Download Hybrid tryptophan aporepressor containing ligand binding sites

US005190873A
United States Patent [191
[11] Patent Number:
Lernhardt et a1.
[45]
[54] HYBRID TRYPTOPHAN APOREPRESSOR
CONTAINING LIGAND BINDING SITES
[75] Inventors: Waldemar Lernhardt, Solana Beach;
[73] Assignee:
Mario Bourdon, San Diego; Phil
Youderian, Ramona, all of Calif.
California Institute of Biological
Research, La Jolla, Calif.
[21] Appl. No: 720,222
[22] Filed:
Jun. 21, 1991
[51]
Int. Cl.5 .................... .. C07K 13/00; C07K 17/00;
[52]
US. Cl. ................................. .. 435/177; 435/697;
C07K 17/02; C12P 21/00
435/691; 530/350; 530/812; 930/250
[58]
Field of Search ..................... .. 435/91, 69.7, 69.1,
[56]
435/177; 530/350, 812; 930/250
References Cited
U.S. PATENT DOCUMENTS
4,703,039 10/1987
l-lawiger et a1. .................... .. 514/21
FOREIGN PATENT DOCUMENTS
9100912
1/1991
PCT lnt’l Appl. .
OTHER PUBLICATIONS
Botstein, et al. Science 229:1193-1201 1985.
Bass, et a]. Science 242:240-245 1988.
Gunsalus, et al. PNAS 77(12) 7117-7121 1980.
Date of Patent:
5,190,873
Mar. 2, 1993
Joachimiak et al., J. Biol. Chem, 258:12641-12643
(1983).
Lawson et al., Nature, 333:869-871 (1988).
Luisi et al., Biochim. Biophys. Acta, 1048:113-126 (1990).
Marrnorstein et al., J. Biol. Chem, 262:4922-4927
(1987).
Pfau et al., Nucl. Acids Res, 18:6165 (1990).
Schevitz et al., Nature, 317:782-786 (1985).
Arrowsmith et al., Biochem, 29:6332-6341 (1990).
Arvidson et al., J. Biol. Chem, 261:238-243 (1986).
Arvidson et al., Genetics, 128:29-35 (1991).
Bourdon et al., J. Cell Biol., 108:1149-1155 (1989).
Joachimiak et al., Proc. Natl. Acad. Sci. USA,
80:668-672 (1983).
Zhang et al., Nature, 327:591-597 (1987).
Engel et al., Biochem. 30: 3161—3169 (1991).
Primary Examiner-David L. Lacey
Assistant Examiner—-Donald E. Adams
Attorney, Agent, or Firm-Thomas Fitting
[57]
ABSTRACT
Hybrid proteins containing repressor proteins and sub‘
stituted receptor binding sites, amino acid and DNA
sequences encoding the hybrid proteins are provided.
Methods for preparing the hybrid proteins are also
described.
19 Claims, 6 Drawing Sheets
US. Patent
Mar. 2, 1993
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1
5,190,873
2
of the B cell cycle of human and murine preactivated
blasts (Melchers, supra (1985); Bohnsack and Cooper J.
HYBRID TRYPTOPHAN APOREPRESSOR
CONTAINING LIGAND BINDING SITES
Immunol, 141:2569 (1988)). CR2 is also phosphorylated
during B cell stimulation, a common property of
This invention was made with government support 5 growth factor receptors (Changelian and Fearon J.
under government contract 5 29 CA45506 awarded by
Exp. Med, 161:101 (1986)).
the National Institutes of Health. The government has
CR2 occurs on normal and malignant B lymphocytes
certain rights in the invention.
(Cooper et al., supra (1988); Hatzfeld et al , J. Immunol,
140:170 (1988)), on epithelial cells (Young et al., The
FIELD OF THE INVENTION
10 Lancet, 240 (1986)), and, to a lesser extent, on immature
This invention relates to hybrid proteins constructed
from prokaryotic repressor proteins and receptor bind
ing sites, and to the methods for producing the hybrid
proteins.
