U.S. patent application number 11/038652 was filed with the patent office on 2005-08-04 for oligomerization of hepatitis delta antigen.
This patent application is currently assigned to President and Fellows of Harvard College. Invention is credited to Elkin, Carl, Filman, David, Hogle, James M., Zuccola, Harmon J..
Application Number | 20050170337 11/038652 |
Document ID | / |
Family ID | 33566958 |
Filed Date | 2005-08-04 |
United States Patent
Application |
20050170337 |
Kind Code |
A1 |
Hogle, James M. ; et
al. |
August 4, 2005 |
Oligomerization of hepatitis delta antigen
Abstract
This invention relates to HDAg peptides, including mutants,
derivatives fragments and fusion molecules, including fusion
proteins, coiled-coil oligomers, nucleic acid molecules, vectors
comprising HDAg nucleic acid molecules, cells comprising said
molecules, methods of multivalent expression and association of
binding moieties of HDAg fusion molecules, and methods of use
involving the molecules. The molecules are particularly useful as a
framework for multivalent display via formation of C-terminal
and/or N-terminal fusion proteins or via chemical coupling to
chemically reactive sidechains, e.g. cysteine residues.
Inventors: |
Hogle, James M.; (Newton,
MA) ; Zuccola, Harmon J.; (Brookline, MA) ;
Filman, David; (Auburndale, MA) ; Elkin, Carl;
(Cambridge, MA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
President and Fellows of Harvard
College
Cambridge
MA
|
Family ID: |
33566958 |
Appl. No.: |
11/038652 |
Filed: |
January 18, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11038652 |
Jan 18, 2005 |
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09347175 |
Jul 1, 1999 |
|
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6844171 |
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60091609 |
Jul 2, 1998 |
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Current U.S.
Class: |
435/5 ;
435/235.1; 435/325; 435/456; 435/69.3; 530/350; 536/23.72 |
Current CPC
Class: |
C07K 2299/00 20130101;
C12N 2760/10122 20130101; C07K 2319/00 20130101; C07K 14/005
20130101 |
Class at
Publication: |
435/005 ;
435/069.3; 435/456; 435/325; 435/235.1; 530/350; 536/023.72 |
International
Class: |
C12Q 001/70; C07H
021/04; C07K 014/02; C12N 015/86 |
Goverment Interests
[0002] The invention was made with Government support from the
National Institutes of Health under grant A132480 and R01-HL37974
and the U.S. Public Health Service (GM42031) and the Giovanni
Armorisse Harvard Center for Structural Biology. The Government has
certain rights in the invention.
Claims
What is claimed is:
1. An isolated nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of: a) a nucleotide
sequence depicted in FIG. 9, nucleotides 37-150 of FIG. 9,
nucleotides 37-186 of FIG. 9, FIG. 10, nucleotides 1421-1566 of
FIG. 10, nucleotides 1457-1566 of FIG. 10, FIG. 15 and FIG. 16; b)
a complementary strand of the sequence of a); c) DNA sequences that
hybridize to the sequence of a) or b); and d) RNA sequences
transcribed from the sequences of a), b) or c), or a fragment or
mutation thereof, which encodes a coiled-coil oligomer.
2. An isolated nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of: a) a nucleotide
sequence encoding a polypeptide comprising an amino acid sequence
depicted in a row of FIG. 1, amino acids 12-48 of a row of FIG. 1,
the top row of FIG. 3C, FIG. 9, amino acids 12-48 of a row of FIG.
9, FIG. 10, amino acids 12-88 of FIG. 10, FIG. 11 and FIG. 17; b)
the complementary strand of the sequence of a); c) RNA sequences
transcribed from the sequences of a) or b), or a fragment or
mutation thereof, which encodes a coiled-coil oligomer.
3. An isolated nucleic acid molecule encoding a fusion molecule
comprising a fusion molecule comprising HDAg and at least one
binding moiety.
4. A fusion gene comprising a nucleic acid molecule of claim 1
operably linked to a nucleic acid molecule encoding a heterologous
peptide.
5. A fusion gene comprising a nucleic acid molecule of claim 2
operably linked to a nucleic acid molecule encoding a heterologous
peptide.
6. A vector comprising a nucleic acid molecule which encodes a
subunit of an HDAg coiled-coil octamer.
7. A vector comprising a nucleic acid molecule of claim 1.
8. A vector comprising a nucleic acid molecule of claim 2.
9. A vector comprising a nucleic acid molecule of claim 3.
10. A vector comprising a nucleic acid molecule encoding a fusion
molecule comprising HDAg and at least one binding moiety.
11. A vector comprising a nucleic acid molecule encoding HDAg and
at least one multiple cloning site.
12. The vector of claim 11 wherein at least one multiple cloning
site is located 3' to the nucleic acid molecule encoding HDAg.
13. The vector of claim 11 wherein at least one multiple coding
site is located 5' to the nucleic acid molecule encoding HDAg.
14. The vector of claim 11 wherein there are at least two multiple
coding sites, wherein at least one multiple coding site is located
in a flanking region 3' to the nucleic acid molecule encoding HDAg
and at least one multiple coding site is located in a flanking
region 5' to the nucleic acid molecule encoding HDAg.
15. A vector comprising a nucleic acid molecule of claim 1 and at
least one multiple cloning site.
16. A vector comprising a nucleic acid molecule of claim 2 and at
least one multiple cloning site.
17. The vector of claim 16 further comprising a nucleic acid
molecule encoding a nuclear localization signal.
18. The vector for expression of the fusion molecule comprising
HDAg and at least one binding moiety wherein a first heterologous
gene encodes a first binding moiety and a second heterologous gene
encodes a second binding moiety.
19. A vector of claim 11 which further comprises a nucleic acid
molecule which encodes a heterologous gene.
20. A host cell which comprises a nucleic acid molecule which
encodes a fusion molecule comprising HDAg and at least one binding
moiety.
21. A host cell which comprises a nucleic acid molecule of claim
1.
22. A host cell which comprises a nucleic acid molecule of claim
2.
23. A method of manufacturing a host cell comprising a nucleic acid
molecule encoding a fusion molecule comprising HDAg and at least
one binding moiety comprising introducing a vector of claim 10 into
the host cell.
24. A method of expressing a high valency display of at least one
binding moiety comprising introducing into a cell a vector
comprising a nucleic acid molecule encoding HDAg and a nucleic acid
molecule encoding the binding moiety and culturing the cell under
conditions sufficient to permit expression of a fusion molecule
comprising the binding moiety and HDAg.
25. A method for delivering molecules to a cell comprising
contacting them with a fusion molecule comprising HDAg and at least
one binding moiety.
26. The method of claim 25 wherein the binding moiety is an
oligonucleotide.
27. The method of claim 25 wherein the oligonucleotide hybridizes
to a nucleic acid molecule in the cell.
28. The method of claim 26 wherein said fusion molecule further
comprises a double-stranded nuclease.
29. The method of claim 25 wherein the fusion molecule comprises a
first binding moiety and a second binding moiety wherein the first
binding moiety interacts with a binding partner and the second
binding moiety functions as an effector.
30. The method of claim 29 wherein the first binding moiety
interacts with a cell surface receptor and the second binding
moiety can kill the cell.
31. A method of amplifying a signal in a solid phase assay
comprising coupling an HDAg octamer with at least one copy of a
domain which interacts with a ligand and at least two copies of a
label.
32. A method of claim 31 wherein the label is selected from the
group consisting of alkaline phosphatase, a radiolabel,
streptadavin and green fluorescent protein.
33. The method of claim 31 wherein the solid phase assay is an
ELISA assay.
34. A method of facilitating exchange of substrates and products
comprising coupling an HDAg oligomer to at least two enzymes which
function in a linked pathway.
35. A method of enhancing a reaction between binding partners
comprising coupling the binding partners to an HDAg oligomer.
36. A method of enhancing a reaction between two binding partners
comprising coupling one binding partner to an HDAg oligomer and
contacting the oligomer to a second binding partner.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 09/347,175, filed Jul. 1, 1999, which claims priority to U.S.
Provisional Application No. 60/091,609 filed Jul. 2, 1998. The
entire teachings of the above applications are incorporated herein
by reference.
BACKGROUND OF THE INVENTION
[0003] The hepatitis D virus (HDV) is a small satellite virus of
hepatitis B virus (HBV). Coinfection with HBV and HDV causes severe
and sometimes fatal liver disease in humans. The HDV genome encodes
a single known protein, the hepatitis delta antigen (HDAg).
SUMMARY OF THE INVENTION
[0004] This invention is based on the discovery of the high
resolution crystal structure of a synthetic peptide corresponding
to residues 12-60 of the hepatitis delta antigen (HDAg). This
peptide includes a coiled-coil region believed to be important for
dimerization of HDAg. The peptide forms an antiparallel coiled coil
with hydrophobic residues near the termini of each peptide forming
an extensive hydrophobic core with residues C-terminal to the
coiled-coil domain in the dimer protein. The crystal structure
shows how HDAg forms dimers, but, surprisingly, also shows the
dimers forming an octameric structure that forms a large 50 .ANG.
ring lined with basic sidechains.
[0005] The dimers associate further to form octamers through
residues in the coiled-coil domain that are not involved in a
heptad repeat, as well as through residues C-terminal to this
region. The crystal structure of the peptide and cross-linking
hydrodynamic studies which show that the full-length recombinant
protein also forms octamers suggest that the structure of the delta
antigen represents a previously unseen organization of a viral
nucleocapsid protein. This N-terminal octamer can serve as a
convenient high-valency framework for linking a variety of
functional peptides and domains.
[0006] The invention includes HDAg proteins, including derivatives,
mutants and fragments, and nucleic acid molecules encoding HDAg.
Derivatives of HDAg protein include fusion molecules. In one
embodiment, the fusion molecule comprises HDAg and at least one
binding moiety bound, for example, to the HDAg through the C
terminus, N terminus and/or other amino acid. The binding moiety
can be selected from the group consisting of an antigen, an
antibody, a ligand, a receptor, an enzyme, a ligand interaction
peptide, a chemical, an effector, an oligonucleotide, a signal
amplification peptide, an enhancer recognition protein, a promoter
binding protein, a label, a growth factor, a cytokine, a nuclease,
a small organic molecule, a test substance, a cytotoxic agent, a
substrate, a solid substrate, a drug, or a fragment thereof. The
fusion molecules of the invention can also comprise two binding
moieties which are binding partners. The fusion molecule can be a
fusion protein. The HDAg and the binding moiety can be chemically
linked or the HDAg and the binding moiety can be expressed as a
single unit.
[0007] The invention also relates to coiled-coil oligomers
comprising at least two such fusion molecules. The coiled-coil
oligomer can be an octamer. In the coiled-coil oligomer, the two
fusion molecules can be the same or different.
[0008] The invention also relates to nucleic acid molecules. For
example, a nucleic acid molecule can comprise a nucleotide sequence
depicted in FIG. 9, nucleotides 37-150 of FIG. 9, nucleotides
37-186 of FIG. 9, FIG. 10, nucleotides 1421-1566 of FIG. 10,
nucleotides 1457-1566 of FIG. 10, FIG. 15 or FIG. 16. The nucleic
acid molecule can also comprise a nucleotide sequence which encodes
a polypeptide comprising an amino acid sequence depicted in a row
of FIG. 1, amino acids 12-48 of a row of FIG. 1, the top row of
FIG. 3C, FIG. 9, amino acids 12-48 of a row of FIG. 9, FIG. 10,
amino acids 12-88 of FIG. 10, FIG. 11 or FIG. 17. Also included are
complementary strands of these sequences, DNA sequences that
hybridize to the sequences, RNA sequences transcribed from the
sequences, or a fragment or mutation thereof, which encodes a
coiled-coil oligomer.
[0009] An isolated nucleic acid molecule can be a fusion molecule
described herein. The invention also includes fusion genes
comprising an HDAg nucleic acid molecule operably linked to a
nucleic acid molecule encoding a heterologous (non-HDAg)
peptide.
[0010] Also encompassed in the scope of the invention are isolated,
purified and/or recombinant peptides and molecules comprising
peptides. In one embodiment, a polypeptide comprises an amino acid
sequence encoded by an HDAg nucleic acid molecule. The molecules
can comprise a polypeptide having an amino acid sequence selected
from the group consisting of an amino acid sequence depicted in a
row of FIG. 1, amino acids 12-48 of a row of FIG. 1, amino acids
12-60 of a row of FIG. 1, the top row of FIG. 3C, FIG. 9, amino
acids 12-48 of FIG. 9, amino acids 12-60 of FIG. 9, FIG. 10, FIG.
11 and FIG. 17, or a fragment or derivative thereof which forms a
coiled-coil oligomer. The peptide can be a derivative peptide
wherein a serine residue is substituted with cysteine. The
molecules can comprise a polypeptide comprising an amino acid
sequence of amino acids 12-88 of HDAg, or a fragment or derivative
thereof which forms a coiled-coil oligomer and nuclear localization
signal. The polypeptides can be encoded by fusion genes comprising
HDAg. It is possible that the molecule can be larger than the 12-48
or 12-60 or 12-88 amino acids, for example. It may be desirable to
make a 12-65 or 10-93 peptide, for example.
[0011] The invention also includes vectors which can express HDAg.
The vectors can comprise a nucleic acid molecule which encodes a
subunit of an HDAg coiled-coil octamer. The nucleic acid molecule
can comprise a sequence listed above. The nucleic acid molecule can
encode a fusion molecule. The vector can be a nucleic acid molecule
encoding HDAg and at least one multiple cloning site. The multiple
cloning site(s) can be located 3' to the nucleic acid molecule
encoding HDAg or 5' to the nucleic acid molecule encoding HDAg.
There can be two or more multiple coding sites, wherein at least
one multiple coding site is located in a flanking region 3' to the
nucleic acid molecular encoding HDAg and/or at least one multiple
coding site is located in a flanking region 5' to the nucleic acid
molecule encoding HDAg. The vector can further comprise a nucleic
acid molecule encoding a nuclear localization signal. A vector can
further comprise a nucleic acid molecule which encodes a
heterologous gene. The vector can express a fusion molecule of HDAg
wherein a first heterologous gene encodes a first binding moiety
and a second heterologous gene encodes a second binding moiety.
[0012] The invention also encompasses host cells which comprise a
nucleic acid molecule which encodes a molecule of HDAg, including a
fusion molecule.
[0013] The invention also includes methods of manufacturing such a
host cell comprising a nucleic acid molecule encoding a fusion
molecule comprising HDAg and at least one binding moiety, by
introducing a vector of the invention into the host cell.
[0014] The invention also relates to methods of using the
molecules, i.e., peptides, nucleic acids, and vectors of the
invention. One method comprises expressing a high valency display
of at least one binding moiety comprising introducing into a cell
with a vector comprising a nucleic acid molecule encoding HDAg and
a nucleic acid molecule encoding the binding entity and culturing
the cell under conditions sufficient to permit expression of the
binding moiety and HDAg.
[0015] The invention also encompasses a method of enhancing
interaction between binding partners comprising contacting a fusion
molecule of HDAg with a second binding moiety wherein the first and
second moieties are binding partners. The fusion molecule can
present the first and second moieties. The interaction between
ligands can occur in solution, on membranes or on surfaces. The
fusion molecule can be a subunit of a coiled-coil oligomer, e.g.,
an octamer, and the first and second moieties are bound to the
oligomer. In one embodiment, fusion of a first cell and a second
cell is enhanced.
[0016] The invention also includes a method for delivering
molecules to a cell comprising contacting them with an HDAg fusion
molecule. In one embodiment, the binding moiety is an
oligonucleotide. The oligonucleotide can hybridize to a nucleic
acid molecule in the cell. The fusion molecule can further comprise
a double-stranded nuclease. In one embodiment, the fusion molecule
comprises a first binding moiety and a second binding moiety
wherein the first binding moiety interacts with a binding partner
and the second binding moiety functions as an effector. The first
binding moiety can interact with a cell surface receptor on a cell
and the second binding moiety can kill the cell.
[0017] The invention also includes a method of amplifying a signal
in a solid phase assay comprising coupling an HDAg octamer with at
least one copy of a domain which interacts with a ligand and at
least two copies of a label. The label can be, for example,
alkaline phosphatase, a radiolabel, streptadavin, and green
fluorescent protein. In one embodiment, the solid phase assay is an
ELISA assay. The invention also encompasses a method of
facilitating exchange of substrates and products comprising
coupling an HDAg oligomer to at least two enzymes which function in
a linked pathway.
[0018] The invention also encompasses a method of enhancing a
reaction between at least two binding partners comprising coupling
the binding partners to an HDAg oligomer. In a different
embodiment, the method of enhancing a reaction between two binding
partners comprises coupling one binding partner to an HDAg oligomer
and contacting the oligomer to a second binding partner.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0020] FIG. 1 depicts sequence alignment of 11 serotypes of
hepatitis delta antigen (HDAg) between amino acids 12 to 60.
Asterisks, *, indicate residues which make up the a and d positions
in the heptad repeat in the predicted coiled-coil region. Bold pink
and purple indicate residues involved in the hydrophobic
interactions in the dimer between the two termini. The "ds"
indicate residues involved in the dimer-dimer interface. A region
(pink), B (green), C (purple).
[0021] FIG. 2 depicts the final atomic model superimposed upon a
portion of the final 1.8 A resolution 2F.sub.O-F.sub.C map. The map
is contoured at 1.2.sigma. and shows the residues involved in the
interaction between monomers at the A and C regions. Orientation is
similar to that in FIG. 4. Yellow indicates carbon, red indicates
oxygen and blue indicates nitrogen. The figure was produced using
BOBSCRIPT.
[0022] FIG. 3A depicts C.alpha. trace of the peptide
.delta.12-60(Y). A region pink, B region green, and C region
purple. The individual helix takes a sharp bend at proline 49
(Pro49). FIG. 3B is a ribbon diagram of the view in FIG. 3A rotated
90.degree. along the horizontal axis. The sidechains have been
added and the C region of the peptide (residues 50-60(Y)) has been
removed for clarity. Sidechains are colored as follows: hydrophobic
gray, polar yellow, acidic red and basic blue. FIG. 3C is the amino
acid sequence of the long helix formed from residues 12 to 48
displayed in the antiparallel orientation of the peptide. The
letters above the amino acid sequence represent the heptad repeat
(abcdefg).sub.n where the a and d residues tend to be hydrophobic.
The residues involved in the heptad repeat at the a and d positions
are shown in bold.
[0023] FIG. 4 depicts the monomer-monomer interactions. The regions
are colored as follows: A region pink, B region green, and C region
purple. The white row of X's indicates a hydrogen bond between the
sidechain of Glu45 (E45) and the indole nitrogen of Trp20 (W20).
This figure was produced using RIBBONS, Carson, M. & Bugg, C.
E., J. Mol. Graphics, 4:121-122 (1986).
[0024] FIG. 5 depicts the interactions of dimers in the
P2.sub.12.sub.12 unit cell. The unit cell is outlined in black and
the directions of the A and B axes are shown. The two independent
copies of the dimer in the asymmetric unit are colored orange and
blue. The view is looking down the crystallographic 2-fold axis.
This figure was produced using the program MOLSCRIPT (Kraulis, P.
J. J. Appl. Cryst. 24:946-950 (1991)).
[0025] FIG. 6 depicts the dimer-dimer interface. FIG. 6A
illustrates that the dimer-dimer interface is composed of a
four-helix bundle made of the N and C termini of two dimers, one
from across the crystallographic two-fold axis. One (unlabeled)
dimer is colored yellow and the other (labeled) dimer is colored
according to the scheme used in FIG. 1. FIG. 6B depicts the view in
FIG. 6A rotated 90.degree. around the y axis. This figure was
created with RIBBONS (Carson, M. & Bugg, C. E., J. Mol. Graph.
4:121-122 (1986)).
[0026] FIG. 7A is a GRASP electrostatic potential surface of the
octameric .delta.12-60(Y) peptide contoured at=10 kT/e (positive
potential blue) and=10 kT/e (negative potential in red) (K is
Boltzman's constant and T is temp .degree. K. The edges and the
lining of the large 50 .ANG. hole are basic. FIG. 7B illustrates
the hole formed by the octamer.
[0027] FIG. 8 graphically illustrates MALDI-TOF mass spectrometry
analysis of recombinant small delta antigen (r-HDAg-S) (FIG. 8A),
and the glutaraldehyde cross-linked protein (FIG. 8B).
[0028] FIG. 9 depicts the sequence of a synthetic gene for
optimized expression of HDAg-S in E. coli. The synthetic gene has
been modified such that the codon usage which is unusual in the
natural gene is assistant with the known preferences for codon
usage in E. coli. The underlined sequences correspond to the eight
primers used in the first round of PCR. The primers used in the
second round are indicated with a dotted underline. The amino acid
sequence is shown above the DNA sequence by the one-letter amino
acid code. The restriction sites used in cloning are shown in
italics.
[0029] FIG. 10 depicts the complete sequence of human HDV cDNA, and
the predicted amino-acid sequence of human HDV delta antigen.
[0030] FIG. 11 depicts synthetic peptides from the multimer-forming
domain of HDAg. (A) Structural organization of HDAg. The lightly
stippled region is the multimer-forming domain (amino acid residues
12-60), the solid regions are the RNA-binding domains, and the
heavily stippled region is the C-terminal extension of large HDAg.
Hydrophobic residues contributing to the heptad repeat are shown in
boldface type, (B) Amino acid sequences of three HDAg peptides.
[0031] FIG. 12 depicts use of the delta antigen as a scaffold. A
construct containing eight appended protein domains on the
octameric framework. FIG. 12A depicts an oligomerization domain and
spheres which represent potential effectors/ligands/nucleic acid
binding domains etc. that have been fused to the C-terminus of the
oligomerization domain of the delta antigen. FIG. 12B depicts
binding domain which is a multimer, specifically a tetramer. A
similar fusion of up to eight effector/legands/nucleic acid binding
domains could be made at the N-terminus, and constructs with fusion
domains at up to eight N-termini and up to eight C-termini could
also be made.
