 bp fragment which in turn was ligated to pVC31 to create
PE-alpha-TGF fusion gene. When expressed in E. coli B121, this plasmid
produces a protein that reacts with antibodies to alpha-TGF and to PE and
has a molecular weight of 51,000.
Example 8
Construction of IL-2-PE Fusion Gene
pVC8 was cut with AvaII at position 1190, the 3.6 fragment isolated, its
single stranded ends filled with Klenow enzyme, and dephosphorylated to
produce fragment I. Clone PST-5 [Gallo et al, PNAS, 81:2543-2547 (1984)]
was cut with PstI to produce a 1 kD fragment of IL-2 which was then cut
with Bsp 1286 at positions 105 and 669 and treated with T4 polymerase to
fill up the ends. The 564 bp fragment was ligated to fragment I to create
pHL-1 and transformed into BL21 expression cells. Upon induction, a 60 kD
protein was produced that reacts with antibodies to IL-2 and to PE, and
contains ADP-ribosylating activity.
TABLE 1
______________________________________
Summary of the Amount and Activity of the Recombinant
Toxins Measured By SDS-PAGE, ADP-ribosylation, and Cell
Killing Experiments
Amount of PE per mg of Cellular Protein
Measured by Measured by Measured by
SDS-PAGE ADP-ribosylation*
Cell Killing
(mg) (units) (units)
Total Sup. Pellet Sup. Pellet
______________________________________
pJH1 0.20.sup.a N.D. N.D. N.D. N.D.
pJH2 0.20.sup.a N.D. N.D. N.D. N.D.
pJH3 degraded <0.001 <0.001 N.D. N.D.
pJH4 0.20.sup.a 0.10 0.20 <0.001 0.2
pJH5 degraded 0.15 0.02 N.D. N.D.
pJH6 degraded <0.001 <0.001 N.D. N.D.
pJH7 0.25.sup.o,a
<0.001 <0.001 <0.001 <0.001
pJH8 0.04.sup.o,n
0.66 0.04 <0.001 <0.001
pJH9 0.15.sup.a 0.40 0.20 <0.001 <0.001
pJH10 0.15.sup.a 0.40 0.15 <0.001 0.001
pJH11 0.25.sup.a <0.001 <0.001 <0.001 <0.001
pJH12 <0.01.sup.o,n
<0.001 <0.001 N.D. N.D.
pJH13 0.01 0.25 0.20 <0.001 <0.001
pJH14 0.30.sup.a <0.001 <0.001 <0.001 <0.001
pJH15 0.10.sup.a 0.18 0.30 <0.001 <0.001
pJH16 < 0.01.sup.n
0.02 N.D. N.D. N.D.
pJH17 0.03.sup.o,n
0.06 <0.001 <0.001 <0.001
pJH18 <0.01.sup.n 0.02 N.D. N.D. N.D.
______________________________________
*1 unit of ADPribosylation or cell killing activity is equivalent to the
activity from 1 mg of native PE.
N.D. = not determined
.sup.a aggregated
.sup.o positive by Western
.sup.n not visible on SDS PAGE
TABLE 2
______________________________________
Construction Domain Present
Protein Size
______________________________________
pJH4 met, I, II, III
68 kd
pJH7 III 28 kd
pJH8 II, Ib, III 45 kd
pJH13 I, half of II, III
63 kd
pJH14 Ia 32 kd
pJH17 20 a.a. of Ib, III
31 kd
______________________________________
TABLE 4
______________________________________
Toxin Dose (ug)
Deaths
______________________________________
PE 50 2/3
PE 20 0/3
PE 5 0/3
PE 10 2/3
PE 1.0 3/3
PE 0.3 3/3
PE 0.2 1/2
PE 0.1 0/3
______________________________________
TABLE 3
______________________________________
3H--leucine in
corporation (cpm .times. 10.sup.3)
with PE
Domain Tested without PE
(I, II, III)
______________________________________
none 11.2 .+-. .1
2.3 .+-. .2
Ia 11.5 .+-. .15
9.4 .+-. .3
II, Ib, & III 11.7 .+-. .15
2.4 .+-. .2
I, 1/2 II, III 10.7 .+-. .15
9.2 .+-. .2
8 M urea (10 ul)
11.1 .+-. .1
2.9 .+-. .2
______________________________________
Statement of Deposit
The following plasmids have been deposited in the American Type Culture
Collection in Rockville, Md., under the respective ATCC numbers, prior to
the filing of this application, and at issuance of this application into a
patent will be maintained for a term of thirty (30) years from the date of
deposit or five (5) years after the last request for such deposit or for
the effective life of the patent, whichever is longest. The deposits will
be replaced if the cultures mutate or become nonviable during the term of
the deposit:
______________________________________
pJH12 ATCC 67205
pHL-1 ATCC 67206
pVC33 ATCC 67207
pJH8 ATCC 67208
pJH14 ATCC 67209
______________________________________
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