'
I
BACKGROUND OF THE INVENTION
Receptors are molecules, typically proteins or glyco
proteins, found on the surface of cells, including mam
malian cells, that possess speci?c af?nity for other mole
thymocytes and follicular dendritic cells (Tsoukas and
Lambris, Eru. J. Immunol. 18:1299 (1988); Reynes et
al., J. Immunol, 135:2687 (1985)). The primary struc
ture of CR2 has been deduced from the DNA sequence
15 of its clone. Human CR2 is a membrane glycoprotein of
145 kd, and has sequence similarity to other members of
the family of complement binding proteins.
The recognition sites on CR2 for C3d and EBV have
been located on the N-terminal part of this longitudinal
cules known as ligands. Ligands may be small or large
molecule. The sequence motifs on C3 and EBV coat
(macro) molecules such as proteins. Binding of ligands
protein, gp350, that mediate binding to CR2 receptor
to receptors on the surface of mammalian cells elicits
dramatic responses or “signals” in the cells such as
have also been de?ned (Lambris et al., Proc. Natl. Acad.
Sci. USA, 8224235 (1985); Nemerow et al., Cell 56:369
proliferation and adhesion. These cellular responses
(1989)) (Table 1). Synthetic hexapeptides with the se
involve protein-protein interactions and intercellular 25 quence of the CR2 binding site on C3 inhibit human and
interactions that regulate important physiological pro
murine B cell proliferation (Lernhardt et al., Immunol.
cesses, such as the humoral immune response. Defects
in receptor structure and function may interfere with
Rev., 99:239 (1987)). Thus CR2 receptor can bind both
monomeric C3d and aggregated C3d as ligands, as well
recognition of ligands by the cell-bound receptors re
sulting in disease or dysfunction and death.
as the major epitope of EBV capsid protein.
Many cell surface receptors and their corresponding
ligands have been identi?ed and characterized structur
CR2 ligands act in concert with other B cell growth
modulators, including growth factors, lymphokines,
and cytokines. Thus, the growth-inducing effect of
anti-CR2 monoclonal antibody OKB7 is T cellsdepend
ent, and requires T cell-derived B cell growth factors
only short stretches of amino acids, e.g. at least three 35 (Cooper et al., supra (1988)). It has been shown that
amino acids, on the surfaces of mammalian proteins are
optimal cell cycle progression and cell division occurs
both necessary and suf?cient to bind speci?c receptors
only in the presence of both anti-lg antibodies and IL-2
and thereby elicit dramatic cellular responses.
or IL-5.
Examples of characterized receptors include comple
The CR2 receptor is of clinical interest, because it is
ment receptor type 2 (CD21 or “CR2") (Weigle et al., 40 the receptor for Epstein-Barr virus (EBV) (Frade supra
In Complement, Muller-Eberhard and Miescher, Eds,
(1985)). EBV is the causative agent of infectious mono
Springer-Verlag, Berlin, p. 323 (1985)). The clonal ex
nucleosis (Huang et al., Int. J. Cancer 14:580 (1974)),
pansion of mature, antigen-reactive B lymphocytes in
and possibly is a human cancer virus, because its pres
the humoral immune response is regulated both by di
ence is correlated with nasopharyngeal carcinoma and
rect intercellular interactions (with T helper lympho 45 Burkitt’s lymphoma (Henle et al., Science, 157:1064
cytes and accessory cells), and by interactions with
(1967)). In addition, EBV may play a role in the onset of
soluble growth factors (Unanue, Adv. Immunol, 15:95
B cell neoplasia observed in a substantial fraction of
(1972)). These soluble factors include B cell growth
AIDS patients (Y archoan et al., J. Clin. Invest, 78:439
factors, interleukins, interferons, and components of the
(1986)). At the least, a substantial fraction of AIDS
complement system (O'Garra et al., Immunol. Today, 50 patients have chronic EBV infections. Exposure of
8:45 (1988); Weigle et al., supra). Members of the latter
pregnant women to individuals infected with and shed
two classes of proteins bind speci?c cell surface recep
ding EBV poses a signi?cant risk to fetal development.
tors, including CR2. CR2 is the B lymphocyte receptor
It would be useful to better understand the mechanism
for the proteolytic activation products C3bi, C3dg and
of CR2 ligand action and to design and engineer prote
C3d of complement component C3 (Cooper et al., Ann. 55 ins that function as recombinant inhibitors of EBV in
fection and lymphoma proliferation.
Rev. Immunol, 6:85 (1988); Aggregated C3b and C3d
induce B cell proliferation (Erdei et al., Eur. J.