[0032] FIG. 13 depicts plasmids comprising HDAg for expression in
bacteria. "DAg" refers to a delta antigen sequence (with or without
nuclear localization sequence); "MCS1" refers to a multiple cloning
site for insertion of a heterologous gene at N-terminal end of
delta antigen; "MCS2" refers to a multiple cloning site for
insertion of a heterologous gene a C-terminal end of delta antigen;
"Ori" refers to the origin of replication for the bacteria; "drug"
refers to a drug marker; "f1 ori" refers to origin of replication
of single-stranded DNA by a bacteriophage; "promoter" refers to
bacterial promoter. FIG. 13A depicts MCS1 3' to HDAg; FIG. 13B
depicts MCS1 5' to HDAg; and FIG. 13C depicts MCS1 3' and MCS2 5'
to HDAg.
[0033] FIG. 14 depicts a plasmid comprising HDAg for expression in
eukaryotic cells. "DAg refers to a delta antigen sequence (with or
without nuclear localization sequence). "MCS1" refers to plasmids
comprising replication of plasma in bacteria; "Ori" refers to
origin of expression or replication of the plasmid in bacterial
cells; "drug1" refers to a drug marker (e.g. ampicillin, kanamycin)
for propagation of plasmid in E. coli; "drug2" refers to a drug
marker or eukaryotic drug resistance (e.g. neomycin, zeocin,
hybromycin), for propagation of plasmid in a eukaryotic cell; "f1
ori" refers to origin of replication of single-stranded DNA by
bacteriophage; "promoter" refers to eukaryotic promoter.
[0034] FIG. 15 is a comparison of the wildtype nucleotide sequence
of HDAg-S and the sequence of the synthetic HDAg gene for optimized
expression in E. coli.
[0035] FIG. 16 is the nucleotide sequence of the synthetic open
reading frame (ORF) for the synthetic HDAg.
[0036] FIG. 17 is a comparison of the protein amino acid sequence
encoded by the wildtype ORF and the synthetic ORF, showing complete
(100%) identity.
[0037] FIG. 18 depicts the nucleotide sequences of the primers used
for the two polymerase chain reactions (PCR) to create the
synthetic gene. Primer1-primer8 were used in the first round of PCR
and primer9-primer10 were used in the second round of PCR.
DETAILED DESCRIPTION OF THE INVENTION
[0038] As set forth above, the present invention relates to the
discovery of the oligomeric structure of the hepatitis delta
antigen (HDAg) which serves as a convenient high-valency framework
for linking a variety of binding partners, including functional
peptides and domains. The structure of the antigen includes a
doughnut-shaped octamer comprising N-terminal antiparallel
coiled-coil domains and stabilizing C-terminal domains. The
invention includes HDAg proteins, including derivatives, mutants
and fragments, and nucleic acid molecules encoding HDAg. It also
includes an altered HDAg gene for the capsid protein wherein the
codons conform to use preferences for E. coli. Included in the
derivatives are fusion molecules, e.g., fusion proteins, in which
one or more binding moieties are attached to one or both termini of
a monomer and coiled-coil oligomers (e.g., octamers) formed from
the monomers. Coiled-coil oligomers of the present invention can
comprise one or more fusion molecules as described herein. The
binding moieties can be, for example the same (homologous) or
different (heterologous) binding partners. The invention also
includes vectors and cassette expression systems which can be used
to produce the fusion molecules. The vectors comprise HDAg and one
or more binding moieties which are operably linked to HDAg. The
invention also relates to cells comprising HDAg nucleic acid, e.g.
cells transformed with such vectors, and to methods of producing
such cells. The invention also includes therapeutic and diagnostic
methods involving HDAg.
[0039] HDAg Peptides
[0040] HDAg, as defined herein, includes both the large and small
delta antigens (HDAg-S and HDAg-L). HDAg encompasses native ("wild
type") proteins and also includes derivatives, mutations, and
functional protein or polypeptide fragments of the native protein
and/or proteins or polypeptides where one or more amino acids have
been deleted, added or substituted.
[0041] HDAg can be isolated and/or purified, or it can be
recombinant or prepared by synthetic techniques described herein or
known to those of skill in the art. HDAg proteins or fragments can
be isolated from the cell of origin or produced synthetically or
recombinantly. In a preferred embodiment, the protein is isolated
to the substantial absence of conspecific proteins. A conspecific
protein is a protein other than HDAg which can be obtained from the
cell of origin for the protein or its nucleic acid. The proteins
(and nucleic acids) described herein can be preferably isolated, by
known methods, to a purity of at least about 50% by weight, more
preferably at least about 75% and most preferably to substantial
homogeneity. "Substantial homogeneity" refers to the substantial
absence of conspecific proteins.
[0042] An HDAg peptide can, e.g., include all or a portion of the
amino acids depicted FIGS. 1, 3C, 10, 11 or 17. An HDAg peptide can
be encoded by an isolated and/or purified or recombinant nucleic
acid molecule or a fusion gene (nucleic acid molecule) such as
those described herein. In a preferred embodiment, an isolated and
purified polypeptide has an amino acid sequence depicted in a row
of FIG. 1, amino acids 12-48 of a row of FIG. 1, amino acids 12-60
of a row of FIG. 1, a row of FIG. 3C, FIG. 9, amino acids 12-48 of
FIG. 9, amino acids 12-60 of FIG. 9, FIG. 10, FIG. 11, FIG. 17 or a
fragment or derivative thereof, which forms a coiled-coil
oligomer.
[0043] In another embodiment, an isolated and purified polypeptide
has the amino acid sequence of amino acids 12-88 of HDAg, or a
fragment or derivative thereof, which forms a coiled-coil oligomer
and nuclear localization signal.
[0044] "Homology" is defined herein as sequence identity.
Preferably, the protein or polypeptide shares at least about 50%
sequence identity or homology and more preferably at least about
75% identify or at least about 90% identity with the corresponding
sequences of the native protein, for example, with FIG. 10. The
phrase "substantially the same sequence" is intended to include
sequences which bind the viral protein and possess a high
percentage of (e.g., at least 90%, preferably at least about 95%)
amino acid sequence identity with the native sequence. For example,
a derivative, e.g., a mutant or variant can possess substantially
the same amino acid sequence as the native protein.
[0045] The modifications to the amino acid sequence (substitutions)
can be conserved or non-conserved, natural or unnatural amino
acids. The residues that function to form or stabilize the
coiled-coil domain or binding sites thereof can be substituted,
e.g., conservatively, or they can be maintained. Amino acids of the
native sequence for substitution, deletion or conservation can be
identified, for example, by a sequence alignment between proteins
from different serotypes from related species or other related
proteins. In one embodiment, the amino acids which are deleted,
added or substituted are amino acids which are not "conserved"
between serotypes or species, for example, the amino acids so
identified in the sequence alignment exemplified in FIG. 1.
Conserved amino acids may also be substituted. In one embodiment
they are substituted conservatively, for example, substituted by
structurally similar amino acids. The phrase "conservative amino
acids substitutions" is intended to mean substitutions of amino
acids which possess similar side chains (e.g., hydrophobic,
hydrophilic, basic acidic, aromatic, and aliphatic) as is known in
the art. See, for example, Hermanson, G.I. Bioconjugate Techniques,
Academic Press, Inc. San Diego, Calif. (1996). Conservative
substitutions include amino acid substitutions of one hydrophobic
amino acid for another, for example within the following grouping:
W, F, A, P, L, M, I, V. Acidic amino acids include E and D; basic
amino acids include K, R, and H. Polar amino acids include S, T, N,
Q and G and amide residues include Q and N. An example of a
suitable derivative or mutant of the HDAg protein is a protein
possessing a consensus sequence of the originating species.
[0046] In one embodiment, the derivative does not contain
substitutes of the residues of Arg13, Leu17, Trp20, Arg24, Trp50 or
Leu51. In another embodiment, it contains only conservative
substitutions in this region. Hydrophobic residues, for example
Ile16, Leu17, Trp20, Trp50 and Leu51 can be maintained, or they can
be replaced with other hydrophobic amino acids, for example, those
from the group consisting of Trp, Phe, Ala, Pro, Leu, Met, Ile and
Val. In another example, the residues Glu31, Lys38, Trp20 and Glu45
are not substituted or are substituted conservatively. In addition,
Arg13 and Arg24 can be maintained (not substituted) or substituted
conservatively. In another embodiment, the residues of FIG. 1 which
are involved in hydrophobic interactions are substituted with other
hydrophobic residues. In FIG. 3A, hydrophobic residues can be
substituted for other hydrophobic residues, polar residues can be
substituted for other polar residues, acidic residues can be
substituted for other acidic residues, and/or basic residues for
other basic residues. In one example, the residues labeled in FIG.
3A can be maintained (not substituted) or can be replaced with
amino acids with similar characteristics. The amino acids at the
`a` and `d` positions of the heptad repeat (for example, those
indicated with an asterisk in FIG. 1 or those listed in bold in
FIG. 3C), can be conserved (maintained), or they can be substituted
conservatively, e.g., replaced with hydrophobic amino acids. The
residues involved with the dimer-dimer interface (e.g., residues
marked with a `d` in FIG. 1 or residues labeled in FIG. 6) can be
maintained. The residues indicated in FIG. 4 can be maintained. The
derivatives, e.g. mutant and wild-type peptides, can crystallize
isomorphously. In one preferred embodiment, at least one serine
residue, e.g., Ser22, is replaced with a cysteine. In another
embodiment, Trp20 is replaced with Ala20.
[0047] A variety of substitutions based on amino acid
characteristics can be made. For example, the polar amino acid
residues can be substituted and the hydrophobic amino acids can be
maintained. In addition the nonhydrophobic residues can be
substituted and the hydrophobic residues can be maintained. In one
embodiment, a derivative can comprise substitutions of any or all
of the amino acid residues in the following positions: 14, 15, 18,
19, 22, 24, 25, 26, 28, 29, 31, 32, 33, 35, 36, 38, 39, 40, 42, 43,
45, 46 and 47. The nonhydrophobic residues can be substituted such
that acidic amino acids are alternated with basic amino acids. The
hydrophilic residues in the C terminal region can be substituted to
optimize stability of the helix, for example by presenting one or
more amino acids which form disulfide bonds, strong ionic bonds or
cross-linked moieties, with the corresponding amino acid of another
subunit. In one embodiment, residues 53, 55 and 60 are
substituted.
[0048] Especially preferred are derivatives, e.g., coiled-coil
subunits, which improve (e.g., optimize) stability of the
coiled-coil structure or which improve cross-linkage involving the
structure, or which improve the ability of the structure to be
immobilized on a solid substrate.
[0049] The term derivative is also intended to include proteins
which have been labeled, such as with a radioactive or calorimetric
label. Such derivatives are more readily detected in an assay. In
one embodiment, a peptide is synthesized that corresponds to
residues 12-60 of HDAg, and includes a C-terminal tyrosine,
enabling the peptide to be labeled, e.g., with I.sup.25, for use in
a radioimmunoassay. In one embodiment, the peptide is
.delta.12-60(Y). Yet other derivatives are proteins which consist
essentially of the amino acid sequence of a given protein (e.g.,
possess the relevant sequence and, optionally, other amino acids
residing at the termini which do not significantly alter or detract
from the properties of the protein).
[0050] A "functional" fragment, derivative, mutant, or allelic
variant is of sufficient length and/or structure as to possess one
or more biological activities of the protein. One example of such a
biological activity of the protein is formation of a coiled-coil
oligomer, e.g., an octamer, for example, an octamer doughnut-shaped
structure. In one embodiment, the protein derivative is conserved
within the coiled-coil regions but is lacking in or mutated within
one or more other regions (e.g., sequences not within the
coiled-coil. Examples of suitable fragments include peptides
lacking fragments which encode or stabilize the coiled coil, for
example, amino acids 12-48, or the peptides depicted in FIG. 11.
One example includes fragments which lack all or part of the region
C-terminal to the proline bend (e.g. C-Terminal to Pro49). Another
fragment includes the coiled coil and nuclear localization signal
(e.g., amino acids 12-88); or solely the nuclear localization
signal (amino acids 68-88). Xia et al., J. Virol. 66:914-21 (1992).
Yet another example includes HDAg which encodes the coiled-coil
region but is lacking all or a portion of the nuclear localization
signal. In one embodiment, all or a portion of one or both termini
of a monomer is absent or mutated. For example, the C region of the
peptide (e.g., residues 50-60) can be mutated or all or a portion
can be eliminated. Yet another example of derivatives includes
peptides which possess amino acid modifications or additions which
are characterized by a functional group which can react with a
compound substituted by a "binding moiety", such as those described
above or with a cross-linking agent.
[0051] Yet other biologically activities are the ability of HDAg-S
to function as a trans activator of replication and the ability of
HDAg-L to act as an inhibitor of replication. In yet another
embodiment, the biological activity of the protein is antigenic or
immunogenic activity.
[0052] Fragments of the protein can possess at least about 10 amino
acids from the 12-48 amino acid region, preferably at least about
20 amino acids. In other embodiments, the fragment possesses
essentially all of the amino acids of the full-length protein
(e.g., at least about 85%, or at least about 95%).
[0053] HDAg includes monomers and oligomers, e.g., dimers and
octamers, comprising the monomers as subunits. The invention
encompasses HDAg coiled-coil oligomers, e.g. octamers.
[0054] Fusion Molecules
[0055] Fusion molecules are intended to be included within the
definition of HDAg derivatives and can be made by linking one or
more binding moieties, e.g., chemicals or peptides, to the HDAg
protein or fragment, for example, through a covalent bond or
preferably a peptide bond or cysteine group. As such, derivatives,
such as fusion proteins, can comprise the amino acid sequence of
the HDAg protein or fragment and a binding moeity such as a given
protein, e.g., a native protein.
[0056] A "binding moiety," as the term is defined herein, includes
a chemical entity which is bound to HDAg. The binding can be via a
covalent bond (e.g. through a cysteine group), ionic bonding,
hydrogen bonding or other mechanism. The binding moiety and the
HDAg can be expressed as a single unit. The binding moiety can be a
peptide (including post-translationally modified proteins, such as
amidated, demethylated, glycosylated or phosphorylated proteins),
sugar, lipid, steroid, nucleic acid, small molecule, anion or
cation, drug, chemical or combination thereof which binds the
specified binding partner (e.g., a target molecule). Preferably,
the binding will possess a high affinity. Examples of high affinity
can have a dissociation constant of 10.sup.-5M (preferably
10.sup.-8M) or lower. Examples of binding moieties include an
antigen, an antibody, a ligand, a receptor, an enzyme, a (ligand)
interaction peptide, a chemical, an effector, an oligonucleotide, a
signal amplification peptide, an enhancer recognition protein, a
promoter binding protein, a label, a growth factor, a cytokine, a
nuclease, a small organic molecule, a test substance, a cytotoxic
agent, a substrate, a solid substrate, a drug or a fragment
thereof.
[0057] Where there are two or more binding moieties, the binding
moieties can be the same (homologous) or different (heterologous).
The binding moieties can be binding partners. Examples of binding
partners include, but are not limited to, antigen-antibody and
ligand-receptor. First and second binding moieties can also include
the following pairs: enzyme1-enzyme2, (ligand) interaction
peptide-effector peptide (or chemical), oligonucleotide-nuclease,
interaction agent (e.g. peptide signal amplification agent, (e.g.
peptide), enhancer recognition agent (e.g.
protein)-promoter-binding agent (e.g. protein), enhancer
recognition agent-promoter binding agent, ligand-label, test
substance-label, targeting agent--effector agent, drug (or hormone)
-label, or any other combination.
[0058] In one embodiment, a first binding moiety binds, a target
molecule on a target cell (e.g. a surface protein) and the binding
partner is the surface protein or target cell. The "target cell" is
defined as the cell which is intended to be contacted by the fusion
cell. Typically, the target cell is of animal origin and can be a
stem cell or somatic cell. Suitable animal cells for use on the
claimed invention can be of, for example, mammalian and avian
origin. Examples,of mammalian cells include human, bovine, ovine,
porcine, murine, rabbit cells. The cell may be an embryonic cell,
bone marrow stem cell or other progenitor cell. Where the cell is a
somatic cell, the cell can be, for example, an epithelial cell,
fibroblast, smooth muscle cell, blood cell (including a
hematopoietic cell, red blood cell, T-cell, B-cell, etc.), tumor
cell, cardiac muscle cell, macrophage, dendritic cell, neuronal
cell (e.g., a glial cell or astrocyte), or pathogen-infected cell
(e.g., those infected by bacteria, viruses, virusoids, parasites,
or prions).
[0059] Typically, cells isolated from a specific tissue (such as
epithelium, fibroblast or hematopoietic cells) are categorized as a
"cell-type." The cells can be obtained commercially or from a
depository or obtained directly from an animal, such as by biopsy.
Alternatively, the cell need not be isolated at all from the animal
where, for example, it is desirable to deliver the vector to the
animal in gene therapy.
[0060] Cells can typically be characterized by markers expressed at
their surface that are termed "surface markers". These surface
markers include surface proteins or target molecules, such as
cellular receptors, adhesion molecules, transporter proteins,
components of the extracellular matrix and the like. These markers,
proteins and molecules also include specific carbohydrates and/or
lipid moieties, for example, conjugated to proteins. In one
embodiment, a binding moiety on a fusion molecule can bind to one
or more surface proteins on the target cell. Surface proteins can
be tissue- or cell-type specific (e.g. as in surface markers) or
can be found on the surface of many cells. Typically, the surface
marker, protein or molecule is a transmembrane protein with one or
more domains which extend to the exterior of the cell (e.g. the
extracellular domain). Where cell-type specific delivery is desired
(as in in vivo delivery of a drug), the surface protein selected
for the invention is preferably specific to the tissue. By
"specific" to the tissue, it is meant that the protein be present
on the targeted cell-type but not present (or present at a
significantly lower concentration) on a substantial number of other
cell-types. While it can be desirable, and even preferred, to
select a surface protein which is unique to the target cell, it is
not required for the claimed invention. It is to be appreciated,
however, that specific delivery may not be required where the cell
or cells are contacted with the viral vector in pure or
substantially pure form, such as can be the case in an in vitro
gene transfer. As such, the surface protein or targeted protein for
the first binding moiety may be present on many different
cell-types, specific or even unique to the targeted cell-type.
[0061] As set forth above, the surface protein can be a cellular
receptor or other protein, preferably a cellular receptor. Examples
of cellular receptors include receptors for cytokines, growth
factors, and include, in particular epidermal growth factor
receptors, platelet derived growth factor receptors, interferon
receptors, insulin receptors, proteins with seven transmembrane
domains including chemokine receptors and frizzled related proteins
(Wnt receptors), immunoglobulin-related proteins including MHC
proteins, CD4, CD8, ICAM-1, etc., tumor necrosis factor-related
proteins including the type I and type II TNF receptors, Fas, DR3,
DR4, CAR1, etc., low density lipoprotein receptor, integrins, and,
in some instances, the Fc receptor.
[0062] Other examples of surface proteins which can be used in the
present invention include cell-bound tumor antigens. Many of these
surface proteins are commercially available and/or have been
characterized in the art, including the amino acid and nucleic acid
sequences, which can be obtained from, for example, GENBANK, as
well as the specific binding characteristics and domains. Cytokine
and chemokine receptors are reviewed for example, in Miyama, et al.
Ann. Rev. Immunol., 10:295-331 (1992), Murphy, Ann. Rev. Immunol.
12:593-633 (1994) and Miller et al. Critical Reviews in Immunol.
12:17-46 (1992).
[0063] The binding moiety can be selected or derived from native
ligands or binding partners to the surface protein of the target
cell. In the case of a cellular receptor, for example, for a
cytokine or growth factor, the binding moiety can be a polypeptide
comprising at least the receptor-binding portion of the native
ligand. A "native ligand" or "native binding partner" is defined
herein as the molecule naturally produced by, for example, the
animal or species which binds to the surface protein in nature.
Preferably, the binding moiety is a polypeptide or protein. As
such, the native ligand of a cytokine receptor can be the native
cytokine. In another embodiment, the binding moiety can comprise a
binding fragment of an antibody, such as the variable region or a
single chain antibody.
[0064] Where a binding moiety comprises a binding fragment of an
antibody, many antibodies to surface proteins are known or are
commercially available, as are the amino acid sequences which are
responsible for binding. Alternatively, novel antibodies can be
prepared by methods known in the art, such as by Harlow and Lane,
"Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory
(1988). The binding fragment can comprise an antibody fragment, for
example, the constant region or, the variable region (e.g., Fc
fragment or FAb' fragment).
[0065] A binding moiety can be a polypeptide ligand to a cellular
receptor. Examples of preferred ligands are growth factors,
epidermal growth factor, interleukins, GM-CSF, G-CSF, M-CSF, EPO,
TNF, interferons, and chemokines. In one embodiment, the receptor
is a transferon receptor.
[0066] The binding moiety can have an amino acid sequence which is
the same or substantially the same as an amino acid sequence of at
least the receptor-binding portion of a native ligand for the
cellular receptor. Similar to cellular receptors, many of the
corresponding ligands have been identified, sequenced and
characterized, including the portions thereof which bind to the
receptor. The binding moiety can, therefore, include the same or
substantially the same sequence of the entire native ligand.
Alternatively, binding moiety comprises the receptor binding
portion of the native ligand, eliminating, in some cases, the
effector function of the ligand.