Prokaryotic repressors are small, multimeric proteins
that are easy to manipulate genetically. Prokaryotic
Immunol, 15:184 (1985); Melchers et al., Nature,
317:264 (1985)). CR2 is also the receptor for Epstein
repressors bind short stretches of DNA called oper'a
Barr virus (EBV), a potent polyclonal B cell activator 60 tors. Aporepressor proteins bind operators poorly and
(Fingeroth et al., Proc. Natl. Acad. Sci. USA, 81:4510
must complex with other small molecules called core
pressors, such as tryptophan or S-adenosylmethionine
(1985); Frade et al., Proc. Natl. Acad. Sci. USA, 82:1490
(1985)). CR2 plays a central role in signalling B cell
to form active repressor complexes, or simply, repres
proliferation (Cooper et al., supra (1988)). Several
sors. Corepressors act as “keystones” that ?t into and
monoclonal antibodies and polyclonal anti-CR2 antisera 65 stabilize the hydrophobic cores of their aporepressors.
stimulate T cell-dependent B cell proliferation (Cooper
The E. Coli Tryptophan (Trp) aporepressor mono
et al., supra (1988)). Furthermore, ligand binding to
mer is a peptide 108 amino acids long (M,= 12,356
CR2 is necessary for the transition from G1 to S phase
daltons) (Gunsalus and Yanofsky, Proc. Nat. Acad. Sci.
ally and biochemically. The study of protein-mediated
intercellular signalling yields the surprising result that
3
5,190,873
USA, 77:7117-7121 (1980)) that assembles as a dimer
4
(Joachimiak et al., Proc. Natl. Acad. Sci. USA,
80:668-672 (1983); Arvidson et al., J. Biol. Chem,
tially disordered, and the residues organized as D in the
crystal do not form an a-helix, but rather comprise
some sort of surface loop (the “D Loop”) (Arrowsmith
261:238-243 (1986)). Trp aporepressor binds DNA
poorly in the absence of the corepressor ligand, L-tryp
et al., supra (1990)). The binding of the corepressor,
L-tryptophan, restricts the motion of the D loop; amide
tophan (or the analog 5-methyltryptophan, S-MT). Ap
protons of residues in D become less solvent-accessible
in the presence of corepressor. However, corepressor
binding does not elicit a coil-to-helix transition, because
these protons remain uninvolved in H-bond formation
orepressor assembles with tryptophan or S-MT to form
active Trp repressor complex, a global repressor that
binds operator sites to regulate the initiation of tran
scription from at least three different E. coli promoters. 10 in the repressor complex and in the speci?c repressor
In addition, aporepressor can form inactive Trp
/operator complex [Arrowsrnith and Jardetsky, supra.
pseudorepressor complexes with indole-3-propionic
Trp aporepressor is usually stable, and may be puri
?ed in large quantities (Arvidson eta1., In Protein Puri
acid (IPA) or indole-rr-acrylic acid (IAA); these pseu
do-repressor complexes bind operator DNA more
poorly than aporepressor (Doolittle and Yanofsky, J.
Bacteriol, 95:1283-1294 (1968); Baker and Yanofsky,
Proc. Natl. Acad. Sci USA, 60:313-320 (1968)).
?cation: Micro to Macro, UCLA Symp. Mol. Cell Biol
(Ed. Burgess), Alan R. Liss, NY; (1986); Smith et al.,
Proc. Natl. Acad. Sci, USA. 82:6104-6108 (1985)). In
addition, to understand how particular amino acids
Trp aporepressor controls three operons that com
contribute to the structure and function of Trp repres
prise a system to maintain the concentration of L-tryp
sor, methods have been developed for both mismatch
tophan in E. coli homeostatically, within levels neces 20 primer (Arvidson et al., Genetics, 128 (1991) and cas
sary for efficient protein synthesis. When concentra
sette-style (Pfau and Youderian, Nuc. Acids Res,
tions of intracellular tryptophan are low, TrpR exists
18:6165 (1990) mutagenesis of either single or multiple
predominantly as an aporepressor that cannot bind trp
adjacent codons of TroR. Mutagenesis may be coupled
operator DNA, and 'the trpEDCBA biosynthetic genes
with a rapid screen for Trp repressor function; this
are expressed maximally. When L-tryptophan levels are 25 screen depends on the color of colonies made by a strain
high, a substantial fraction of TrpR is active repressor,
of bacteria, CGl03, which overproduces Trp repressor.
and tryptophan biosynthesis slows (Cohen and Jacob,
Comparisons of the NMR structures of Trp apore
CR. Acad. Sci. Paris, 248:3490-3492 (1959); Yanofsky,
pressor and repressor suggests that the binding of indole
J. Amer. Med. Assoc, 218:1026-1035 (1971); Bennet et
analogs results in subtle changes in the orientation of D
al., Proc. Natl. Acad. Sci. USA, 73:2351-2355 (1976); 30 and E relative to the stationary hydrophobic core of the
Zurawski et al., J. Mol. Biol, 145:47-73 (1981); Ya
protein. The TrpR dimer has two identical, independent
nofsky et al., J. Bacteriol, 158: 1018-1024 (1984)). Trp
binding sites for corepressor (Arvidson et al., supra
aporepressor regulates a biosynthetic pathway in re
(1986); Marmorstein et al., J. Biol. Chem,
sponse to the amount of an end product; thus, it func
262:4922-4927 (1987)); surprisingly, these are formed
tions as a rheostat, rather than an on/off switch. In 35 by the side chains of residues from both monomers in a
contrast, y. and other phage repressors control binary
dimer (Schevitz et al., supra (1985)). The interactions
that each corepressor is predicted to make with apore
developmental decisions, and are not known to respond
to small ligands. Other ligand-activated DNA-binding
proteins have been studied to lesser extents.
The X-ray crystal structures of two forms of Trp
repressor (Schevitz et al., Nature, 317:782-786 (1985);
Lawson et al., Proteins, 3:18-31 (1988)), aporepressor
(Zhang et al., Nature, 327:591-597 (1987)), and
pseudorepressor (Lawson et al., Nature, 333:869-871
(1988)) have been determined, and show that, when 45
pressor are primarily hydrophobic. Presumably, the
binding of corepressor restricts the ensemble of pre
ferred conformations of the DNA-binding domains of
an aporepressor to a subset of conformations that bind
DNA with lower free energies (pseudorepressor bind
ing restricts aporepressor conformations to a subset that
binds DNA with higher free energies).
crystallized, the peptide monomer is a bundle of six
Attempts have been made to construct hybrids be
tween structural proteins and receptor binding sites.
a-helices with a disordered, ll-residue N-terminal arm.
For example, hybrids between proteins having highly
The TrpR dimer has a remarkable subunit interface, in
repetitive sequences such as silk-like protein (SLP) and
which four of each subunit’s six a-interface, helices (A,
the ten-residue RGD motif of ?bronectin have been
B, C, and F) are interlocked. The amino acid sequence 50 described (Cappello and Crissman, Chemical and Engi
of the two ?exible a-helices, D and E, resembles the
neering News, pp. 26-32 (July 16, 1990)). Although the
conserved “helix-turn-helix” DNA-binding motif char
acteristic of many prokaryotic repressors, and pairs of
the 2° substructures formed by the D loop and E are
hybrid protein is active in vitro, its highly repetitive
gene is unstable.
It would be advantageous to provide a method for
positioned on the surface of Trp repressor to contact 55
producing hybrid proteins containing receptor binding
successive major grooves of trp operator DNA. Ge
netic analyses of mutant TrpR genes show that residues
from both D and E are critical for DNA-binding (Bass
et al., Science, 242:240-245 (1988); Kelly et al., Proc.
Natl. Acad. Sci USA, 79:3120-3124 (1982)).
sites, that are active as ligands for mammalian cell re
ceptors to design reagents for a variety of applications
including treatment of diseases resulting from recep
tor/ligand dysfunction.