[0067] In another embodiment, the binding moiety is selected or
derived from native ligands or binding partners to a cellular
surface molecule of a target cell. A "cellular surface molecule" as
defined herein can be a peptide (including post-translationally
modified proteins, such as amidated, demethylated, methylated,
prenylated, palmitoylated, glycosylated, myristylated, acetylated
or phosphorylated proteins), sugar, lipid, steroid, anion or
cation, or a combination thereof which binds the first binding
moiety. Preferably, the binding of the cellular surface molecule to
the binding moiety of the bifunctional molecule will be of high
affinity. Examples of high affinity have a dissociation constant of
10.sup.-5M (preferably 10.sup.-8M) or better.
[0068] The cellular surface molecule need not be
.differential.specific" for the target cell. However, the cellular
surface molecule is specific for a desired viral vector. For
example, specific delivery of Influenza A viral vectors can employ
sialic acid cellular surface molecules for entry into a target cell
whereas targeting of VSV viral vectors can employ a phospholipid as
the surface molecule. As such, the cellular surface molecule for
the first binding moiety can be present on many different
cell-types, specific or even unique to the target cell.
[0069] In other embodiments, the effector function can be
desirable, thereby stimulating or modulating the cellular activity
of the target cell which can enhance therapy. An example of where
such a therapy can be desirable is in the delivery of a negative
selection marker or suicide protein to a tumor where the target
cell is a lymphokine and the ligand is a cytokine. Where the
lymphokine is stimulated, the cell, can also possess therapeutic
value in the recruitment of an endogenous immune response against
the tumor, thereby increasing the therapeutic benefit of the
therapy.
[0070] The phrase "substantially the same sequence" is intended to
include sequences which bind the surface protein and possess a high
percentage of (e.g., at least about 90%, preferably at least about
95%) sequence identity with the native sequence. The modifications
to the sequence can be conserved or non-conserved, natural and
unnatural, amino acids and are preferably outside of the binding
domain. Amino acids of the native sequence for substitution,
deletion, or conservation can be identified, for example, by a
sequence alignment between proteins from related species or other
related proteins.
[0071] In addition to the first binding moiety, there can be a
second binding moiety which is a chemical entity which binds to
HDAg. The binding can be via a covalent bond, ionic bonding,
hydrogen bonding or other mechanism. The second binding moiety can
be the same or different from the first. For example, it can be a
peptide, sugar, lipid, steroid, nucleic acid, small molecule, anion
or cation, or combination thereof which binds the HDAg. In one
embodiment, the second binding moiety of the fusion protein is also
a polypeptide. One embodiment of the second binding moiety
comprises an antigen-binding fragment of an antibody which
recognizes and binds to an antigen.
[0072] Ligand receptors which are cellular receptors can be
transmembrane proteins comprising intracellular, transmembrane
(characterized by highly hydrophobic regions in the sequence) and
extracellular domains. In one embodiment, the second binding moiety
can comprise the native extracellular domain of a receptor
molecule.
[0073] Fusion proteins can be made conveniently through known
methods, e.g. recombinantly. The binding moieties can be directly
bonded to HDAg or can be bonded to HDAg through a linking moiety.
Where one or both of the moieties are polypeptides, a peptide bond
or peptide linker may be preferred, thereby obtaining a "fusion
protein". The "fusion protein" of the HDAg and one or more moieties
can be expressed by a single nucleic acid construct in series. One
or more moieties and HDAg alternatively be linked directly or
indirectly other than via a peptide bond or peptide linker, thereby
obtaining a "conjugate".
[0074] Where the moieties and HDAg are directly bonded to each
other, the bond can be covalent, as in a peptide bond, ionic bond
or hydrogen bond. Where the bond is a peptide bond, a binding
moiety can be bonded to the N terminus of HDAg via the C terminus,
or vice versa or both. It is acknowledged that one fusion protein
may possess greater activity than a second fusion protein due to
conformational or steric considerations. The binding moieties can
be, for example, monomers, dimers and tetramers.
[0075] Where one or more of the binding moieties are not
polypeptides, they can be joined via chemical reaction through
functional groups present on each moiety which, under the
appropriate conditions, will react with each other. For example,
acid groups (or activated derivatives thereof) can be reacted with
amines, alcohols or thiols to form amide or ester bonds, as is
known in the art.
[0076] Alternatively and advantageously, a linking moiety is
employed to link the binding moieties, e.g. binding partners, to
HDAg. The linker can preferably be a flexible linker and sufficient
in length to separate the moieties in space, thereby not
restricting the ability of the fusion molecule to bind
independently and maintain the proper conformation. Again, where
both moieties are polypeptides, the linker moiety will generally be
a peptide, polypeptide, or a "pseudopeptide". A "pseudopeptide" is
a bifunctional linker which contains at least one non-amino acid
and reacts to form a peptide bond, or other bond, with the terminal
amine or carboxyl group of the moiety. For example, a peptide
characterized by substitution of the terminal amine for a carboxyl
group can function to react with the amine terminus of each moiety.
Such as linker is considered to be a "pseudopeptide." Similarly, a
peptide characterized by substitution of the terminal carboxyl for
an amine group can function to react with the carboxyl terminus of
each moiety.
[0077] Generally, however, the linker will be a peptide linker
which will link the amine terminus of a moiety to the carboxyl
terminus of HDAg or vice versa. One advantage to such a molecule is
the ability to express the fusion protein in a recombinant host
cell with a single nucleic acid construct.
[0078] Peptide linkers can be obtained from immunoglobulin hinge
regions, such as a proline-rich region. Also, linkers can be
characterized by little steric hindrance, thereby permitting
maximal independent movement of the two moieties, such as with a
polyglycine linker. Alternatively, the linker selected to be
reactive to or inert to cellular proteases can be desirable. In
another embodiment, the linker can be selected to avoid or minimize
an immune response against the fusion molecule. The length of the
linker also is not particularly critical. Typically, the length of
the linker can be between about 2 and about 20 amino acids. As can
be seen, the selection of the particular linking group is not
critical to the invention.
[0079] In yet another embodiment, the linker can be a bifunctional
compound which will react with other functional groups on the
binding moieties or HDAg, such as in the reaction of acids and
amines or alcohols (as present in peptides, carbohydrates and
lipids, for example) in the formation of amides or esters.
[0080] A preferred combination of the above first and second
binding moieties includes one binding partner, e.g. a polypeptide
ligand to a cell-type specific cellular receptor linked, via a
peptide linker, through a terminus of the ligand to the terminus of
HDAg. A second binding partner, e.g. a extracellular domain of a
cellular receptor or a mutant thereof, can be linked to the same or
different HDAg subunit in the same manner.
[0081] For example, the C terminus of a binding polypeptide is
linked to the N terminus of HDAg via the polypeptide linker or the
N terminus of the first binding polypeptide is linked to the C
terminus of the HDAg via the polypeptide linker.
[0082] In another aspect of the invention, peptidomimetics
(molecules which are not polypeptides, but which mimic aspects of
their structures to bind to the same site) that are based upon the
above-described polypeptides, can also be used. For example,
polysaccharides can be prepared that have the same functional
groups as the polypeptides of the invention, and which interact
with binding partners in a similar manner. Peptidomimetics can be
designed, for example, by establishing the three=dimensional
structure of the polypeptide in the environment in which it is
bound or will bind to the binding partner. The peptidomimetic can
comprise at least two components, a binding entity or entities and
a backbone or supporting structure entity.
[0083] The binding entities of the peptidomimetic are the chemical
atoms or groups which will react or complex (as in the formation of
a hydrogen or covalent bond) with a binding partner. In general,
the binding entities in a peptidomimetic are the same as the
polypeptide moieties. Alternatively, the binding entities can be an
atom or chemical group which will react with the binding partner in
the same or similar manner as the polypeptide. Examples of binding
entities suitable for use in designing a peptidomimetic for a basic
amino acid in a polypeptide are nitrogen containing groups, such as
amines, ammoniums, guanidines and amides or phosphoniums. Examples
of binding entities suitable for use in designing a peptidomimetic
for an acidic amino acid in a polypeptide can be, for example,
carboxyl, lower alkyl carboxylic acid ester, sulfonic acid, a lower
alkyl sulfonic acid ester or a phosphorous acid or ester
thereof.
[0084] The supporting structure is the chemical entity that, when
bound to the binding moiety or moieties, provides the three
dimensional configuration of the peptidomimetic. The supporting
structure can be organic or inorganic. Examples of organic
supporting structures include polysaccharides and polymers (such
as, polyvinyl alcohol or polylactide). It is preferred that the
supporting structure possess substantially the same size and
dimensions as the polypeptide backbone or supporting structure.
This can be determined by calculating or measuring the size of the
atoms and bonds of the polypeptide and peptidomimetic. For example,
the nitrogen of the peptide bond can be substituted with oxygen or
sulfur, thereby forming a polyester backbone. Likewise, the
carbonyl of the peptide bond can be substituted with a sulfonyl
group or sulfonyl group, thereby forming a polyamide. Reverse
amides of the peptide can be made (e.g., substituting one or more
--CONH-- groups for a --NHCO-- group). In addition, the peptide
backbone can be substituted with a polysilane backbone.
[0085] These peptidomimetic compounds can be manufactured by
art-known and art-recognized methods. For example, a polyester
corresponding to a given peptide can be prepared by the
substituting a hydroxyl group for each corresponding amine group on
the amino acids, thereby preparing a hydroxyacid and sequentially
esterifying the hydroxyacids, optionally blocking the basic side
chains and acids to minimize side reactions. Determining an
appropriate chemical synthesis route can generally be readily
identified upon determining the chemical structure using no more
than routine skill.
[0086] The fusion molecules can be manufactured according to
methods generally known in the art. For example, where one or both
of the binding moieties is a nonpeptide, the fusion molecule can be
manufactured employing known organic synthesis methods useful for
reacting a functional or reactive group on the moiety with a
functional or reactive group on the other moiety or, preferably, a
linker. In carrying out the synthesis, derivation or inactivation
of the functional group(s) required for binding to the moiety's
binding partner should be avoided. Appropriate syntheses are highly
dependent upon the chemical nature of the binding moiety and,
generally, can be selected from an organic chemistry text, such as
March et al. Advanced Organic Chemistry, 3rd Edition (1985) John E.
Wiley & Sons, Inc., New York, N.Y., or other known methods.
[0087] Where the binding moieties are polypeptides, the fusion
molecule can be a conjugate or a fusion protein and manufactured
according to known methods. Where a fusion protein is desired, the
molecule can be manufactured according to known methods of
recombinant DNA technology. For example, the fusion protein can be
expressed by a nucleic acid molecule comprising sequences which
code for both moieties, such as by a fusion gene (nucleic acid
molecule). Thus, the invention further relates to nucleic acid
molecules, including fusion genes, which encode HDAg fragments,
mutants and derivatives.
[0088] Nucleic Acid Molecules
[0089] Recombinant or isolated nucleic acid molecules of the
invention, in one embodiment, encode an HDAg protein (including the
e.g., native proteins, fragments, derivatives, mutants and allelic
variants) as defined herein. A nucleic acid molecule of the present
invention can be double-stranded or single-stranded and can be a
DNA molecule, such as cDNA or genomic DNA, or an RNA molecule. The
nucleic acid molecule can be placed in a construct, which can be
inserted into a vector. As such, the nucleic acid molecule can
include one or more exons, with or without, as appropriate,
introns. In one embodiment, the nucleic acid molecule contains a
single open reading frame which encodes HDAg and one or more
binding moieties and, optionally, a signal sequence and/or a
polypeptide linker, when present. By way of example in a multi-exon
construct, the nucleic acid molecule contains a first exon which
begins with an ATG, encodes a binding moiety, and optionally the
polypeptide linker, and ends with a splice donor site. The
construct would also contain an HDAg-coding nucleic acid sequence
and would further would contain an intron followed by a second exon
which begins with a splice acceptor site and, optionally, a
polypeptide linker, coding sequences for a second binding moiety
and ending with a stop codon. Alternative combinations of these
elements would be apparent to the person of skill in the art.
[0090] As such, the nucleic acid molecule can include sequences
which encode HDAg, and one or more moieties, as well as one or more
of the following optional sequences, in a functional relationship:
regulatory sequences (as will be discussed in more detail below) a
start codon, a signal or leader sequence, splice donor sites,
splice acceptor sites, introns, a stop codon, transcription
termination sequences, 5' and 3' untranslated regions,
polyadenylation sequences, negative and/or positive selective
markers, and replication sequences.
[0091] The coding regions of the nucleic acid molecule code for
HDAg and the binding moeity or moieties and any polypeptide linkers
present. Where the binding moiety is a native ligand or cellular
surface protein (e.g. a cellular receptor), or a binding fragment
thereof, the nucleic acid molecule coding regions can correspond to
the native sequences which encode a binding moiety. Because many
amino acids are encoded by a plurality of codons, the coding
sequence can be mutated to result in the same amino acid sequence.
This may be advantageous where a codon is preferred by the selected
host cell. In one embodiment, the HDAg gene can be altered such
that the codons conform to the known codon use preferences for E.
coli. See FIG. 9 and FIGS. 15-17. The gene can be inserted into a
convenient expression vector which allows production of several
forms of the capsid protein including residues 1-84 (terminated in
the middle domain), the short isoform and the long isoform. Dingle
et al., J. Virol, (1998). All three forms express well. Preferably,
the nucleic acid molecule comprises the or corresponding coding
nucleotide sequence of FIG. 9, 10, 15-16, or substantially the same
sequences thereof, or the complement thereof. In another
embodiment, the nucleic acid molecule does not possess the
nucleotide sequence of GenBank Accession #M28267. The nucleic acid
molecule can be, for example, isolated and/or purified or
recombinant.
[0092] In a preferred embodiment, the nucleic acid molecule
comprises the nucleotide sequence depicted in FIG. 9, nucleotides
37-150 of FIG. 9, nucleotides 37-186 of FIG. 9, FIG. 10,
nucleotides 1421-1566 of FIG. 10 or nucleotides 1457-1566 of FIG.
10, FIG. 15, FIG. 16; or a fragment or mutation thereof, which
encodes a coiled-coil oligomer. In another preferred embodiment,
the nucleic acid molecule comprises a nucleotide sequence encoding
a polypeptide comprising an amino acid sequence depicted in a row
of FIG. 1, amino acids 12-48 of a row of FIG. 1, FIG. 3C, FIG. 9,
amino acids 12-48 of a row of FIG. 9, FIG. 10, amino acids 12-88 of
FIG. 10, FIG. 9, or .delta.12-60(Y). Also encompassed in the
invention are complementary strands of these sequences, DNA
sequences that hybridize to these sequences and RNA sequences
transcribed from these sequences. Also included are fragments or
mutations thereof, which encode a coiled-coil oligomer.
[0093] In one embodiment, the nucleic acid molecule encodes a
polypeptide of a fusion molecule described herein.
[0094] Also included are fusion nucleic acid molecules (e.g. fusion
genes) comprising an HDAg nucleic acid molecule operably linked to
a heterologous nucleic acid molecule ("heterologous gene"), which
encodes a peptide which is not HDAg and not derived therefrom
(i.e., "heterologous protein"). Where the binding moiety is a
mutation or variant of a native sequence, as provided above,
generally, the nucleic acid sequence can be mutated
correspondingly. It may also be preferred for ease of manufacture
of the nucleic acid sequence to maintain as much of the native
sequence as possible. In one embodiment, the nucleic acid molecule
shares at least about 50% sequence identity with the corresponding
native sequence such as the coding region, for example, the
coiled-coil region, e.g., amino acids 12-48 or amino acids 12-60.
In one embodiment, the sequence identity is at least about 65%,
more preferably, 75%. In a more preferred embodiment, the percent
sequence identity is at least about 90%, and still more preferably,
at least about 95%.
[0095] Recombinant nucleic acid molecules meeting these criteria
comprise nucleic acids having sequences identical to sequences of
naturally occurring genes, including polymorphic or allelic
variants, and portions (fragments) thereof, or variants of the
naturally occurring genes. Such variants include mutants differing
by the addition, deletion or substitution of one or more residues,
modified nucleic acids in which one or more residues are modified
(e.g., DNA or RNA analogs), and mutants comprising one or more
modified residues.
[0096] Many nucleic acid molecules coding for suitable binding
moieties are known in the art and can be obtained from, for
example, GENBANK. Alternatively, other sequences can be employed,
such as homologs of known genes.
[0097] Such homologous nucleic acids, including DNA or RNA, can be
detected and/or isolated by hybridization (e.g., under high
stringency conditions or moderate stringency conditions).
"Stringency conditions" for hybridization is a term of art which
refers to the conditions of temperature and buffer concentration
which permit hybridization of a particular nucleic acid to a second
nucleic acid in which the first nucleic acid may be perfectly
complementary to the second, or the first and second may share some
degree of complementarity which is less than perfect. For example,
certain high stringency conditions can be used which distinguish
perfectly complementary nucleic acids from those of less
complementarity. "High stringency conditions" and "moderate
stringency conditions" for nucleic acid hybridizations are
explained on pages 2.10.1-2.10.16 (see particularly 2.10.8-11) and
pages 6.3.1-6 in Current Protocols in Molecular Biology (Ausubel,
F. M. et al., eds., Vol. 1, containing supplements up through
Supplement 29, 1995), the teachings of which are hereby
incorporated by reference. The exact conditions which determine the
stringency of hybridization depend not only on ionic strength,
temperature and the concentration of destabilizing agents such as
formamide, but also on factors such as the length of the nucleic
acid sequence, base composition, percent mismatch between
hybridizing sequences and the frequency of occurrence of subsets of
that sequence within other non-identical sequences. Thus, high or
moderate stringency conditions can be determined empirically.
[0098] By varying hybridization conditions from a level of
stringency at which no hybridization occurs to a level at which
hybridization is first observed, conditions which will allow a
given sequence to hybridize (e.g. selectively) with the most
similar sequences in the sample can be determined.
[0099] Exemplary conditions are-described in Krause, M. H. and S.
A. Aaronson, Methods in Enzymology, 200:546-556 (1991). Also, see
especially page 2.10.11 in Current Protocols in Molecular Biology
(supra), which describes how to determine washing conditions for
moderate or low stringency conditions. Washing is the step in which
conditions are usually set so as to determine a minimum level of
complementarity of the hybrids. Generally, starting from the lowest
temperature at which only homologous hybridization occurs, each
.degree. C. by which the final wash temperature is reduced (holding
SSC concentration constant) allows an increase by 1% in the maximum
extent of mismatching among the sequences that hybridize.
Generally, doubling the concentration of SSC results in an increase
in T.sub.m of .about.17.degree. C. Using these guidelines, the
washing temperature can be determined empirically for high,
moderate or low stringency, depending on the level of mismatch
sought. The following table provides an example of each condition
of stringency.
1 % Allowed .degree. C. Stringency mismatch Temperature % Formamide
High 6.6 52 50 Medium 13 45 50 Low 27 45 22
[0100] "Selective isolation", or "selective hybridization", is
defined herein as embracing the isolation of a sufficiently few
number of molecules (preferably one) as to readily permit the
identification of the nucleic acid of interest.
[0101] The nucleic acid molecule also preferably comprises
regulatory sequences. Regulatory sequences include cis-acting
elements that control transcription and regulation such as,
promoter sequences, enhancers, ribosomal binding sites, and
transcription binding sites. Selection of the promoter will
generally depend upon the desired route for expressing the protein.
For example, where the molecule will be introduced (e.g.
transformed) into a cell by a viral vector, e.g. a plasmid,
preferred promoter sequences include viral, such as retroviral or
adenoviral, promoters. Examples of suitable promoters include the
cytomegalovirus immediate-early promoter, the retroviral LTR, SV40,
and TK promoter. Where the molecule is to be expressed in a
recombinant eukaryotic or prokaryotic cell, the selected promoter
is recognized by the host cell. In one embodiment the construct is
a cassette expression system. A suitable promoter which can be used
can include the native promoter for the binding moiety which
appears first in the construct.
[0102] The elements which comprise the nucleic acid molecule can be
isolated from nature, modified from native sequences or
manufactured de novo, as described, for example, in the
above-referenced texts. The elements can then be isolated and fused
together by methods known in the art, such as exploiting and
manufacturing compatible cloning or restriction sites.
[0103] Vectors and Host Cells
[0104] The nucleic acid molecules can be inserted into a construct,
e.g. a vector, such as a plasmid or cassette expression system,
which can, optionally, replicate and/or integrate into a
recombinant host cell, by known methods.
[0105] The vectors of the present invention comprise a nucleic acid
molecule which encodes HDAg (e.g. an HDAg monomer). The monomer can
be a subunit of an HDAg coiled-coil oligomer, e.g. an octamer. The
oligomer can comprise an HDAg polypeptide as described herein. The
nucleic acid molecule thus includes any of the nucleic acid
molecules described herein, for example, a native (wild type)
nucleic acid, or a fragment, mutant or derivative. Especially
preferred are nucleic acids encoding full-length HDAg (e.g. HDAg-S
or HDAg-L) or a fragment or derivative thereof, (e.g. a functional
fragment) capable of forming a coiled-coil octamer (e.g. an
N-terminal coiled-coil octamer). Preferred vectors comprise a
nucleic acid molecule comprising nucleotide sequence depicted in
FIG. 9, nucleotides 37-150 of FIG. 9, nucleotides 37-186 of FIG. 9,
FIG. 10, nucleotides 1421-1566 of FIG. 10 or nucleotides 1457-1566,
FIG. 10, FIG. 15 and FIG. 16. Preferred vectors also comprise a
nucleic acid comprising a nucleotide sequence encoding a
polypeptide comprising an amino acid sequence depicted in a row of
FIG. 1, amino acids 12-48 of a row of FIG. 1, the top row of FIG.
3c, FIG. 9, amino acids 12-48 of a row of FIG. 9, FIG. 10, amino
acids 12-88 of FIG. 10, FIG. 11, FIG. 17 or 8 12-60(Y). Other
preferred vectors comprise nucleic acids comprising sequences which
are the complementary strands of the above, DNA sequences which
hybridize to these sequences, RNA sequences transcribed from these
sequences, and fragments and mutations thereof, which encode a
coiled-coil oligomer, e.g. an octamer. Vectors can also comprise
fusion molecules comprising HDAg and at least one binding moiety,
as described herein.
[0106] In a preferred embodiment, the vector additionally comprises
at least one multiple cloning site. A multiple cloning site
comprises a cleavage sites for commonly used restriction sites to
facilitate incorporation of foreign (non-HDAg) gene, e.g. a
cassette. A multiple cloning site can be located 3' or 5' to the
nucleic acid molecule encoding HDAg. There can be multiple cloning
sites, for example, there can be a multiple, cloning site 3' of the
HDAg nucleic acid molecule and another multiple cloning site 5' to
the HDAg nucleic acid molecule. The multiple cloning site can be
located in a flanking region.
[0107] The vector can further comprise nucleic acid encoding a
nuclear localization signal, e.g. an HDAg nuclear localization
signal, for example, amino acids 68-88 of HDAg, as shown in FIG. 9.
The vector can also comprise an HDAg nucleic acid molecule
comprising a sequence encoding a coiled coil and a nuclear
localization signal.
[0108] The vectors of the invention can be used for the expression
of a fusion molecule as described herein. A heterologous (non-HDAg)
nucleic acid molecule, e.g., a gene, encoding a binding moiety of a
fusion molecule can be inserted into a vector comprising HDAg,
e.g., into a multiple cloning site. A vector can comprise one
heterologous gene or more than one heterologous gene. The genes can
be the same or different. A first heterologous gene can encode a
first binding moiety and a second heterologous gene can encode a
second binding moiety. The first and the second binding moieties
can be binding partners as described herein (for example, single
chain antibody and antigen, ligand and receptor, components of a
linked pathway, etc). The heterologous gene or genes and the
nucleic acid encoding HDAg can be operably linked, e.g., in the
same open reading frame. Where HDAg nucleic acid and a heterologous
gene encoding a binding moiety are operably linked, they are
expressed as a single protein unit, i.e., a fusion molecule.
[0109] In one embodiment, a vector comprises an HDAg nucleic acid
molecule (e.g. a nucleic acid cassette) encoding a monomer capable
of being a unit of a coiled-coil octamerization scaffold and a
heterologous gene encoding a binding moiety, wherein the expressed
binding moiety is bound to one terminal of the monomer, e.g. the N
terminus or the C terminus. Where there are two expressed
heterologous genes, each end of the monomer can be bound to an
expressed binding moiety.
[0110] Vectors can additionally comprise a nucleic acid molecule
encoding a nuclear localization signal, which can transport protein
expressed by the vector to the nucleus of a cell.
[0111] The vectors described herein can express nucleic acid, e.g.
a fusion gene, in a host cell, e.g. a procaryotic or eukaryotic
cell. In one embodiment, the vector can be expressed in a bacteria
cell, for example, Escherischia, e.g. E. coli. The nucleic acid in
the vector can also be expressed in Bacillus. It can also be
expressed in baculoviruses, pichia expressions systems, and animal
tissue or cells, for example insect, mammal, e.g., a human, or
yeast (such as Saccharomyces). Examples of specific cells include
somatic or embryonic cells, HeLa cells, human 293 cells, monkey
COS-7 cells, etc.
[0112] The vector can comprise a number of other components. For
example, the vector can comprise a marker, for example a positive
or negative selection marker, e.g. ampicillin or kanamycin. The
vector can comprise two markers, wherein the first marker is
capable of detecting propagation of a vector (e.g. a plasmid) in a
bacterial cell and a second marker is capable of detecting
propagation of the vector in a eukaryotic cell. The vector can
comprise an origin of replication for bacteria and an origin of
replication that is capable of mediating production of a
single-stranded DNA by a bacteriophage, such as f1 phage or M13
phage.
[0113] The vector can comprise a promoter. In one embodiment, the
promoter is a viral promoter, such as a retroviral or adenoviral
promoter. Examples of suitable promoters include T7, lac, trc, tac,
CMV, SV40, the cytomegalovirus immediate-early promoter, the
retroviral LTR and the TK promoter. In a preferred embodiment, the
promoter can be selected for high-level expression. A promoter can
be selected for optimal expression in bacteria, (e.g. T7, lac, trc,
tac etc.) or in a eukaryotic cell (e.g. CMV or SV40).
[0114] The vector can also comprise enhancers, ribosomal binding
sites and transcription binding sites. In one embodiment is a
vector depicted in FIG. 13A, B or C or FIG. 14. In one embodiment,
a vector comprises HDAg nucleic acid (with or without a nuclear
local signal), a heterologous gene, a marker, an origin of
replication for a host cell, an origin of replication capable of
mediating production of single-stranded DNA by a bacteriophage, a
promoter, and a ribosome binding site. In one embodiment, the
origin of replication is selected for maintaining plasmid
expression is E. coli.
[0115] Especially preferred is a vector for overexpression of
hepatitis delta antigen in E. coli, for example, a vector produced
by the method as herein described in Example 2, below, e.g. a
vector comprising a nucleic acid molecule sequence optimized for
expression of HDAg in E. coli. The gene can be inserted into a
vector which allows production of several forms of the capsid
protein including residues 1-84 (terminated in the middle domain),
the short isoform and the long isoform. In a preferred embodiment,
the vector is pR5.delta.V5. Another preferred embodiment is a
cassette expression system which allows any expressed sequence or
sequences (e.g. a binding moiety) to be appended to the N-terminus
of C-terminus of the HDAg octamerization scaffold. In a preferred
embodiment, the HDAg gene is mutated such that in the expressed
peptide, a serine residue of HDAg is replaced with (substituted by)
a cysteine to allow for convenient chemical cross-linking of the
octamerization domain, e.g., to an inert support matrix (e.g.
polyethylene glycol), to a synthetic peptide, to an
oligosaccharide, to a small organic molecule, or to lipids. FIG. 12
(A and B) depicts a representation of a construct containing eight
appended protein domains on an HDAg octameric framework.
[0116] The vector can be viral. Viral vectors include baculovirus,
retrovirus, adenovirus, parvovirus (e.g., adeno-associated
viruses), coronavirus, negative strand RNA viruses such as
orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies
and vesicular stomatitis virus), paramyxovirus (e.g. measles and
Sendai), positive strand RNA viruses such as picomavirus and
alphavirus, and double stranded DNA viruses including adenovirus,
herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr
virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and
canarypox). Other viruses include Norwalk virus, togavirus,
flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis
virus, for example. Examples of retroviruses include: avian
leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses,
HTLV-BLV group, lentivirus, spumavirus. Other examples include
murine leukemia viruses, murine sarcoma viruses, mouse mammary
tumor virus, bovine leukemia virus, feline leukemia virus, feline
sarcoma virus, avian leukemia virus, human T-cell leukemia virus,
baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer
monkey virus, simian immunodeficiency virus, simian sarcoma virus,
Rous sarcoma virus and lentiviruses. Fundamental Virology, Third
Edition, edited by B. N. Fields, D. M. Knipe, P. M. Howley, et al.
Lippincott-Raven Publishers, Philadelphia (1996) and additional
examples of viruses are described in detail in Fields Virology,
Third Edition edited by B. N. Fields, D. M. Knipe, P. M. Howley et
al., Lippincott-Raven Publishers, Philadelphia, Pa. (1996).
[0117] A nucleic acid molecule described herein can be introduced
(incorporated or inserted) into the host cell, by known methods.
Such cells, comprising such nucleic acid molecules, are encompassed
in the invention. The host cell can be a eukaryotic or prokaryotic
cell and includes, for example, baculoviruses, Pichia expression
systems, yeast (such as Saccharomyces), bacteria (such as,
Escherichia or Bacillus), animal cells or tissue, including insect
or mammalian cells (such as somatic or embryonic human cells,
Chinese hamster ovary cells, HeLa cells, human 293 cells and monkey
COS-7 cells, etc.). Examples of suitable methods of transfecting or
transforming cells include calcium phosphate precipitation,
electroporation, microinjection, infection, lipofection and direct
uptake. Methods for preparing such recombinant host cells are
described in more detail in Sambrook et al., "Molecular Cloning: A
Laboratory Manual," Second Edition (1989) and Ausubel, et al.
"Current Protocols in Molecular Biology," (1992), for example.
[0118] The host cell is then maintained under suitable conditions
for expression and recovering the molecule, e.g. a fusion molecule.
Generally, the cells are maintained in a suitable buffer and/or
growth medium or nutrient source for growth of the cells and
expression of the gene product(s). The growth media are not
critical to the invention, are generally known in the art and
include sources of carbon, nitrogen and sulfur. Examples include
Dulbeccos modified eagles media (DMEM), RPMI-1640, M199 and Grace's
insect media. Again, the selection of a buffer is not critical to
the invention. The pH which can be selected is generally one
tolerated by or optimal for growth for the host cell.
[0119] The cell is maintained under a suitable temperature and
atmosphere. Anaerobic host cells are generally maintained under
anaerobic conditions. Alternatively, the host cell is aerobic and
the host cell is maintained under atmospheric conditions or other
suitable conditions for growth. The temperature should also be
selected so that the host cell tolerates the process and can be for
example, between about 300 and 40.degree. C. for mammilian cells
and between 20 and 40.degree. C. for bacteria, yeast and insect
cells.
[0120] The recombinant molecules, including fusion molecules,
produced by the processes described herein can be isolated and
purified by known means. Examples of suitable purification and
isolation processes are generally known and include ammonium
sulfate precipitation, dialysis, gel filtration, immunoaffinity,
chromatography, electrophoresis, ultrafiltration, microfiltration
or diafiltration.
[0121] In addition the fusion molecule can incorporate commonly
used sequence tags e.g. his, tag or fla to facilitate purification
via ligand affinity chromatograph. The fusion molecule is
preferably purified substantially prior to use, particularly where
the protein will be employed as an in vivo therapeutic, although
the degree of purity is not necessarily critical where the molecule
is to be used in vitro. In one embodiment, the bifunctional
molecule can be isolated to about 50% purity (by weight), more
preferably to about 80% by weight or about 95% by-weight. It is
most preferred to employ a molecule which is essentially pure
(e.g., about 99% by weight or to homogeneity).
[0122] Fusion molecules which are prepared according to the above
method can be used directly in the disclosed methods or can be
screened for an activity prior to use. To screen the fusion
molecule for activity, for example, in vitro, the fusion molecule
(or mixtures of fusion molecules) can be contacted with, for
example, the binding partner of a binding moiety of the fusion
molecule under conditions suitable for binding and then assayed for
binding. For example, a fusion molecule comprising a ligand can be
screened for the ability to bind the ligand's receptor, or the
binding protein of the ligand's receptor, in vitro, by contacting
the receptor (or portion thereof) and the fusion molecule under
conditions suitable for binding and detecting binding.
[0123] Methods
[0124] The HDAg molecules of the invention are useful in a variety
of methods. The N-terminal octamer may serve as a convenient high
valency framework for linking, presenting or delivering a variety
of binding moieties, e.g. as described above. For example, the
molecules are useful in the delivery of one or more therapeutic
agents, such as drugs, proteins or polynucleotides (e.g., genes) or
products thereof to a patient. The polynucleotide or the product
thereof can be a therapeutic agent. In one embodiment, therapeutic
polynucleotide includes RNA (e.g., ribozymes) and antisense DNA
that prevents or interferes with the expression of an undesired
protein in the target cell. The polynucleotide can also encode a
heterologous therapeutic protein. A heterologous protein or
polynucleotide is one which is not HDAg. Examples of therapeutic
proteins include antigens or immunogens such as a polyvalent
vaccine, cytokines, tumor necrosis factor, interferons,
interleukins, adenosine deaminase, insulin, T-cell receptors,
soluble CD4, epidermal growth factor, human growth factor, blood
factors, such as Factor VIII, Factor IX, cytochrome b,
glucocerebrosidase, ApoE, ApoC, ApoAI, the LDL receptor, negative
selection markers or "suicide proteins", such as thymidine kinase
(including the HSV, CMV, VZV TK), anti-angiogenic factors, Fc
receptors, plasminogen activators, such as t-PA, u-PA and
streptokinase, dopamine, MHC, tumor suppressor genes such as p53
and Rb, monoclonal antibodies, antigen binding fragments or
constant regions thereof, drug resistance genes, ion channels, such
as a calcium channel or a potassium channel, and adrenergic
receptors, etc.
[0125] Also encompassed by the present invention are the use of
HDAg fusion molecules for high through put screening assays, such
as for detecting ligand and cell specific receptor binding pairs.
The ligand and/or receptor can be peptides (including
post-translationally modified proteins) and/or small molecules
(including sugars, steroids, lipids, anions or cations). The
ligands and ligand-cell specific receptors can be known or unknown.
Where the ligand is known and the receptor is unknown, ligand-cell
specific receptors can be identified, for example, by screening for
host cells transfected with nucleotides encoding potential
receptors. For example, the ligands can be secreted (such as
chemokines) or non-secreted (such as the extracellular domains of
chemokines receptors) proteins.
[0126] A library of host cells displaying putative ligand-cell
surface receptors can be obtained by transfecting suitable host
cells with nucleic acid constructs, including but not limited to
cDNA or genomic libraries, under appropriate regulatory control to
result in the expression of cell-surface receptors on the host
cell. The fusion molecule with ligand is added to the population of
host cells under conditions suitable for introduction. Introduction
can be detected, for example, with a label. A similar approach can
be used to select unknown ligands in the case where the ligand-cell
specific receptors are known and the ligand is unknown. In this
embodiment, a library of fusion molecules with putative ligands
(e.g., chemokines) can be obtained and contacted with one or more
host cells displaying cell surface receptors.
[0127] A similar approach can be used to identify unknown ligands,
test substances, drugs wherein the cell surface receptor is known,
where the fusion molecule comprises a binding moiety which is a
receptor which binds a surface molecule. The host cell expresses a
distinct ligand or a collection of recombinant ligands.
Ligand-receptor binding can be detected following introduction of
the molecule to the host cell.
[0128] The invention is also particularly useful for vaccine
delivery. In this embodiment, an antigen or immunogen can be
expressed heterologously (e.g., by recombinant insertion of a
nucleic acid sequence which encodes the antigen or immunogen
(including antigenic or immunogenic fragments) into a vector
comprising HDAg). Alternatively, the antigen or immunogen and HDAg
can be expressed in a live attenuated, pseudotyped virus vaccine,
for example. Generally, the methods can be used to generate humoral
and cellular immune responses, e.g. via expression of heterologous
pathogen-derived proteins or fragments thereof in specific target
cells.
[0129] The dosage administered (e.g., the effective amount) will,
of course, vary depending upon known factors such as the
pharmacodynamic characteristics of the particular agent, e.g., the
therapeutic binding entity, and its mode and route of
administration; age, health, and weight of the recipient; nature
and extent of symptoms, kind of concurrent treatment, frequency of
treatment, and the effect desired.
[0130] It can be administered one to several times per day,
depending on the mode of administration. Effective doses can be
determined by those of skill in the art. An effective dose of an
agent is an amount sufficient to relieve the individual of the
symptoms of the disorder which the agent is intended to treat.
[0131] Methods of introduction of the agent at the site of
treatment include, but are not limited to, intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous, oral,
intranasal, gene therapy, cellular implantation or particle
bombardment. Other suitable methods include or employ biodegradable
devices and slow release polymeric devices.
[0132] Because proteins are subject to being digested when
administered orally, parenteral administration, e.g., intravenous,
subcutaneous, or intramuscular, would ordinarily be used to
optimize absorption.
[0133] For parenteral administration, particularly suitable are
injectable, sterile solutions, preferably oily or aqueous
solutions, as well as suspensions, emulsions, or implants,
including suppositories. The molecule comprising the agent can be
administered in a solution, suspension, emulsion or lyophilized
powder in association with a pharmaceutically acceptable parenteral
vehicle. Examples of such vehicles are water, saline, Ringer's
solution, dextrose solution, and 5% human serum albumin. Liposomes
and nonaqueous vehicles such as fixed oils can also be used. The
vehicle or lyophilized powder can contain additives that maintain
isotonicity (e.g., sodium chloride, mannitol) and chemical
stability (e.g., buffers and preservatives). The formulation is
sterilized by commonly used techniques. Suitable pharmaceutical
carriers are described in the most recent edition of Remington's
Pharmaceutical Sciences, A. Osol, a standard reference text in this
field of art. Ampules are convenient unit dosages. Formulations for
transdermal or transmucosal administration generally include
penetrants such as fusidic acid or bile salts in combination with
detergents or surface-active agents. The formulation can then be
manufactured as aerosols, suppositories, or patches.
[0134] Oral agents may be administered if formulated as to be
protected from digestive enzymes. If administered orally, the SCR-P
will be administered in a therapeutic composition which may also
include an appropriate carrier (e.g., a physiologically compatible
carrier), a flavoring agent and a sweetener.
[0135] Suitable pharmaceutical carriers include, but are not
limited to water, salt solutions, alcohols, polyethylene glycols,
gelatin, carbohydrates such as lactose, amylose or starch,
magnesium stearate, talc, silicic acid, viscous parafin, fatty acid
esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc. The
pharmaceutical preparations can be sterilized and, if desired,
mixed with auxiliary agents, e.g., lubricants, preservatives,
stabilizers, wetting agents, emulsifiers, salts for influencing
osmotic pressure, buffers, coloring, and/or aromatic substances and
the like which do not deleteriously react with the active
compounds. They can also be combined where desired with other
active agents, e.g., enzyme inhibitors, to reduce metabolic
degradation.
[0136] Using procedures similar to those described above, HDAg
molecules (e.g. fusion molecules) and vectors (e.g. cassette
expression systems) comprising nucleic acid molecules, such as the
vectors described herein, can be used for a variety of purposes.
For example, the vector comprising all or part of a nucleic acid
sequence of FIG. 9 or FIG. 15 (synthetic) can be used to
overexpress thee hepatitis delta antigen in bacteria.
[0137] In a preferred embodiment, multiple copies of a binding
moiety (e.g. a peptide or domain) can be expressed using the
vectors described herein, e.g., by inserting a nucleic acid
cassette encoding the moiety into the vector, transforming a host
cell with the vector and culturing the cell under conditions
sufficient for expression of the moiety. Up to sixteen copies of
the moiety (eight at the C terminus of the HDAg monomer and eight
at the N terminus) can be made in this way. In a preferred
embodiment, the vector is one depicted in a Figure selected from
the group consisting of FIG. 13A, 13B, 13C and 14.
[0138] In one embodiment, a vector or molecule described herein can
be used for a high valency expression of a binding moiety, for
example, a peptide or protein domain, e.g., an antigen. In another
embodiment, the vector can be used to express a high valency
display of at least two different peptides or protein domains, to
enhance interaction between ligands. The interaction between
ligands can occur in solution, on membranes or on surfaces.
[0139] In another embodiment, interaction (e.g., fusion) between
two cells (or two cell types) can be mediated or enhanced by, for
example, associating an HDAg octamer expressing multiple copies of
a domain which interacts with a ligand on the surface of cell type
one and multiple copies of a domain which interacts with a ligand
on the surface of cell type two. This method could work for
embodiments involving more than two cells or cell types as well. A
fusion molecule, e.g., an octamer construct of the present
invention, which can be coupled to a surface, for example, by
chemical cross-linking or by inclusion of at least one copy of a
second domain which interacts with a ligand displayed on a surface,
can be used to display multiple copies of a domain on a surface. In
another embodiment, the octamer can be used to express different
enzymes from a linked pathway on a single framework, e.g., for
facilitating rapid exchange of substrates and products between
enzymes from a single pathway. The enzymes implicated Krebs in the
can be cycle.
[0140] In another embodiment, the octamer can also be used to link
a first binding moiety, e.g. a peptide or domain mediating
(specifying) an interaction (an "interaction" domain) and a second
binding moiety, which mediates an effect (an "effector" domain). In
another embodiment, the effector domain is a chemical, e.g. a drug,
linked to the octamer via a free --SH group on the octamer. In a
preferred embodiment, the interaction domain mediates interaction
with a specific receptor on a cell surface, and the effector domain
generates a specific function, such as cell killing.
[0141] In another embodiment, the octamer can contain one or more
copies of an domain for interaction with a ligand, and one or more
copies of a domain which is a label (e.g. alkaline phosphatase,
radiolabel, streptodevice, and green fluorescent protein) for
amplifying a signal in a solid phase assay, e.g. an ELISA
assay.
[0142] In another embodiment, an oligomer construct can be used to
couple an oligonucleotide which interacts (e.g. hybridizes) with a
nucleic acid molecule in the cell (e.g. a specific complementary
DNA or RNA sequence) and one or more copies of an effector to
target specific DNA or RNA sequences for cleavage. In a preferred
embodiment, the effector is a double-stranded nuclease. In a
preferred embodiment, the octamer is a mutant with a free
sulfhydryl (--SH) group. An octamer construct can be used to couple
multiple copies of a ligand in such a way that the interaction of
the octamer with a cell triggers signaling or internalization by
pathways which depend on the multimerization of a receptor or
ligand on the cell surface. The vectors can be used to promote
interaction between intracellular components in a signal
transduction pathway, for example components which are upstream or
downstream from each other.
[0143] In another embodiment, the system can be used to mediate
efficient (drive) gene expression by coupling (e.g. covalently) an
enhancer recognition protein and at least one promoter binding
protein.
[0144] In another embodiment, the system can be used as a high
valency trap to identify the vector can be used as a diagnostic. In
one embodiment, the binding moiety is Sp120 and the molecule is
used to test for the presence of Human Immunodeficiency Virus. In
another embodiment, at least one binding moiety is a drug which has
a therapeutic effect when administered to an animal. In another
embodiment, a molecule of the present invention is used to screen
test substances for an effect. In another embodiment, an agent can
be administered to a patient, wherein the agent will inhibit
formation of the coiled coil HDAg oligomer. Peptides (e.g.
proteins) and nucleic acids, as discussed above, the inventions
described herein are based upon the discovery that the HDAg protein
oligomerizes to a coiled-coil olctamer. The HDAg protein is derived
from the Hepatitis D virus which bind to peptides or protein
domains.
[0145] In another embodiment, at least one binding moiety is a drug
which has a therapeutic effect when administered to an animal.
[0146] In another embodiment, a molecule of the present invention
is used to screen test substances for an effect.
[0147] In another embodiment, an agent can be administered to a
patient, wherein the agent will inhibit formation of the coiled
coil HDAg oligomer.
[0148] As discussed above, the inventions described herein are
based on the discovery that the HDAg protein oligomerizes to a
coiled-coil octamer. The HDAg protein is derived from the Hepatitis
D virus.
[0149] Whereas Hepatitis B virus infection alone generally causes
mild, sometimes chronic, hepatitis, coinfection of hepatitis D
virus (HDV) with hepatitis B virus (HBV) causes severe, and often
fatal, liver disease in humans, and is the most common cause of
fulminant viral hepatitis, Hoofnagle, J. H., J. Am. Med. Assoc.,
261:1321-1325 (1989). The virus is an obligatory subviral satellite
of HBV, requiring the hepatitis B surface antigen (HBsAg) for
assembly and cell-to-cell transmission. Rizzetto, M. et al., Proc.
Natl. Acad. Sci. USA, 77:6124-6128 (1980). However, the viral
genome can replicate in the absence of HBV. Kuo, M. Y. P et al., J.
Virol. 63:1945-1950 (1989). Hepatitis delta encodes all of the
information required to direct replication of its RNA genome by the
host RNA Pol II. Efficient transmission of hepatitis delta virus
requires that the viral RNA and the capsid protein be encapsidated
within the hepatitis B virus surface antigen. The viral genome is a
1.7 kilobase single-stranded circular RNA, which is approximately
70% complementary to itself, Wang, K. S. et al., Nature,
323:508-513 (1986), and forms a rod-like structure, Kos., A. et
al., Nature, 323:558-560 (1986). The virus is believed to replicate
by a double rolling-circle mechanism in infected cells, Taylor, J.,
Cell, 61:371-373 (1990). Both the genomic and antigenomic strands
of the virus contain ribozymes, Wu, H. et al., Proc. Natl. Acad.
Sci. USA 86:1831-1835 (1989), Wu, H. N. et al, Science 243:652-654
(1989), Sharmeen, L. et al., J. Virol., 62:2674-2679 (1988), Kuo,
M. et al., J. Virol. 62:4439-4444, which are responsible for
reducing multimeric viral genomes into unit length and for
directing the religation of the linear genomes, Sharmeen, L. et
al., J. Virol. 63:1428-1430 (1989). The antigenomic strand of the
genome encodes the only viral protein known to be associated with
HDV, the hepatitis delta antigen (HDAg) (also known as delta virus
capsid protein). Wang, K. S. et al., Nature 323:508-513 (1986),
Makino, S. et al. Nature, 329:343-346.
[0150] HDAg exists in two isoforms. Early in the life cycle of the
virus, HDAg is expressed as a 195-amino acid protein, the small
hepatitis delta antigen (s-HDAg), which functions as a
transactivator of HDV RNA replication. This form predominates early
in infection. Kuo, M. Y. P, et al., J. Virol. 63:1945-1950 (1989).
Later in the life cycle of the virus, there is an RNA editing event
that changes the UAG stop codon of the HDAg-S to a UGG codon,
encoding a tryptophan. This allows translation to proceed for an
additional 19 amino acids, resulting in a 214-amino acid residue
form of the protein, the large delta antigen (HDAg-L). The 19 amino
acids include a stop signal which allows the large isoform to be
farnesylated at its terminus. HDAg-L is a potent inhibitor
(dominant repressor) of HDV replication, Chao, M. et al., J. Virol.
64:5066-5069 (1990), Glenn, J. S. & M. J. Virol. 65:2357-2361
(1991), and is also involved in packaging the viral RNA, Chang, F.
L. et al. Proc. Natl. Acad. Sci. USA 88:8490-8494 (1991), Wang, C.
J. et al., J. Virol. 65:6630-6636 (1991), Ryu, W. S., et al., J.
Virol. 66:2310-2315 (1992), and coencapsidation, i.e., the
copackaging of the small antigens into the viral particle. It also
directs association with the hepatitis B antigen. Chang, F. L. et
al. Proc. Natl. Acad. Sci. USA 88:8490-8494 (1991), Ryu, W. S. et
al., J. Virol. 66:2310-2315 (1992), Chang, M. F. et al., J. Virol.,
68:646-653 (1994). Both the large and small antigens are highly
specific RNA-binding phosphoproteins. Chang, M. F. et al., J.
Virol. 62:2403-2410, Lin, J. H. et al., J. Virol. 64:4051-4058
(1990) and have been shown to recognize specifically the viral
rod-like structure of the HDV viral genomes, Chao, M. et al., J.
Virol. 65:4057-4062 (1991). Crosslinking studies have shown that
both proteins can exist as either homomultimers (all small antigen
or all large antigen) or as heteromultimeric structures (a mixture
of small and large antigen) Xia, Y. P. & Lai, M. M. C., J.
Virol. 66:6641-6848 (1992), Wang, J. G. & Lemon, S. M., J.
Virol. 67:446-454 (1993), Chang, M. F. et al., J. Virol,
67:2529-2536 (1993).
[0151] There have been a number of structure-function studies of
both the large and small delta antigens. The N-terminal third of
the small delta antigen contains a putative coiled-coil sequence,
Xia, Y. P. & Lai, M. M. C., J. Virol. 66:6641-6848 (1992),
Wang, J. G. & Lemon, S. M., J. Virol. 67:446-454 (1993), Chang,
M. F. et al., J. Virol, 67:2529-2536 (1993), comprising heptad
repeats, which is followed by a linker domain which contains
bipartite nuclear localization signal. Xia, Y. P. et al., J. Virol.
66:914-921 (1992). The middle portion of HDAg contains two
arginine-rich motifs that have been shown to bind to the viral RNA.
Lee, C. Z. et al., J. Virol., 67:2221-2227 (1993). The C-terminal
segment of s-HDAg is proline- and glycine-rich. Lazinski, D. W.
& Taylor, J. M. J. Virol., 67:2672-2680 (1993). L-HDAg is
prenylated at the extreme C terminus and it is believed that this
part of the molecule interacts with HBsAg and the membranes of the
endoplasmic reticulum. Hwang, S. B. & Lai, M. M. C. J. Virol.,
67:7659-7662 (1993), de Bruin, W. et al., Virus. Res. 31:27-37
(1994). There is also some evidence that common segments of the
large and small antigens may have subtly different conformations.
Hwang, S. B. & Lai, M. M. C. Virology 193:924-931 (1993), Hwang
S. B. & Lai, M M C, J. Virol. 68:2958-2964 (1994).
[0152] The coiled-coil domain has been shown to be required for a
number of the functions of both small and large delta antigens.
Mutations that destroy or alter the coiled-coil domain either
greatly reduce or totally eliminate the ability of the HDAg-S to
function as a trans activator of replication, Chang, M. F. et al.,
J. Virol., 68:646-653 (1994), Chang, M. F. et al., J. Virol.
62:2403-2410 (1998), Lin, J. H. et al., J. Virol. 64:4051-4058
(1990), Chao, M. et al., J. Virol. 65:4057-4062 (1991), Xia, Y. P.
et al., J. Virol., 66:6641-6648 (1992). These same mutations also
prevent the HDAg-L from inhibiting HDV RNA replication and inhibit
its function in mediating the copackaging of the small antigen,
Chang, M. F. et al., J. Virol., 68:646-653 (1994), Chang, M. F. et
al., J. Virol. 62:2403-2410, Lin, J. H. et al., J. Virol.
64:4051-4058 (1990), Chao, M. et al., J. Virol. 65:4057-4062
(1991). Transfection of cells undergoing HDV replication with a
plasmid containing just the N-terminal one-third of the delta
antigen (which contains the coiled-coil domain) inhibited HDV
replication, Xia, Y. P. & Lai, M. M. C., J. Virol.,
66:6641-6648 (1992). However, removal of the coiled-coil domain
does not prevent the delta antigen from binding the viral RNA, Lin,
J. H. et al., J. Virol., 64:4051 -4058 (1990) nor does it prevent
the HDAg-L from packaging the viral RNA, Chen, P. J., et al., J.
Virol., 66:2853-2859 (1992). A "black sheep" model has been
proposed for the mechanism of inhibition of the HDV replication.
HDAg-L is believed to disrupt the homo-oligomeric small antigen
multimers, essentially poisoning the HDAg-S complex. Xia, Y. P.
& Lai, M. M. C., J. Virol., 66:6641-6648 (1992). While the
precise role of HDAg-S in replication of HDV is unknown, the
protein is not a polymerase, and RNA amplification is thought to be
mediated by host cell RNA polymerase II, MacNaughton, T. G. et al.,
Virology 184:387-390 (1991), Fu., T. B. & Taylor, J. et al., J.
Virol. 67:6965-6972 (1993).
[0153] Biophysical studies were undertaken to examine the
coiled-coil domain of HDAg. Rozzelle, J. E., Jr. et al., Proc.
Natl. Acad. USA, 92:382-386 (1995). As described in Example 1, a
peptide was synthesized that corresponded to residues 12 to 60 of
the .delta.12-60(Y). This region includes the N-terminal heptad
repeats. The peptide also included a C-terminal tyrosine so that
the peptide could be labeled with I.sup.125 for use in a
radioimmunoassay. The peptide sequence was conceptually divided
into three segments based on the presence of two potential helix
breakers Gly23 (G23) and Pro49 (P49); segments A (residues 12-24),
B (residues 25-49), and C (residues 50-60) (FIG. 1). The
full-length peptide .delta.12-60(Y) and two shorter peptides that
corresponded to regions A+B and B+C were synthesized. A number of
biophysical experiments, including circular dichroism (CD), mass
spectrometry, and analytical ultracentrifugation, clearly showed
that the .delta.12-60(Y) peptide was largely helical and formed a
coiled coil Rozzelle, J. E., Jr. et al., Proc. Natl. Acad. USA,
92:382-386 (1995). The shorter peptides formed much less stable
structures and were considerably less helical than .delta.12-60(Y).
Human polyclonal antibodies from hemophilic patients who were
chronic carriers of HBV and HDV reacted with the .delta.12-60(Y)
peptide, in both an ELISA and in a sandwich radioimmunoassay.
Rozzelle, J. E., Jr. et al., Proc. Natl. Acad. USA, 92:382-386
(1995), Wang, J. G. et al. J. Virol. 64:1108-1116. Subsequent
studies indicated that monoclonal antibodies against the peptide
recognized a conformational epitope only presented by the
full-length peptide and not the shorter, extensively overlapping
peptides, Rozzelle, J. E., Jr. et al., Proc. Natl. Acad. USA,
92:382-386 (1995).
[0154] Described herein for the first time is the crystal structure
of the peptide .delta.12-60(Y) to 1.8 .ANG. resolution. The
structure reveals that the capsid protein dimerizes as an unusual
antiparallel coiled coil. In the crystal structure, the dimers
further oligomerize to form an octamer. The octamer forms an open,
square planar structure with an antiparallel dimer forming each
side of the square. Crosslinking and hydrodynamic studies suggest
that both the peptide and the full-length short isoform exist as
stable octamers in solution.
[0155] The structure of the peptide lends new insights into the
mechanism by which HDAg dimerizes and further associates into
higher ordered structures. The structure also explains why residues
C-terminal to the predicted coiled-coil domain, and the
helix-breaking proline residues are important for the stabilization
of the coiled-coil structure. The peptide structure has important
consequences for the in vivo oligomerization of HDAg. The unique
octameric structure which is observed in the crystal structure also
suggests that the N-terminus of the molecule may have a previously
undetermined function.
[0156] When the HDAg open reading frame was originally examined,
amino acids from residue 13 to 47 were identified as possibly
forming a coiled coil. Glutaraldehyde cross-linking studies of
full-length HDAg, as well as of the peptide, confirmed the
formation of dimers, tetramers and higher-ordered structures, Wang,
J. G. & Lemon, S. M., J. Virol., 67:446-454 (1993), Rozzelle,
J. E., Jr. et al., Proc. Natl. Acad. USA, 92:382-386 (1995). The
crystal structure of the peptide clearly shows how monomers come
together to form antiparallel dimers as well as a higher-ordered
octameric structure. The structure of .delta.12-60(Y) also agrees
well with previous circular dichroism studies of the peptide, which
indicated that the two ends of the peptide (regions A and C) were
important for the structural stability of the coiled coil.
Rozzelle, J. E., Jr. et al., Proc. Natl. Acad. USA, 92:382-386
(1995). Shorter synthesized peptides that were missing either the A
or C regions (A+B and B+C), were significantly less helical than
the full-length peptide (A+B+C; 37%, 45% and 84% respectively at
37.degree. C.). The peptide structure shows that hydrophobic
residues from the N terminus of one monomer (region A), not
involved in the heptad repeat, interact with residues outside of
the predicted coiled-coil domain near the C terminus of the other
monomer (region C) to form a hydrophobic core Trp20 (W20), Leu24
(L24), Trp50 (W50), Leu51 (L51) sandwiched between Arg13 (R13) and
Arg24 (R24). This may stabilize the structure by keeping the ends
of the helix from fraying. An additional stabilizing feature is a
hydrogen bond between the sidechain of Glu45 (E45) and the indole
nitrogen of Trp20 (W20). These hydrophobic residues, as well as the
glutamic acid residue, are highly conserved in the 10 different
strains of HDV identified to date (FIG. 1). In fact, they are more
conserved than those residues in the heptad repeat making up the
hydrophobic core of the long helix (FIG. 1).
[0157] As described in Example 3, cross-linking studies of
full-length recombinant small delta antigen (r-HDAg-S or
r-.delta.Ag-S) also demonstrated that the recombinant protein forms
octamers in solution. This indicates that the octamer form seen in
the crystal may not be an artifact of crystallization, but rather
may represent the true state of the oligomerization of the delta
antigen. A study by Chang and colleagues found that a deletion in
the HDAg-L, just C terminal to the coiled-coil domain (residues 50
to 75), prevented the HDAg-S from being copackaged with the HDAg-L,
Chang, M. F. J. Virol., 66:6019-6027 (1992). HDAg-L with this same
deletion could not inhibit HDV replication, whereas a deletion in
L-DHAg of residues 65 to 75 could. This suggested that the
coiled-coil domain alone is not sufficient for the interaction
between the large and small antigens, and that a subdomain between
residues 50 and 65 is also necessary for this interaction. The
crystal structure of .delta.12-60(Y) indeed shows the importance of
residues between 50 and 60 in the formation of the peptide
oligomer. They are not only involved in stabilizing the
.delta.12-60(Y) dimer Trp50 (W50) and Leu51 (L51) but are also
involved in the formation of the dimer-dimer interface Trp50 (W50),
Ile54 (I54), and Ile58 (I58).
[0158] Prior to the studies described here, the overall
organization of the HDAg oligomer was unknown. The structure of the
.delta.12-60(Y) peptide suggests a number of interesting
considerations about the function of the coiled-coil domain of the
hepatitis delta antigen. For example, Lai and coworkers, Xia, Y.
P., et al., J. Virol. 66:6641-6648 (1992), inferring from previous
data that showed that as little as 12% of HDAg-L is needed to
inhibit 90% of viral activity, Chao, M., et al., J. Virol.,
64:5066-5069 (1990), proposed that as little as one part of HDAg-L
in eight parts of HDAg-S could inhibit viral replication. Their
"black sheep model" proposed that the HDAg-L either disrupted the
conformation of the oligomer of HDAg-S, therefore preventing it
from binding to host factors, or that the presence of HDAg-L in the
complex prevents the complex from interacting with host factors.
This would seem in agreement with the peptide structure of
octameric .delta.12-60(Y) and the results of the MALDI-TOF mass
spectrometry analysis. If HDAg-L does disrupt the conformation of
the oligomer of HDAg-S it probably does not do so directly through
the multimerization domain, given that the large and small delta
antigen share the same sequence within this region. Rather, it is
possible that this .alpha..sub.7 or .alpha..sub.n.beta..sub- .m
structure can no longer interact with host factors. Also, since the
C terminus of the L-HDAg interacts with the endoplasmic reticulum
(ER) membrane and with HBsAg for assembly, it could redirect the
complex elsewhere in the cell, preventing the nuclear translocation
of s-HDAg which is required for HDV replication.
[0159] Discovery of the organizational structure also provides
information regarding possible undetermined functions of the N
terminus. The octamer that is formed by the peptide is reminiscent
of proteins that form clamps around DNA, such as PCNA. Talluru, S.
R., et al., Cell 79:1233-1243 (1994). The 50 .ANG. hole formed by
the octameric structure is lined with basic side chains, suggesting
that the N terminus of the protein not only may act as a
dimerization/oligomerization domain, but also that it may function
either as a clamp around the viral RNA or other nucleic acid or
perhaps even function as a spool for nucleic acid. There is a
report that peptides corresponding to the extreme N-terminal
portion of the HDAg residues 2 to 27 and 2 to 17 can bind the viral
RNA Poisson, F., et al., J. Gen. Virol. 74:2473-2478 (1993),
Poisson, F. et al., J. Virol Methods 55:381-389 (1995). Since the
.delta.12-60(Y) structure is missing residues 2 to 11, it is
impossible to say what role they play in binding the viral RNA. Of
the remaining residues, only Lys25 (K25) and Lys26 (K26), which
point into the hole of the octamer, seem likely to play a role in
binding RNA by potentially binding the phosphate backbone of the
viral RNA.
[0160] The large size of the hole may be necessary to accommodate
the viral RNA which is only 70% self complementary, and would
possess a number of regions of bulged out single-stranded sequence,
increasing the radius of gyration of the RNA as well as bending the
RNA. Lilley, D. M. J., Proc. Natl Acad. Sci. USA, 92:7140-7142
(1995). The octameric structure also implies that there may be as
many as four RNA-binding domains on each side of the octamer. This
portion of the molecule may also bind another protein, especially
one that is acidic, such as the recently discovered delta antigen
interacting protein A (dipA), a cellular protein which has been
found to interact with the HDAg Brazas, R. et al., Science
274:90-94 (1996) and; based on its amino acid sequence, would have
an isoelectric point of 4.9.
[0161] Many investigators have referred to the putative coiled-coil
domain of the delta antigen as a leucine zipper-like region.
Experiments involving mutations in this region were interpreted
assuming the coiled-coil domain of the delta antigen would resemble
the parallel coiled-coil of the bZIP family of transcription
factors, such as GCN4. HDAg dimerizes through an antiparallel
coiled-coil domain, rather than a standard parallel coiled
coil.
[0162] Although algorithms have been designed to determine the
oligomerization state of a coiled coil, Woolfson, D. N., et al.,
Protein Sci. 4:1596-1607 (1995), Wolf, E., et al., Protein Sci.
6:1179-1189 (1997), they cannot determine the orientation of the
predicted coiled coil. The discovery that this region forms an
antiparallel coiled coil demonstrates that additional biochemical
or genetic evidence, such as provided herein, is necessary to
determine whether a predicted coiled-coil domain adopts a parallel
or antiparallel conformation. Along with the structure of the
.delta.12-60(Y) peptide, there are other examples of molecules that
dimerize through antiparallel coiled-coil domains, such as the
Escherichia coli regulatory protein AraC, Soisson, S. M., et al.,
Science 276:421-425 (1997) and the replication terminator protein
from Bacillus subtilis, Bussiere, D. E. et al., Cell 80:651-660
(1995).
[0163] The hepatitis delta antigen (HDAg), the sole protein made by
the hepatitis delta virus (HDV), is essential for viral replication
in vivo. Oligomerization of the protein is necessary for both the
transactivating function of the small delta antigen (HDAg-S) and
the trans dominant inhibitory effect of the large delta antigen
(HDAg-L). The structure of the peptide .delta.12-60(Y) that
corresponds to the predicted coiled-coil domain of the hepatitis
delta antigen HDAg suggests that delta antigen HDAg not only
dimerizes through an antiparallel coiled coil, but also forms
octamers. Interestingly, the coiled coil is stabilized by
hydrophobic residues C terminal to the coiled-coil domain. These
C-terminal residues interact with hydrophobic residues in the N
terminus of the coiled-coil region. The hydrophobic core of the
dimer is extended by further hydrophobic interactions at the
interface between dimers in the octameric structure. In contrast to
the rather promiscuous interactions between the coiled-coil domain,
these unique interactions at the termini of the monomer and dimer
interfaces might provide a good target for antivirals against HDV,
since disruption of oligomerization can prevent replication in
vivo.
[0164] The surprising octameric structure of the peptide suggests
that the capsid of the delta antigen (HDAg) will look very
different from the known structures of other viral nucleocapsid
proteins. The octameric structure also suggests important
implications for binding of HDAg to the viral RNA, since as many as
four of the arginine-rich RNA-binding domains might be needed for
binding to the viral RNA. The very basic hole in the octamer
suggests that this portion of the molecule may act as a sort of
"clamp" around an acidic molecule, such as viral RNA, another
nucleic acid or a cellular factor.
[0165] The exact function of HDAg in viral replication is unclear.
The protein may only function as a shuttle, binding to the viral
RNA and transporting it into the nucleus of the infected cell. It
is possible that HDAg functions to recruit host cell
transcriptional machinery to the viral RNA. The discovery of the
structure enables the design of experiments to determine whether
the N terminus of the molecule has RNA-binding capabilities and
investigate the mechanism of oligomerization and inhibition of
small antigen by the large antigen. A systematic examination of the
amino acids involved in dimerization and oligomerization would
allow the determination of the mechanism by which HDAg-L inhibits
HDAg-S. Furthermore, the unique interactions at the termini of the
coiled-coil region provide a new framework to be exploited in the
de novo design of stable antiparallel coiled coils.
[0166] The examples presented below are provided as further
guidance and are not to be construed as limiting the invention in
any way.
EXAMPLE 1
Synthesis of .delta.12-60(Y) Peptide
[0167] Materials and Methods
[0168] PEPTIDE SYNTHESIS: The .delta.12-60(Y) peptide was obtained
by Erickson, B. and Lemon, S. M. and was synthesized and purified
as described previously in Rozzelle, J. E. et al., Proc. Natl.
Acad. Sci. USA, 92:382-386 (1995), incorporated herein by reference
in its entirety.
[0169] The peptide (FIG. 11B) was assembled by
fluorenylmethoxycarbonyl chemistry and purified by reversed-phase
HPLC. It was N.sub..alpha.-acetylated and C.sub..alpha.-amidated.
Crude peptide in 0.05% trifluoroacetic acid was separated on an
octyl-silica column [C.sub.8, Applied Biosystems, 250 mm.times.10
mm (i.d.), 300-.ANG. pore size] by elution at 3 ml/min over 50 min
with a linear gradient of 20-42% acetonitrile in 0.05%
trifluoroacetic acid. Peptide .delta.12-60(Y) was eluted at 36%
acetonitrile (monitored at 230 nm). The homogeneity of the
individual fractions was determined on an analytical octyl-silica
column. The expected mass of the peptide was confirmed by
electrospray ionization (ESI) mass spectrometry: peptide
.delta.12-60(Y), m/z 6034.1.+-.1.2 (calcd. 6033.7).
[0170] The peptide from the 12-60 region of HDAg was synthesized
(FIG. 11B). Peptide .delta.12-60(Y) included segments A, B, and C.
Segment B contains three heptads in which the first and fourth
heptad positions are occupied by five leucines and one isoleucine,
and is probably part of an .alpha.-helical coiled coil. A tyrosine
residue, (Y), was added, Lys.sup.60, to the C terminus of
.delta.12-60(Y) to permit radioiodination.
[0171] CD SPECTROSCOPY. The .alpha.-helicity and the temperature at
the midpoint of thermal denaturation (T.sub.m) of the peptides were
determined by CD spectroscopy. All three peptides had high
.alpha.-helicity in PBS at 5.degree. C. The ratio of the mean
residue ellipticity of the negative bands near 222 nm and 208 nm
([.theta..sub.222])/([.theta..sub.208]) is an indicator of
coiled-coil formation. Values close to 1.0 indicate an
.alpha.-helical coiled coil and values near 0.8 indicate isolated
.alpha.-helices. At 5.degree. C., this ratio was 0.98 for
.delta.12-60(Y). At 37.degree. C., this ratio was 0.94 for
.delta.12-60(Y), consistent with persistence of a coiled-coil
structure. In contrast, at 37.degree. C. this ratio was only 0.79
for .delta.2-49 and 0.76 for 625-60(Y), inconsistent with a
coiled-coil structure.
EXAMPLE 2
Synthetic Gene for Optimized Expression of HDAg-S Materials and
Methods
[0172] EXPRESSION PLASMIDS: pR5.delta.V5 was constructed for the
high-level expression of HDAg-S in Escherichia coli. The protein
sequence of the American strain with the HDAg-S (GenBank accession
no. M28267) was back-translated with the program BACKTRANSLATE.
(This program was from the Wisconsin Package, versions 9.0
[Genetics Computer Group, Madison, Wis.], with E. coli codon
frequencies obtained from
gopher://weeds.mgh.harvard.edu:70/Oftp%3Aweeds.mgh.harvard.edu@/pub/codon-
/eco.cod.) With the sequence obtained as shown in FIG. 9 and FIG.
18, the plasmid pR5.delta.V5 was constructed by a two-step PCR
method, as described previously. Casimuro, D. R. et al.,
Biochemistry, 26:6640-6648(1995), with the exception that Vent
polymerase (New England BioLabs) was used instead of Taq
polymerase. Eight overlapping synthetic primers were synthesized
(FIG. 9 and FIG. 18). Changes in the back-translated sequences were
made so that the overlaps of the PCR primers would have
approximately the same melting temperature. Primers were
electrophoresed into a 10% sequencing gel, visualized by UV
shadowing, and excised from the gel. The primers were then purified
with a Waters Sep-Pak column.
[0173] The first PCR contained 4 pmol of each of the eight primers
in a 100-.mu.l reaction mixture. Ten microliters of the first PCR
was added to a second reaction mixture that contained an upstream
primer (5'-GGGCATATGAGCCGTAGCGA) and a downstream
(5'-GCGCCATGGTTTACGGAAAG) primer designed to amplify the desired
full-length product. Both reactions involved a hot start at
94.degree. C. followed by 30 cycles of 1 min. at 94.degree. C., 1
min at 57.degree. C., and 1 min at 72.degree. C., with a final
5-min extension at 72.degree. C.
[0174] The PCR product from the second reaction was cloned into the
vector pCR-Blunt (Invitrogen), which allows selection based on
disruption of a toxic gene. Plasmids isolated from colonies were
checked for the insert by restriction digest mapping. The open
reading frame of HDAg-S was subdloned into expression vector pRSETb
(Invitrogen). The sequence of the resultant plasmid, pR5.delta.V5,
was verified by dye termination sequencing.
[0175] PROTEIN PURIFICATION: Recombinant HDAg-S (.delta.Ag-S) was
expressed and purified as follows. Plasmid pR5.delta.V5 was
transformed into BL21 (DE3)pLysS cells (Novagen). A single colony
was used to inoculate a 100-ml overnight culture. Ten milliters of
this overnight culture was used to inoculate a 1-liter culture. At
an optical density of between 0.4 and 0.6, the cells were induced
with 3 ml of 100 mM IPTG
(isopropyl-.beta.-D-thiogalactopyranoside). Cell growth was
continued for 3 h, and then cells were pelleted at 5,000.times.g
for 10 min. The cells were resuspended in 15 ml of 50 mM HEPES (pH
7.5)--250 mM NaCl--1 mM MgC.sub.12 and stored at -20.degree. C.
until needed.
[0176] The frozen cells (45 ml corresponding to three 1-liter
cultures) were thawed, and one Complete Protease Inhibitor tablet
(Boehringer Mannheim) was added, along with RNase A and DNase I, to
a final concentration of 50 .mu.g/ml. Cells were lysed by
sonication and pelleted at 10,000.times.g for 30 min. The lysate
was diluted threefold with 50 mM HEPES buffer (pH 7.5) and then
applied to a 10.times.1.5-cm Fast SP Sepharose column (Pharmacia)
equilibrated with 50 mM HEPES buffer (pH 7.5) and eluted with a
salt gradient from 0 to 1 M NaCl in 50 mM HEPES (pH 7.5). The
fractions containing .delta.Ag-S were applied to a Superdex S-200
column (Pharmacia) equilibrated with 50 mM HEPES (pH 7.5), 500 mM
NaCl, and 5% glycerol. The HDAg-S obtained was >85% pure as
judged by Coomassie blue staining of a sodium dodecyl sulfate
gel.
[0177] Proteins with a histidine tag were purified as follows.
Proteins expressed in E. coli were affinity purified with a Talon
column according to the recommendations of the manufacturer
(Clontech). Proteins expressed in mammalian cells were purified by
the Invitrogen Xpress System. In both cases, the fractions
containing the purified protein were identified by sodium dodecyl
sulfate gel electrophoresis, pooled, dialyzed, and
concentrated.
[0178] TRANSFECTION: Plastic 16-mm-diameter tissue culture wells
(Costar) were seeded with approximately 0.1.times.10.sup.6 Huh7
cells, Nakabayoshi, H. et al. Cancer Res., 42:3858-3863 (1982). For
transfections with assembled RNP, 0.25 to 900 ng of HDAg-S and 500
ng of genomic HDV RNA in 125 .mu.l of Opti-MEM were combined with
2.7 .mu.l of lipofectamine (2 mg/ml) in 125 .mu.l of Opti-MEM,
incubated for 30 min at room temperature, and applied to cells that
had been washed with Opti-MEM (Hawley-Nelson, P. et al., Focus,
15:73-79(1993)). In control transfections, either HDAg-S or HDV RNA
was omitted. For cDNA transfections, 500 ng of plasmid DNA was
used. At 5 h after transfection, the transfection mixture was
changed to Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum. At 4 days after transfection, cells were reseeded
into a 30-mm-diameter dish containing a glass coverslip; at 8 days,
the cells were examined by immunofluorescence microscopy. Eight
days length was chosen for three reasons: (I) to avoid detection of
the transfected HDAg-S; (ii) In the immunofluorescence assays, 8
days corresponded to the peak signal for a cell undergoing
RNP-iniated replication; (iii) At 8 days, HDAg-L, created as a
consequence of both RNA editing and genome replication, could be
readily detected (Luo et al. J. Virol., 64:1021-1027 (1990)). In
contrast, for Northern analyses, genome replication was detectable
as early as 2 days.
[0179] Results
[0180] Initial studies with E. coli demonstrated poor expression of
HDAg-S from the wild-type sequence. About 18% of the codons in the
natural HDAg sequence are rarely used by E. coli. Attempted
overexpression of codons that are rare in E. coli not only can
inhibit expression but also can lead to misincorporation (Del Tito,
B. J. et al., J. Bacterial, 177:7086-7091(1995) Therefore, a
nucleotide sequence was designed which maintained the amino acid
sequence, but increased the percentage of codons that were most
favored for expression in E. coli from 26% to 85%. This optimized
sequence (FIG. 9) was used to construct expression plasmid
pR5.delta.V5. Thus, a 40-fold increase in expression was obtained
and the recombinant protein was purified to >85%
homogeneity.
EXAMPLE 3
Structure Determination
[0181] Materials and Methods
[0182] CRYSTALLIZATION AND DATA COLLECTION: The peptide
.delta.12-60(Y) was dissolved in 50 mM acetate, pH 4.8, 50 mM NaCl
and brought to a concentration of 15 mg/ml. The crystals of the
.delta.12-60(Y) peptide were grown at 22.degree. C. by the vapor
diffusion method. The peptide (2 .mu.l of a 15 mg/ml solution) was
mixed with 2 .mu.l of the reservoir solution containing 100 mM
sodium acetate, pH 4.8, and 100 mM sodium citrate, pH 5.6, on a
coverslip and then inverted over the reservoir solution. Crystals
appeared within 3-4 days, and grew as large as
0.5.times.0.3.times.0.3 mm. Crystals belonged to space group
P2.sub.12.sub.12 with unit cell parameters a=109.2 .ANG., b=85.3
.ANG., c=29.4 .ANG., .alpha.=.beta.=.gamma.=90.degree.. When
attempts to find a heavy atom derivative failed, a peptide was
synthesized with serine 22 replaced by a cysteine,
.delta.S22C12-60(Y). The 8S22C12-60(Y) peptide was reacted with an
excess of platinum terpyridine, dialyzed overnight against water,
and then freeze-dried. The peptide was then reconstituted at 15
mg/ml in 50 mM acetate, 50 mM NaCl, 5 mM DTT, pH 4.8, and
crystallized by the same conditions as that of the wild
type-peptide. This peptide crystallized isomorphously with the
.delta.12-60(Y).
[0183] The coverslips containing the crystals were inverted and
cryosolvent (reservoir solution containing 30% glycerol) was slowly
mixed with the drops and continuously replaced until no mixing was
observed. The crystals were mounted in nylon loops and frozen
directly in the nitrogen stream. Crystals used at Brookhaven were
stored in liquid nitrogen until the time of data collection. Two
native data sets were collected at Beamline X12C at the National
Synchrotron Light Source at Brookhaven National Lab using X-rays of
wavelength 1.15 .ANG. (Table 1). The heavy atom data set was
collected on a Siemens rotating anode with a multiwire detector
(Table 1). Data from the native crystals was processed using DENZO,
Otwinowski, Z. SERC Daresbury Laboratory, Warrington, UK:(56-62
(1993)) and SCALEPACK. Data from the heavy atom derivative was
integrated using the program BUDDHA (Blum, M. et al., J. Appl.
Cryst. 20:235-242 (1987)) and processed using ROTAVATA and AGROVATA
from the CCP4 package (CPP4 Acta. Cryst. D., 50:760-763 (1994)).
Structure factors from both data sets were calculated using
TRUNCATE (CPP4, supra). Data from the native and derivative were
scaled together using SCALEIT (CPP4, supra).
[0184] STRUCTURE DETERMINATION AND MODEL BUILDING: The positions of
the heavy atom sites were determined using SHELXS-86 (Sheldrick, G.
M., Acta. Cryst. A. 46:467-473 (1990)). The positions of the heavy
atom sites were refined using MLPHARE (Otwinowski, Z., Proceedings
of the CCP4 Study Weekend, 80-86 SERC Daresbury Laboratory,
Warrington, UK (1991)), and initial SIRAS phases were calculated.
The data was then subjected to a round of solvent flattening with
histogram matching using DM (Zhang, K. Y. J. & Main, P. Acta.
Cryst. A. 46:41-46 (1990). A map was calculated which clearly
showed the position of the two dimers in the asymmetric unit, and
an initial model was built into the initial SIRAS map using the
program O (Jones, T. A. et al. Acta Cryst. A., 47:110-119 (1990)).
The structure was refined using X-PLOR.sub.v3.8.9, Brunger, A. T.,
Yale University Press, New Haven, Conn. (1992). Rounds of
positional refinement, followed by simulated annealing and B-factor
refinement, were carried out with rebuilding of the structure using
O between cycles of refinement. During the initial model building
and refinement, omit maps, which excluded 10 residues at a time,
were used to check the progress of refinement.
[0185] SURFACE AND ELECTROSTATIC CALCULATIONS: Surface calculations
were performed using the surface option in QUANTA version 4.0.
Electrostatic calculations were performed with GRASP version
1.3.
[0186] PROTEIN EXPRESSION AND PURIFICATION: The pR5.delta.V5
plasmid, Dingle, K. et al. J. Virol., 72(6):4783-4788 (1998) which
contains a synthetic gene for the small delta antigen, HDAg-S, was
transformed into BL21 (DE3)pLysS cells (Novagen) and purified as
described previously in Example 2. See also Dingle, K. et al.,
supra. Briefly, 45 ml of frozen cells, corresponding to three 1 L
cultures, were thawed and one protease inhibitor tablet (Boehringer
Mannheim) was added, as well as RNAse A and DNAse I to a final
concentration of 50 .mu.g/ml. Cells lysed by sonication were
pelleted at 10,000.times.g for 30 minutes. The lysate was diluted
three-fold with 50 mM HEPES buffer, pH 7.5, and then applied to a
10.times.1.5 cm Fast SP Sepharose (Pharmacia) column equilibrated
with 50 mM HEPES buffer, pH 7.5, and eluted using a salt gradient
from 0-1M NaCl in 50 mM HEPES, pH 7.5. The fractions containing
recombinant small delta antigen (r.delta.Ag-S [r-HDAg-S]) were
assayed using SDS-PAGE and pooled. The sample was then applied to a
Superdex S-200 column (Pharmacia) equilibrated with 50 mM Hepes, pH
7.5, 500 mM NaCl and 5% glycerol. The elution of the protein from
the column was monitored by UV absorbance at 280 nm.
[0187] Results
[0188] Attempts to find a heavy atom derivative using the peptide
with the wild-type sequence of the American strain of HDAg failed.
Thus, a new peptide was synthesized with a cysteine replacing
serine 22 (Ser22) (this residue demonstrates considerable variation
in different strains of HDV, FIG. 1). The cysteine mutant and
wild-type peptides crystallized isomorphously. The presence of
cysteine 22 (Cys22) allowed the preparation of a platinum
terpyridine derivative, facilitating the determination of the
structure using SIRAS methods (Table 1). Retrospective examination
of the model confirmed that the Pt was bound to the sulfur of
cysteine 22 (Cys22).
[0189] The solvent-flattened map was easily interpretable, and
clearly showed two dimers in the asymmetric unit. Rounds of
positional refinement, simulated annealing, temperature factor
refinement using X-PLOR (Bruinger, A. T., Yale University Press,
New Haven, Conn. (1992)), and manual rebuilding using O (Jones, T.
A. et al., Acta Cryst. A. 47:110-19 (1990)), led to the current
model (Table 2, FIG. 2). The current model has an R factor of 22.5%
and a free R factor of 27% with good geometry (r.m.s.d. bond 0.007
.ANG. and r.m.s.d. bond angles 1.0.degree.). A number of sidechains
exposed to the large solvent channel, as well as the first residue
in the chain and the last residue in one of the chains, are
disordered. The four monomers in the asymmetric unit superimpose
well onto one another, with an average r.m.s.d. for mainchain atoms
of 0.81 .ANG. and for all non-hydrogen atoms 1.51 .ANG.. The main
differences in the monomers are those residues involved in crystal
packing interactions.
[0190] The coordinates have been deposited in the Brookhaven
Protein Data Bank (accession number 1A92).
[0191] Each monomer is composed of a long, N-terminal helix,
approximately 60 .ANG. in length, interrupted by a sharp bend at
proline 49 (Pro49), and continuing on into another short helix. The
long helices of each of two monomers wrap around each other forming
an antiparallel coiled coil (FIG. 3a, FIG. 3b), which straightens
out at the N terminus. Only one of the four possible salt bridges
between Glu31 (E31) and Lys38 (K38) is seen. In the other three
cases, the charged groups are slightly farther apart (3.8 .ANG.,
4.2 .ANG. and 4.4 .ANG. versus 2.9 .ANG.) and the sidechains are
hydrogen bonded to nearby solvent molecules. The sidechain of Glu45
(E45) is hydrogen bonded to the indole nitrogen of Trp20 (W20). The
sidechain of Asn48 (N48), which is located at the C terminus of the
long helix, completes the hydrogen-bonding pattern of the helix by
making a hydrogen bond back to the mainchain oxygen of Leu44 (L44).
The formation of the dimer buries 2650 .ANG..sup.2 of surface area,
approximately 26% of the total surface area.
[0192] Although the majority of residues in the heptad repeat (FIG.
3c) of the predicted coiled-coil region do pack as expected, Trp20
(W20) does not. Even though the C.alpha.-C.beta. vector of Trp20
(W20) points out of the interface as would be expected for a
sidechain in the a position of a heptad repeat, the sidechain of
Trp20 is flipped away from the core of the coiled coil and into a
hydrophobic region formed between segment A (residues 12-24) of one
monomer, and segment C (50-60) of its partner within the peptide
dimer. The dimer shows primarily hydrophobic interactions between
residues in the A and C regions. Ile16 (I16), Leu17 (L17), Trp20
(W20), Trp50 (W50), and Leu51 (L51) are the sidechains primarily
involved in this hydrophobic region, which is capped by the
aliphatic portion of the sidechains of Arg13 (R13) and Arg24 (R24)
(FIG. 4). The primary non-hydrophobic, monomer-monomer interactions
near this region involve the formation of a hydrogen bond between
Trp20 (W20) and Glu45 (E45) (FIG. 4). The heptad repeat is also
unusual in that it contains a glycine at position 23. If the
monomers were oriented in a parallel fashion, a large hole in the
middle of the hydrophobic core of the dimer would result. However,
since the strands are arranged antiparallel, the large sidechain of
Ile41 (I41) packs into the hole formed by Gly23 (G23). The dimer is
stabilized by hydrophobic interactions other than the residues in
the heptad repeat. Residues from the N-termini of each monomer,
Ile16 (I16), Leu17 (L17), Trp20 (W20) from one monomer and Trp50
(W50), Leu51 (L51), and Ile54 (I54) from the other, form a
hydrophobic core which is protected from solvent by the aliphatic
portions of Arg13 (R13) and Arg24 (R24). There is also a hydrogen
bond between the sidechain of Glu45 (E45) and the indole nitrogen
of Trp20 (W20) (O--N distance 2.8 .ANG.).
[0193] In the crystal, each dimer associates with three other
dimers to form a doughnut-like octamer (FIG. 5). The octameric
complex forms a pseudo-centered (C222) cell. The octamer is widely
open with a central "hole", 50 .ANG. in diameter. The open
structure of the octamer is reminiscent of several other proteins,
including Proliferating cell nuclear antigen (PCNA), in which the
hole that is formed is believed to encircle DNA (Talluru, S. R. et
al., Cell, 79:1233-1243 (1994)). It is this octameric structure
which is the translational repeating unit in the crystal (FIG. 5).
The dimer-dimer interface is a four-helix bundle formed across the
crystallographic two-fold axis. The interface of the two dimers
consists of hydrophobic residues in region A of the coiled-coil
domain Leu17 (L17) and Val21 (V21) but also includes residues
C-terminal to the coiled-coil domain, region C, between residues 50
to 60 Trp50 (W50), Ile54 (I54), Ile57 (I57) and Ile58 (I58) (FIG.
6). Thus, hydrophobic residue from both helices pack in the
interface, essentially extending the hydrophobic core mentioned
above. Trp50 is involved in both the formation of the dimer as well
as the octamer. Formation of the octamer buries an additional 800
.ANG..sup.2 of surface area per monomer, which means that
approximately 40% of the total surface area of each monomer is
buried. The 50 .ANG. diameter hole framed by the four dimers is
lined with basic sidechains (FIGS. 7a and b). The hole is large
enough to accommodate an RNA molecule. Residues Lys26 (K26) and
Lys38 (K38) which had been modeled in as alanine were changed to
lysine for this calculation. The electrostatic surface was
calculated using GRASP (Nicholls, A. Columbia University, New York,
N.Y.(1992)), and rendered using RASTER3D (Merritt, E. A. &
Murphy, M. E. P., Acta. Cryst. D. 50:869-873 (1994)).
2TABLE 1 Data Collection Statistics Native* S22C Pt Spacegroup
P2.sub.12.sub.12 P2.sub.12.sub.12 Unit cell (a, b, c) 109.2, 85.3,
29.4 110.3, 86.3, 29.6 Temperature of data -160 -165 collection
(.degree. C.) Resolution (.ANG.) 15-1.73 86-2.5 Number of
reflections 221, 286 44, 362 Number of unique reflections 28, 279
10, 013 Completeness.sup..dagger. (%) 94 (35) 97
I/.sigma..sup..dagger. 51 (7) 6.0 Multiplicity 7.8 4.4
R.sub.sym.sup..dagger..dagger-- dbl..sctn. (%) 4.2 (18) 6.7 [14.0]
R.sub.iso.sup..paragraph. (%) -- 30.5 R.sub.cullis# -- 0.62 (0.52)
R.sub.cullis anon.sup..Yen. -- 0.84 Phasing power** -- 2.2 (1.7)
*Data are from two crystals. .sup..dagger.Numbers in parentheses
represent values in the highest resolutions shell. 1 R sym = ( h ,
k , l ) I ( h , k , l ) - < I ( h , k , l ) > / ( h , k , l )
< I ( h , k , l ) > , where <I.sub.(h,k,l) > represents
the sigma weighted average intensity of symmetry-equivalent
reflections. .sup..sctn.The number in square brackets represents 2
R anom = < I + ( h , k , l ) > - < I - ( h , k , l ) >
/ ( < I + ( h , k , l ) > + < I - ( h , k , l ) > ) ,
where <I+/-.sub.(h,k,l) > represents the statistically
weighted average intensity of symmetry-equivalent reflections. 3
.paragraph. R iso = ( h , k , l ) ( F PH - F P ) / ( F P ) . 4 # R
cullis = ( h , k , l ) F PH - F P + F H / ( h , k , l ) F PH - F P
; number in parentheses represents R.sub.cullis for centric
reflections. 5 .Yen. R cullis anom = ( h , k , l ) F PH + - F PH -
obsvd - F PH + - F PH - calc / ( h , k , l ) F PH + - F PH - obsvd
**Phasing power = <.vertline.F.sub.H.vertline./.vertline.
.vertline.F.sub.PH.vertline. - .vertline.F.sub.P +
F.sub.H.vertline. .vertline.>; number in parentheses is the
power for centric reflections
[0194]
3TABLE 2 Refinement Statistics Resolution range (.ANG.) 15-1.8
R.sub.working*(%) 22.5 R.sub.free.sup..dagger.(%) 27.0 Non-hydrogen
protein atoms 1785 Solvent atoms 114 Rms from ideal geometry bond
lengths (.ANG.) 0.007 bond angles (.degree.) 1.0 dihedral angles
(.degree.) 16.9 impropers (.degree.) 0.59 Average B factor overall
(.ANG..sup.2) 29.3 mainchain 22.5 sidechain 32.4 solvent 34.7 6 * R
working = ( h , k , l ) ( F o - F c ) / ( F o ) , for a working set
composed of 90% of the data. 7 .dagger. R free = ( h , k , l ) ( F
o - F c ) / ( F o ) , for a test set composed of 10% of the data
selected randomly.
EXAMPLE 4
Mass Spectrometry
[0195] Materials and Methods:
[0196] r-HDAg-S was prepared as described above. The samples for
mass spectrometry were prepared as follows: the r-HDAg-S was
dialyzed overnight against water. Cross-linked protein was prepared
by the addition of 5 .mu.l of 0.5% glutaraldehyde to 40 .mu.l of
rHDAg-S for 5 minutes, and quenched by the addition of 5 .mu.l of 1
M ammonium acetate. Mass spectrometry was performed in the BCMP
Biopolymer facility on a Persceptive Biosystems Voyager-DE mass
spectrometer.
[0197] Results
[0198] Previous studies have suggested that both the peptide and
natural HDAg derived from infected liver form multimers in solution
(Wang, J. G. & Lemon S. M. J. Virol, 67:446-454 (1993)),
(Rozzelle, J. E., Jr. et al. Proc. Natl. Sci. USA, 92:382-386
(1995)). In order to investigate the significance of the octamer
formed by the peptide, MALDI-TOF mass spectrometry was used to
determine the mass of monomeric and oligomeric forms of recombinant
small delta antigen, r-HDAg-S. The uncrosslinked protein has a mass
of 2,1832 Da (FIG. 8A), which is the correct mass within 0.01% of
the amino acid sequence of the American strain of the small delta
antigen (HDAg-S) (Genbank accession #-M28267) minus the first
methionine residue. The primary species of the cross-linked rHDAg-S
had a mass of 176,282 Da (FIG. 8B). The M+1 and M+2 peaks of the
octamer were the only significant peaks in the spectrum. The ratio
of the masses of the cross-linked species to the monomer is
8.1:1.
[0199] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the claims.
Sequence CWU 1
1
35 1 49 PRT Hepatitis Delta Virus 1 Gly Arg Glu Asp Ile Leu Glu Gln
Trp Val Ser Gly Arg Lys Lys Leu 1 5 10 15 Glu Glu Leu Glu Arg Asp
Leu Arg Lys Leu Lys Lys Lys Ile Lys Lys 20 25 30 Leu Glu Glu Asp
Asn Pro Trp Leu Gly Asn Ile Lys Gly Ile Ile Gly 35 40 45 Lys 2 49
PRT Hepatitis Delta Virus 2 Gly Arg Glu Glu Val Leu Glu Gln Trp Val
Asn Ser Arg Lys Lys Ala 1 5 10 15 Glu Glu Leu Glu Arg Asp Leu Arg
Lys Thr Lys Lys Lys Ile Lys Lys 20 25 30 Leu Glu Asp Asp Asn Pro
Trp Leu Gly Asn Ile Lys Gly Ile Leu Gly 35 40 45 Lys 3 49 PRT
Hepatitis Delta Virus 3 Gly Arg Glu Glu Val Leu Glu Gln Trp Val Ser
Gly Arg Lys Lys Leu 1 5 10 15 Glu Glu Leu Glu Arg Asp Leu Arg Lys
Val Lys Lys Lys Ile Lys Lys 20 25 30 Leu Glu Asp Glu His Pro Trp
Leu Gly Asn Ile Lys Gly Ile Leu Gly 35 40 45 Lys 4 49 PRT Hepatitis
Delta Virus 4 Gly Arg Glu Glu Val Leu Glu Gln Trp Val Ala Gly Arg
Arg Lys Gln 1 5 10 15 Glu Glu Leu Glu Arg Asp Leu Arg Lys Thr Lys
Lys Lys Ile Lys Lys 20 25 30 Leu Glu Glu Glu Asn Pro Trp Leu Gly
Asn Ile Lys Gly Ile Leu Gly 35 40 45 Lys 5 48 PRT Hepatitis Delta
Virus 5 Thr Arg Glu Glu Thr Leu Glu Lys Trp Ile Thr Ala Arg Lys Lys
Ala 1 5 10 15 Glu Glu Leu Glu Lys Asp Leu Arg Lys Thr Arg Lys Thr
Ile Lys Lys 20 25 30 Leu Glu Glu Glu Asn Pro Trp Leu Gly Asn Ile
Val Gly Ile Ile Arg 35 40 45 6 48 PRT Hepatitis Delta Virus 6 Thr
Arg Glu Glu Thr Leu Glu Lys Trp Ile Thr Ala Arg Lys Lys Ala 1 5 10
15 Glu Glu Leu Glu Lys Asp Leu Arg Lys Ala Arg Lys Thr Ile Lys Lys
20 25 30 Leu Glu Glu Glu Asn Pro Trp Leu Gly Asn Ile Leu Gly Ile
Ile Arg 35 40 45 7 49 PRT Hepatitis Delta Virus 7 Gly Arg Glu Gln
Ile Leu Glu Gln Trp Val Asp Gly Arg Lys Lys Leu 1 5 10 15 Glu Glu
Leu Glu Arg Asp Leu Arg Lys Ile Lys Lys Lys Ile Lys Lys 20 25 30
Leu Glu Glu Glu Asn Pro Trp Leu Gly Asn Val Lys Gly Ile Leu Gly 35
40 45 Lys 8 49 PRT Hepatitis Delta Virus 8 Gly Arg Glu Glu Ile Leu
Glu Gln Trp Val Ala Gly Arg Lys Lys Leu 1 5 10 15 Glu Glu Leu Glu
Arg Asp Leu Arg Lys Thr Lys Lys Lys Leu Lys Lys 20 25 30 Ile Glu
Asp Glu Asn Pro Trp Leu Gly Asn Ile Lys Gly Ile Leu Gly 35 40 45
Lys 9 37 PRT Artificial Sequence Residues 12-48 of delta 12-60 (Y)
9 Gly Arg Glu Asp Ile Leu Glu Gln Trp Val Ser Gly Arg Lys Lys Leu 1
5 10 15 Glu Glu Leu Glu Arg Asp Leu Arg Lys Leu Lys Lys Lys Ile Lys
Lys 20 25 30 Leu Glu Glu Asp Asn 35 10 604 DNA Artificial Sequence
Synthetic Gene for Optimized Expression of HDA g-S in E. Coli 10
gggcat atg agc cgt agc gaa cgt cgt aaa gat cgt ggc ggc cgt gaa 48
Met Ser Arg Ser Glu Arg Arg Lys Asp Arg Gly Gly Arg Glu 1 5 10 gat
att ctg gaa cag tgg gtg agc ggc cgt aag aag tta gag gaa ttg 96 Asp
Ile Leu Glu Gln Trp Val Ser Gly Arg Lys Lys Leu Glu Glu Leu 15 20
25 30 gaa cgt gat ctg cgt aaa ctg aaa aag aag att aag aaa ctg gaa
gaa 144 Glu Arg Asp Leu Arg Lys Leu Lys Lys Lys Ile Lys Lys Leu Glu
Glu 35 40 45 gat aac ccg tgg ttg ggt aat att aaa ggc att att ggc
aag aaa gat 192 Asp Asn Pro Trp Leu Gly Asn Ile Lys Gly Ile Ile Gly
Lys Lys Asp 50 55 60 aaa gat ggc gaa ggc gcg ccg ccg gcg aag aaa
ctg cgt atg gat cag 240 Lys Asp Gly Glu Gly Ala Pro Pro Ala Lys Lys
Leu Arg Met Asp Gln 65 70 75 atg gaa att gat gcg ggc ccg cgt aaa
cgt ccg ctg cgt ggc ggc ttt 288 Met Glu Ile Asp Ala Gly Pro Arg Lys
Arg Pro Leu Arg Gly Gly Phe 80 85 90 acc gat aag gaa cgt cag gac
cat cgt cgt cgt aaa gcg ctg gaa aac 336 Thr Asp Lys Glu Arg Gln Asp
His Arg Arg Arg Lys Ala Leu Glu Asn 95 100 105 110 aaa cgt aaa cag
ctg agc agc ggc ggc aaa tct ctg agc cgt gaa gaa 384 Lys Arg Lys Gln
Leu Ser Ser Gly Gly Lys Ser Leu Ser Arg Glu Glu 115 120 125 gaa gaa
gaa ctg aaa cgt ctg acc gaa gaa gat gaa aaa cgt gaa cgt 432 Glu Glu
Glu Leu Lys Arg Leu Thr Glu Glu Asp Glu Lys Arg Glu Arg 130 135 140
cgt att gca ggt cca tct gtt ggt ggt gtg aac ccg ctg gaa ggc ggc 480
Arg Ile Ala Gly Pro Ser Val Gly Gly Val Asn Pro Leu Glu Gly Gly 145
150 155 agc cgt ggt gca ccg ggc ggt ggc ttt gtg ccg tct atg caa ggt
gtt 528 Ser Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Ser Met Gln Gly
Val 160 165 170 cca gaa agc ccg ttt gcg cgt acc ggc gaa ggc ctg gat
att cgt ggc 576 Pro Glu Ser Pro Phe Ala Arg Thr Gly Glu Gly Leu Asp
Ile Arg Gly 175 180 185 190 agc cag ggc ttt ccg taaaccatgg cgc 604
Ser Gln Gly Phe Pro 195 11 195 PRT Artificial Sequence Amino acid
sequence encoded by synthetic Gene for Optimized Expression of
HDAg-S in E. Coli 11 Met Ser Arg Ser Glu Arg Arg Lys Asp Arg Gly
Gly Arg Glu Asp Ile 1 5 10 15 Leu Glu Gln Trp Val Ser Gly Arg Lys
Lys Leu Glu Glu Leu Glu Arg 20 25 30 Asp Leu Arg Lys Leu Lys Lys
Lys Ile Lys Lys Leu Glu Glu Asp Asn 35 40 45 Pro Trp Leu Gly Asn
Ile Lys Gly Ile Ile Gly Lys Lys Asp Lys Asp 50 55 60 Gly Glu Gly
Ala Pro Pro Ala Lys Lys Leu Arg Met Asp Gln Met Glu 65 70 75 80 Ile
Asp Ala Gly Pro Arg Lys Arg Pro Leu Arg Gly Gly Phe Thr Asp 85 90
95 Lys Glu Arg Gln Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys Arg
100 105 110 Lys Gln Leu Ser Ser Gly Gly Lys Ser Leu Ser Arg Glu Glu
Glu Glu 115 120 125 Glu Leu Lys Arg Leu Thr Glu Glu Asp Glu Lys Arg
Glu Arg Arg Ile 130 135 140 Ala Gly Pro Ser Val Gly Gly Val Asn Pro
Leu Glu Gly Gly Ser Arg 145 150 155 160 Gly Ala Pro Gly Gly Gly Phe
Val Pro Ser Met Gln Gly Val Pro Glu 165 170 175 Ser Pro Phe Ala Arg
Thr Gly Glu Gly Leu Asp Ile Arg Gly Ser Gln 180 185 190 Gly Phe Pro
195 12 1679 DNA Hepatitis Delta Virus 12 cttgagccaa gttccgagcg
aggagacgcg gggggaggat cagctcccga gaggggatgt 60 cacggtaaag
agcattggaa cgtcggagaa actactccca agaagcaaag agaggtctca 120
ggaagcggac gagatcccca caacgccgga gaatctctgg aaggggaaag aggaaggtgg
180 aagaaaaagg ggcgggcctc ccgatccgag gggcccaacc tccagatctg
gagagcactc 240 cggcccgaag ggttgagtag cacccagagg gaggaatcca
ctcggagatg agcagagaaa 300 tcacctccag aggacccctt cagcgaacaa
gaggcgcttc gagcggtagg agtaagacca 360 tagcgatagg aggagatgct
aggagtaggg ggagaccgaa gcgaggagga aagtaaagaa 420 agcaacgggg
ctagccggtg ggtgttccgc cccccgagag gggacgagtg aggcttatcc 480
cggggaattc gacttatcgt ccccatctag cgggaccccg gacccccttc gaaagtgacc
540 ggagggggtg ctgggaacac cggggaccag tggagccatg ggatgcccct
cccgatgctc 600 gactccgact ccccccccca agggtcgccc aggaatggcg
ggaccccact ctgcagggtc 660 cgcgttccat cctttcttac ctgatggccg
gcatggtccc agcctcctcg ctggcgccgg 720 ctgggcaaca ttccgagggg
accgtcccct cggtaatggc gaatgggacc cacaaatctc 780 tctagattcc
gatagagaat cgagagaaaa gtggctctcc cttagccatc cgagtggacg 840
tgcgtcctcc ttcggatgcc caggtcggac cgcgaggagg tggagatgcc atgccgaccc
900 gaagaggaaa gaaggacgcg agacgcaaac ctgtgagtgg aaacccgctt
tattcactgg 960 ggtcgacaac tctggggaga aaagggcgga tcggctggga
agagtatatc ccatggaaat 1020 ccctggtttc ccctgatgtc cagcccctcc
ccggtccgag agaaggggga ctccgggact 1080 ccctgcagac tggggacgaa
gccgcccccg ggcgctcccc tcgatccacc ttcgaggggg 1140 ttcacacccc
caaccggcgg gccggctact cttctttccc ttctctcgtc ttcctcggtc 1200
aacctcctga gttcctcttc ttcctccttg ctgaggttct tgcctcccgc cgatagctgc
1260 ttcttcttgt tctcgagggc cttccttcgt cggtgatcct gcctctcctt
gtcggtgaat 1320 cctcccctga gaggcctctt cctaggtccg gagtctacct
ccatctggtc cgttcgggcc 1380 ctcttcgccg ggggagcccc ctctccatcc
ttatccttct ttccgagaat tcctttgatg 1440 ttccccagcc agggattttc
gtcctcaatc ttcttgagtt tcttctttgt cttccggagg 1500 tctctctcga
gttcctctaa cttctttctt ccggccaccc actgctcgag gatctcttct 1560
ctccccccgc ggttcttcct cgactcggac cggctcatct cggctagagg cggcagtcct
1620 cagtactctt actcttttct gtaaagagga gactgctgga ctcgccgccc
gagcccgag 1679 13 1683 DNA Hepatitis Delta Virus 13 atgggccaag
ttccgaacaa ggatccgcgg ggaggacgga tcacctcccg agaggggtaa 60
gtcgctaaag agcattggaa cgtcggagat acaactccca agaaggaaaa aagagaaagc
120 aagaagcgga agaattcccc ataacgctag tgaaactcta ggaagggaaa
agaggtgcga 180 tggaaaaaga ggaggtgggc ctcccgatcc gagggtcccg
gtggccaagt ttggaggaca 240 ctccggcccg aagggttgag gatcccccag
agggaggaag ccacacggag tagaacagag 300 aaatcacctc cagaggaccc
cttcagcgaa cagaggggcg catcgcgaga gggagtagac 360 catagcgatg
ggaggggatg ctaggagtta ggggagaccg aagcgaggag gaaagtaaag 420
agagcagcgg ggctagtcgg tgggtgttcc gccccccgag aggggacgag tgaggcttat
480 cccggggaat tcgacttatc gtccccacat agcagagccc cggaccccct
ttcaaagcga 540 ccgagggggg tgactttgaa cattggggac cagtggagcc
atgggatgct cctcccgatt 600 ccgcccaaac tccttccccc ccaagggtcg
cccaggaatg gcgggacccc actctgcagg 660 gtccgcgttc catcctttct
tacctgatgg ccggcatggt cccagcctcc tcgctggcgc 720 cggctgggca
acattccgag gggaccgtcc cctcggtaat ggcgaatggg acgcacaaat 780
ctctctagct tcccagagag aagcgagaga aaagtggctc tcccttggcc atccgagtgg
840 acgtacgtcc tccttcggat gcccaggtcg gaccgcgagg aggtggagat
gccatgccga 900 cccgaagagg aaagaaggac gcgagacgca aacctgtgag
tggaaacccg ctttattcac 960 tggggtcgac aactctgggg agagaaggga
gggtcggctg ggaagagtat atcccatggg 1020 aatccctggc ttccccttat
gtccagtccc tccccggtcc gagcgaaggg ggactccggg 1080 actccttgca
tgctggggac gaagccgccc ccgggcgctc ccctcgatcc accttcgagg 1140
gggttcacac ccccaaccga cgggccggct attcttcttt cccttttctc gtcttcctcg
1200 gtcaacctct taagttcctc ttcctcctcc ctgctgaggc tctttccccc
cgacgatagc 1260 tgcttcctct tgttctcgag ggccttcctt cgtcggtgat
cctgcctctc cttgtcggtg 1320 aatcctcccc tgagaggcct cttcctaggt
ccggcgtcta tctccatctg gtccatccgg 1380 agcttcttcg ccgggggtgc
cccctctcca tccttatcct tctttccgat tattcctttg 1440 atgtttccca
gccagggatt gtcttcctct agtttcttga ttttcttctt taacttccgg 1500
aggtctctct cgagttcctc taacttcttt cttccgctca cccactgctc gaggatgtct
1560 tccctccccc cgcggtcttt ccttctttcg gaccggctca tcttcgacta
gaggcgacgg 1620 tcctcagtac tcttactctt ttctgtaaag aggagactgc
tggccctgtc gcccaagctc 1680 gag 1683 14 645 DNA Hepatitis Delta
Virus CDS (1)...(642) 14 atg agc cgg tcc gaa aga agg aaa gac cgc
ggg ggg agg gaa gac atc 48 Met Ser Arg Ser Glu Arg Arg Lys Asp Arg
Gly Gly Arg Glu Asp Ile 1 5 10 15 ctc gag cag tgg gtg agc gga aga
aag aag tta gag gaa ctc gag aga 96 Leu Glu Gln Trp Val Ser Gly Arg
Lys Lys Leu Glu Glu Leu Glu Arg 20 25 30 gac ctc cgg aag tta aag
aag aaa atc aag aaa cta gag gaa gac aat 144 Asp Leu Arg Lys Leu Lys
Lys Lys Ile Lys Lys Leu Glu Glu Asp Asn 35 40 45 ccc tgg ctg gga
aac atc aaa gga ata atc gga aag aag gat aag gat 192 Pro Trp Leu Gly
Asn Ile Lys Gly Ile Ile Gly Lys Lys Asp Lys Asp 50 55 60 gga gag
ggg gca ccc ccg gcg aag aag ctc cgg atg gac cag atg gag 240 Gly Glu
Gly Ala Pro Pro Ala Lys Lys Leu Arg Met Asp Gln Met Glu 65 70 75 80
ata gac gcc gga cct agg aag agg cct ctc agg gga gga ttc acc gac 288
Ile Asp Ala Gly Pro Arg Lys Arg Pro Leu Arg Gly Gly Phe Thr Asp 85
90 95 aag gag agg cag gat cac cga cga agg aag gcc ctc gag aac aag
agg 336 Lys Glu Arg Gln Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys
Arg 100 105 110 aag cag cta tcg tcg ggg gga aag agc ctc agc agg gag
gag gaa gag 384 Lys Gln Leu Ser Ser Gly Gly Lys Ser Leu Ser Arg Glu
Glu Glu Glu 115 120 125 gaa ctt aag agg ttg acc gag gaa gac gag aaa
agg gaa aga aga ata 432 Glu Leu Lys Arg Leu Thr Glu Glu Asp Glu Lys
Arg Glu Arg Arg Ile 130 135 140 gcc ggc ccg tcg gtt ggg ggt gtg aac
ccc ctc gaa ggt gga tcg agg 480 Ala Gly Pro Ser Val Gly Gly Val Asn
Pro Leu Glu Gly Gly Ser Arg 145 150 155 160 gga gcg ccc ggg ggc ggc
ttc gtc ccc agc atg caa gga gtc ccg gag 528 Gly Ala Pro Gly Gly Gly
Phe Val Pro Ser Met Gln Gly Val Pro Glu 165 170 175 tcc ccc ttc gct
cgg acc ggg gag gga ctg gac ata agg gga agc cag 576 Ser Pro Phe Ala
Arg Thr Gly Glu Gly Leu Asp Ile Arg Gly Ser Gln 180 185 190 gga ttc
cca tgg gat ata ctc ttc cca gcc gac cct ccc ttc tct ccc 624 Gly Phe
Pro Trp Asp Ile Leu Phe Pro Ala Asp Pro Pro Phe Ser Pro 195 200 205
cag agt tgt cga ccc cag tga 645 Gln Ser Cys Arg Pro Gln 210 15 214
PRT Hepatitis Delta Virus 15 Met Ser Arg Ser Glu Arg Arg Lys Asp
Arg Gly Gly Arg Glu Asp Ile 1 5 10 15 Leu Glu Gln Trp Val Ser Gly
Arg Lys Lys Leu Glu Glu Leu Glu Arg 20 25 30 Asp Leu Arg Lys Leu
Lys Lys Lys Ile Lys Lys Leu Glu Glu Asp Asn 35 40 45 Pro Trp Leu
Gly Asn Ile Lys Gly Ile Ile Gly Lys Lys Asp Lys Asp 50 55 60 Gly
Glu Gly Ala Pro Pro Ala Lys Lys Leu Arg Met Asp Gln Met Glu 65 70
75 80 Ile Asp Ala Gly Pro Arg Lys Arg Pro Leu Arg Gly Gly Phe Thr
Asp 85 90 95 Lys Glu Arg Gln Asp His Arg Arg Arg Lys Ala Leu Glu
Asn Lys Arg 100 105 110 Lys Gln Leu Ser Ser Gly Gly Lys Ser Leu Ser
Arg Glu Glu Glu Glu 115 120 125 Glu Leu Lys Arg Leu Thr Glu Glu Asp
Glu Lys Arg Glu Arg Arg Ile 130 135 140 Ala Gly Pro Ser Val Gly Gly
Val Asn Pro Leu Glu Gly Gly Ser Arg 145 150 155 160 Gly Ala Pro Gly
Gly Gly Phe Val Pro Ser Met Gln Gly Val Pro Glu 165 170 175 Ser Pro
Phe Ala Arg Thr Gly Glu Gly Leu Asp Ile Arg Gly Ser Gln 180 185 190
Gly Phe Pro Trp Asp Ile Leu Phe Pro Ala Asp Pro Pro Phe Ser Pro 195
200 205 Gln Ser Cys Arg Pro Gln 210 16 214 PRT Hepatitis Delta
Virus 16 Met Ser Arg Ser Glu Arg Arg Lys Asp Arg Gly Gly Arg Glu
Asp Ile 1 5 10 15 Leu Glu Gln Trp Val Ser Gly Arg Lys Lys Leu Glu
Glu Leu Glu Arg 20 25 30 Asp Leu Arg Lys Leu Lys Lys Lys Ile Lys
Lys Leu Glu Glu Asp Asn 35 40 45 Pro Trp Leu Gly Asn Ile Lys Gly
Ile Ile Gly Lys Lys Asp Lys Asp 50 55 60 Gly Glu Gly Ala Pro Pro
Ala Lys Lys Leu Arg Met Asp Gln Met Glu 65 70 75 80 Ile Asp Ala Gly
Pro Arg Lys Arg Pro Leu Arg Gly Gly Phe Thr Asp 85 90 95 Lys Glu
Arg Gln Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys Arg 100 105 110
Lys Gln Leu Ser Ser Gly Gly Lys Ser Leu Ser Arg Glu Glu Glu Glu 115
120 125 Glu Leu Lys Arg Leu Thr Glu Glu Asp Glu Lys Arg Glu Arg Arg
Ile 130 135 140 Ala Gly Pro Ser Val Gly Gly Val Asn Pro Leu Glu Gly
Gly Ser Arg 145 150 155 160 Gly Ala Pro Gly Gly Gly Pro Val Pro Ser
Met Gln Gly Val Pro Glu 165 170 175 Ser Pro Phe Ala Arg Thr Gly Glu
Gly Leu Asp Ile Arg Gly Ser Gln 180 185 190 Gly Phe Pro Trp Asp Ile
Leu Phe Pro Ala Asp Pro Pro Phe Ser Pro 195 200 205 Gln Ser Cys Arg
Pro Gln 210 17 214 PRT Hepatitis Delta Virus 17 Met Ser Arg Ser Glu
Ser Arg Lys Asn Arg Gly Gly Arg Glu Glu Ile 1 5 10 15 Leu Glu Gln
Trp Val Ala Gly Arg Lys Lys Leu Glu Glu Leu Glu Arg 20 25 30 Asp
Leu Arg Lys Thr Lys Lys Lys Leu Lys Lys Ile Glu Asp Glu Asn 35 40
45 Pro Trp Leu Gly Asn Ile Lys Gly Ile Leu Gly Lys Lys Asp Lys Asp
50 55 60 Gly Glu Gly Ala Pro
Pro Ala Lys Arg Ala Arg Thr Asp Gln Met Glu 65 70 75 80 Val Asp Ser
Gly Pro Arg Lys Arg Pro Leu Arg Gly Gly Phe Thr Asp 85 90 95 Lys
Glu Arg Gln Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys Lys 100 105
110 Lys Gln Leu Ser Ala Gly Gly Lys Asn Leu Ser Lys Glu Glu Glu Glu
115 120 125 Glu Leu Arg Arg Leu Thr Glu Glu Asp Glu Arg Arg Glu Arg
Arg Val 130 135 140 Ala Gly Pro Pro Val Gly Gly Val Asn Pro Leu Glu
Gly Gly Ser Arg 145 150 155 160 Gly Ala Pro Gly Gly Gly Pro Val Pro
Ser Leu Gln Gly Val Pro Glu 165 170 175 Ser Pro Phe Ser Arg Thr Gly
Glu Gly Leu Asp Ile Arg Gly Asn Gln 180 185 190 Gly Phe Pro Trp Asp
Ile Leu Phe Pro Ala Asp Pro Pro Phe Ser Pro 195 200 205 Gln Ser Cys
Arg Pro Gln 210 18 37 PRT Artificial Sequence Synthetic peptide
from the multimer-forming domain of HDAg 18 Lys Lys Leu Glu Glu Leu
Glu Arg Asp Leu Arg Lys Leu Lys Lys Lys 1 5 10 15 Ile Lys Lys Leu
Glu Glu Asp Asn Pro Trp Leu Gly Asn Ile Lys Gly 20 25 30 Ile Ile
Gly Lys Tyr 35 19 38 PRT Hepatitis Delta Virus Synthetic peptide
from the multimer-forming domain of HDAg 19 Gly Arg Glu Asp Ile Leu
Glu Gln Trp Val Ser Gly Arg Lys Lys Leu 1 5 10 15 Glu Glu Leu Glu
Arg Asp Leu Arg Lys Leu Lys Lys Lys Ile Lys Lys 20 25 30 Leu Glu
Glu Asp Asn Pro 35 20 50 PRT Artificial Sequence Synthetic peptide
from the multimer-forming domain of HDAg 20 Gly Arg Glu Asp Ile Leu
Glu Gln Trp Val Ser Gly Arg Lys Lys Leu 1 5 10 15 Glu Glu Leu Glu
Arg Asp Leu Arg Lys Leu Lys Lys Lys Ile Lys Lys 20 25 30 Leu Glu
Glu Asp Asn Pro Trp Leu Gly Asn Ile Lys Gly Ile Ile Gly 35 40 45
Lys Tyr 50 21 598 DNA Artificial Sequence Synthetic HDAg gene for
optimized expression in E. Coli 21 atg agc cgt agc gaa cgt cgt aaa
gat cgt ggc ggc cgt gaa gat att 48 Met Ser Arg Ser Glu Arg Arg Lys
Asp Arg Gly Gly Arg Glu Asp Ile 1 5 10 15 ctg gaa cag tgg gtg agc
ggc cgt aag aag tta gag gaa ttg gaa cgt 96 Leu Glu Gln Trp Val Ser
Gly Arg Lys Lys Leu Glu Glu Leu Glu Arg 20 25 30 gat ctg cgt aaa
ctg aaa aag aag att aag aaa ctg gaa gaa gat aac 144 Asp Leu Arg Lys
Leu Lys Lys Lys Ile Lys Lys Leu Glu Glu Asp Asn 35 40 45 ccg tgg
ttg ggt aat att aaa ggc att att ggc aag aaa gat aaa gat 192 Pro Trp
Leu Gly Asn Ile Lys Gly Ile Ile Gly Lys Lys Asp Lys Asp 50 55 60
ggc gaa ggc gcg ccg ccg gcg aag aaa ctg cgt atg gat cag atg gaa 240
Gly Glu Gly Ala Pro Pro Ala Lys Lys Leu Arg Met Asp Gln Met Glu 65
70 75 80 att gat gcg ggc ccg cgt aaa cgt ccg ctg cgt ggc ggc ttt
acc gat 288 Ile Asp Ala Gly Pro Arg Lys Arg Pro Leu Arg Gly Gly Phe
Thr Asp 85 90 95 aag gaa cgt cag gac cat cgt cgt cgt aaa gcg ctg
gaa aac aaa cgt 336 Lys Glu Arg Gln Asp His Arg Arg Arg Lys Ala Leu
Glu Asn Lys Arg 100 105 110 aaa cag ctg agc agc ggc ggc aaa tct ctg
agc cgt gaa gaa gaa gaa 384 Lys Gln Leu Ser Ser Gly Gly Lys Ser Leu
Ser Arg Glu Glu Glu Glu 115 120 125 gaa ctg aaa cgt ctg acc gaa gaa
gat gaa aaa cgt gaa cgt cgt att 432 Glu Leu Lys Arg Leu Thr Glu Glu
Asp Glu Lys Arg Glu Arg Arg Ile 130 135 140 gca ggt cca tct gtt ggt
ggt gtg aac ccg ctg gaa ggc ggc agc cgt 480 Ala Gly Pro Ser Val Gly
Gly Val Asn Pro Leu Glu Gly Gly Ser Arg 145 150 155 160 ggt gca ccg
ggc ggt ggc ttt gtg ccg tct atg caa ggt gtt cca gaa 528 Gly Ala Pro
Gly Gly Gly Phe Val Pro Ser Met Gln Gly Val Pro Glu 165 170 175 agc
ccg ttt gcg cgt acc ggc gaa ggc ctg gat att cgt ggc agc cag 576 Ser
Pro Phe Ala Arg Thr Gly Glu Gly Leu Asp Ile Arg Gly Ser Gln 180 185
190 ggc ttt ccg taaaccatgg cgc 598 Gly Phe Pro 195 22 195 PRT
Artificial Sequence Amino acid sequence encoded by synthetic gene
for optimized HDAg expression in E. Coli 22 Met Ser Arg Ser Glu Arg
Arg Lys Asp Arg Gly Gly Arg Glu Asp Ile 1 5 10 15 Leu Glu Gln Trp
Val Ser Gly Arg Lys Lys Leu Glu Glu Leu Glu Arg 20 25 30 Asp Leu
Arg Lys Leu Lys Lys Lys Ile Lys Lys Leu Glu Glu Asp Asn 35 40 45
Pro Trp Leu Gly Asn Ile Lys Gly Ile Ile Gly Lys Lys Asp Lys Asp 50
55 60 Gly Glu Gly Ala Pro Pro Ala Lys Lys Leu Arg Met Asp Gln Met
Glu 65 70 75 80 Ile Asp Ala Gly Pro Arg Lys Arg Pro Leu Arg Gly Gly
Phe Thr Asp 85 90 95 Lys Glu Arg Gln Asp His Arg Arg Arg Lys Ala
Leu Glu Asn Lys Arg 100 105 110 Lys Gln Leu Ser Ser Gly Gly Lys Ser
Leu Ser Arg Glu Glu Glu Glu 115 120 125 Glu Leu Lys Arg Leu Thr Glu
Glu Asp Glu Lys Arg Glu Arg Arg Ile 130 135 140 Ala Gly Pro Ser Val
Gly Gly Val Asn Pro Leu Glu Gly Gly Ser Arg 145 150 155 160 Gly Ala
Pro Gly Gly Gly Phe Val Pro Ser Met Gln Gly Val Pro Glu 165 170 175
Ser Pro Phe Ala Arg Thr Gly Glu Gly Leu Asp Ile Arg Gly Ser Gln 180
185 190 Gly Phe Pro 23 598 DNA Hepatitis Delta Virus CDS
(1)...(598) 23 atg agc cgg tcc gaa aga agg aaa gac cgc ggg ggg agg
gaa gac atc 48 Met Ser Arg Ser Glu Arg Arg Lys Asp Arg Gly Gly Arg
Glu Asp Ile 1 5 10 15 ctc gag cag tgg gtg agc gga aga aag aag tta
gag gaa ctc gag aga 96 Leu Glu Gln Trp Val Ser Gly Arg Lys Lys Leu
Glu Glu Leu Glu Arg 20 25 30 gac ctc cgg aag tta aag aag aaa atc
aag aaa cta gag gaa gac aat 144 Asp Leu Arg Lys Leu Lys Lys Lys Ile
Lys Lys Leu Glu Glu Asp Asn 35 40 45 ccc tgg ctg gga aac atc aaa
gga ata atc gga aag aag gat aag gat 192 Pro Trp Leu Gly Asn Ile Lys
Gly Ile Ile Gly Lys Lys Asp Lys Asp 50 55 60 gga gag ggg gca ccc
ccg gcg aag aag ctc cgg atg gac cag atg gag 240 Gly Glu Gly Ala Pro
Pro Ala Lys Lys Leu Arg Met Asp Gln Met Glu 65 70 75 80 ata gac gcc
gga cct agg aag agg cct ctc agg gga gga ttc acc gac 288 Ile Asp Ala
Gly Pro Arg Lys Arg Pro Leu Arg Gly Gly Phe Thr Asp 85 90 95 aag
gag agg cag gat cac cga cga agg aag gcc ctc gag aac aag agg 336 Lys
Glu Arg Gln Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys Arg 100 105
110 aag cag cta tcg tcg ggg gga aag agc ctc agc agg gag gag gaa gag
384 Lys Gln Leu Ser Ser Gly Gly Lys Ser Leu Ser Arg Glu Glu Glu Glu
115 120 125 gaa ctt aag agg ttg acc gag gaa gac gag aaa agg gaa aga
aga ata 432 Glu Leu Lys Arg Leu Thr Glu Glu Asp Glu Lys Arg Glu Arg
Arg Ile 130 135 140 gcc ggc ccg tcg gtt ggg ggt gtg aac ccc ctc gaa
ggt gga tcg agg 480 Ala Gly Pro Ser Val Gly Gly Val Asn Pro Leu Glu
Gly Gly Ser Arg 145 150 155 160 gga gcg ccc ggg ggc ggc ttc gtc ccc
agc atg caa gga gtc ccg gag 528 Gly Ala Pro Gly Gly Gly Phe Val Pro
Ser Met Gln Gly Val Pro Glu 165 170 175 tcc ccc ttc gct cgg acc ggg
gag gga ctg gac ata agg gga agc cag 576 Ser Pro Phe Ala Arg Thr Gly
Glu Gly Leu Asp Ile Arg Gly Ser Gln 180 185 190 gga ttc cca tgg gat
ata ctc t 598 Gly Phe Pro Trp Asp Ile Leu 195 24 199 PRT Hepatitis
Delta Virus 24 Met Ser Arg Ser Glu Arg Arg Lys Asp Arg Gly Gly Arg
Glu Asp Ile 1 5 10 15 Leu Glu Gln Trp Val Ser Gly Arg Lys Lys Leu
Glu Glu Leu Glu Arg 20 25 30 Asp Leu Arg Lys Leu Lys Lys Lys Ile
Lys Lys Leu Glu Glu Asp Asn 35 40 45 Pro Trp Leu Gly Asn Ile Lys
Gly Ile Ile Gly Lys Lys Asp Lys Asp 50 55 60 Gly Glu Gly Ala Pro
Pro Ala Lys Lys Leu Arg Met Asp Gln Met Glu 65 70 75 80 Ile Asp Ala
Gly Pro Arg Lys Arg Pro Leu Arg Gly Gly Phe Thr Asp 85 90 95 Lys
Glu Arg Gln Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys Arg 100 105
110 Lys Gln Leu Ser Ser Gly Gly Lys Ser Leu Ser Arg Glu Glu Glu Glu
115 120 125 Glu Leu Lys Arg Leu Thr Glu Glu Asp Glu Lys Arg Glu Arg
Arg Ile 130 135 140 Ala Gly Pro Ser Val Gly Gly Val Asn Pro Leu Glu
Gly Gly Ser Arg 145 150 155 160 Gly Ala Pro Gly Gly Gly Phe Val Pro
Ser Met Gln Gly Val Pro Glu 165 170 175 Ser Pro Phe Ala Arg Thr Gly
Glu Gly Leu Asp Ile Arg Gly Ser Gln 180 185 190 Gly Phe Pro Trp Asp
Ile Leu 195 25 195 PRT Hepatitis Delta Virus 25 Met Ser Arg Ser Glu
Arg Arg Lys Asp Arg Gly Gly Arg Glu Asp Ile 1 5 10 15 Leu Glu Gln
Trp Val Ser Gly Arg Lys Lys Leu Glu Glu Leu Glu Arg 20 25 30 Asp
Leu Arg Lys Leu Lys Lys Lys Ile Lys Lys Leu Glu Glu Asp Asn 35 40
45 Pro Trp Leu Gly Asn Ile Lys Gly Ile Ile Gly Lys Lys Asp Lys Asp
50 55 60 Gly Glu Gly Ala Pro Pro Ala Lys Lys Leu Arg Met Asp Gln
Met Glu 65 70 75 80 Ile Asp Ala Gly Pro Arg Lys Arg Pro Leu Arg Gly
Gly Phe Thr Asp 85 90 95 Lys Glu Arg Gln Asp His Arg Arg Arg Lys
Ala Leu Glu Asn Lys Arg 100 105 110 Lys Gln Leu Ser Ser Gly Gly Lys
Ser Leu Ser Arg Glu Glu Glu Glu 115 120 125 Glu Leu Lys Arg Leu Thr
Glu Glu Asp Glu Lys Arg Glu Arg Arg Ile 130 135 140 Ala Gly Pro Ser
Val Gly Gly Val Asn Pro Leu Glu Gly Gly Ser Arg 145 150 155 160 Gly
Ala Pro Gly Gly Gly Phe Val Pro Ser Met Gln Gly Val Pro Glu 165 170
175 Ser Pro Phe Ala Arg Thr Gly Glu Gly Leu Asp Ile Arg Gly Ser Gln
180 185 190 Gly Phe Pro 195 26 93 DNA Artificial Sequence PCR
Primer 26 gggcatatga gccgtagcga acgtcgtaaa gatcgtggcg gccgtgaaga
tattctggaa 60 cagtgggtga gcggccgtaa gaagttagag gaa 93 27 95 DNA
Artificial Sequence PCR Primer 27 atattaccca accacgggtt atcttcttcc
agtttcttaa tcttcttttt cagtttacgc 60 agatcacgtt ccaattcctc
taacttctta cggcc 95 28 94 DNA Artificial Sequence PCR Primer 28
taacccgtgg ttgggtaata ttaaaggcat tattggcaag aaagataaag atggcgaagg
60 cgcgccgccg gcgaagaaac tgcgtatgga tcag 94 29 94 DNA Artificial
Sequence PCR Primer 29 gatggtcctg acgttcctta tcggtaaagc cgccacgcag
cggacgttta cgcgggcccg 60 catcaatttc catctgatcc atacgcagtt tctt 94
30 93 DNA Artificial Sequence PCR Primer 30 ataaggaacg tcaggaccat
cgtcgtcgta aagcgctgga aaacaaacgt aaacagctga 60 gcagcggcgg
caaatctctg agccgtgaag aag 93 31 89 DNA Artificial Sequence PCR
Primer 31 caacagatgg acctgcaata cgacgttcac gtttttcatc ttcttcggtc
agacgtttca 60 gttcttcttc ttcttcacgg ctcagagat 89 32 101 DNA
Artificial Sequence PCR Primer 32 tattgcaggt ccatctgttg gtggtgtgaa
cccgctggaa ggcggcagcc gtggcgcgcc 60 gggcggcggc tttgtgccgt
ctatgcaagg tgttccagaa a 101 33 90 DNA Artificial Sequence PCR
Primer 33 gcgccatggt ttacggaaag ccctggctgc cacgaatatc caggccttcg
ccggtacgcg 60 caaacgggct ttctggaaca ccttgcatag 90 34 20 DNA
Artificial Sequence PCR Primer 34 gggcatatga gccgtagcga 20 35 20
DNA Artificial Sequence PCR Primer 35 gcgccatggt ttacggaaag 20
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