SUMMARY OF THE INVENTION
The present invention provides such method and
Recently, Arrowsmith, Jardetsky and coworkers
have determined the structure of Trp repressor in solu
tion, using lH-NMR spectroscopic methods (Arrow
hybrid proteins. The hybrid proteins are produced from
smith et al., Biochemistry, 2926332 (1990); Arrowsmith
and Jardetsky, submitted for publication (1991). Their
a prokaryotic repressor protein and a peptide segment
results show that the structure of Trp repressor in solu
tion resembles the crystal structures closely, with two
major differences. In solution, the ?rst half of A is par
65
that is heterologous to (not naturally present in) the
repressor. Preferably, the peptide segment de?nes a
binding site from a ligand reactive with a mammalian
cell surfacereceptor. Examples of prokaryotic repres
5
5,190,873
sor protein that may be used include TrpR aporepressor
6
DETAILED DESCRIPTION OF THE
INVENTION
protein, MET aporepressor protein, bacteriophage
lambda, Lac repressor, bacteriophage P22 Arc repres
sor and the like. The invention also provides DNA
DEFINITIONS
sequences encoding the amino acid sequences of the
invention, vectors containing the DNA sequence and
host cells transfected with the vectors.
The receptor binding site can be obtained from inter
chemical digestion (hydrolysis) of a polypeptide at its
peptide linkages. The amino acid residues described
Amino Acid Residue: An amino acid formed upon
herein are preferably in the “L” isomeric form. How
feron alpha, ?brinogen gamma, tenascin, ?bronectin,
and the like. Viral immunogenic pathogen derived pro
teins, such as proteins from HIV, EBV, hepatitis B and
ever, residues in the “D” isomeric form can be substi
tuted for any L-amino acid residue, as long as the de
the like can also be used.
NH; refers to the free amino group present at the amino
terminus of a polypeptide. COOI-I refers to the free
carboxy group present at the carboxy terminus of a
sired functional property is retained by the polypeptide.
The invention also provides a method for preparing a
hybrid protein prokaryotic repressor protein containing
polypeptide. In keeping with standard polypeptide no
menclature (described in J. Biol. Chem, 243:3552-59
(1969) and adopted at 37 CPR. l.822(b)(2)), abbrevia
at least one substituted binding site from a ligand reac
tive with an eukaryotic cell surface receptor by l) mu
tating the DNA codons for at least one selected amino
acid in the amino acid sequence encoding the prokary
tions for amino acid residues are shown in the following
otic repressor to encode a contiguous sequence of 20 Table of Correspondence:
amino acids that encodes a selected receptor binding
TABLE OF CORRESPONDENCE
site from a ligand reactive with an eukaryotic cell re
SYMBOL
ceptor to produce a mutated amino acid sequence; 2 a
l-Letter
B-Letter
AMINO ACID
hybrid protein encoded by the mutated DNA sequence
that has the activity of the selected receptor binding 25
site; and isolating the hybrid protein produced. The
hybrid protein is then puri?ed and tested for biological
activity as a binding site for eukaryotic cell receptors.
The hybrid protein is produced by recombinant means
or by chemical synthesis. A preferred prokaryotic re
pressor protein is Trp aporepressor.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram illustrating the Construction of
pTrpR-TN Plasmids. Single stranded DNA template
35
was prepared by M13 infection of C1236 transformed
with pPYlSO. Site direct mutagenesis was done on sin
gle stranded DNA with oligonucleotides (38-45 mer)
and transformation into CGl03F.Kan. Tac P/O tac
promotor/operator fragment rpoc+: rpoC transcrip
Y
G
F
Tyr
Gly
Phe
tyrosine
glycine
phenylalanine
M
A
S
I
L
T
V
Met
Ala
Ser
Ile
Leu
Thr
Val
methionine
alanine
serine
isoleucine
leucine
threonine
valine
proline
P
Pro
K
Lys
lysine
1-!
His
histidine
Q
Gln
glutamine
E
2
Glu
Glx
glutamic acid
Glu and/or Gln
W
R
D
Trp
Arg
Asp
tryptophan
arginine
aspartic acid
N
B
Asn
Asx
asparagine
Asn and/or Asp
tion terminator.
C
Cys
cysteine
J
Xaa
Unknown or other
FIG. 2 illustrates cell Attachment to TrpR-TNS.
Shown is cell attachment (relative fluorescence) of Di-I
stained U251 human glioma cells to wells elated with
It should be noted that all amino acid residue sequen
increasing amounts of TrpR-TNS as described in Exam 45 ces represented herein by formulae have a left-to-right
ple 7.
orientation in the conventional direction of amino ter
FIG. 3 illustrates cell Attachment to TrpR-TNS and
minus to carboxy terminus. In addition, the phrase
TrpR-TNI. Shown is cell attachment of U251 cells to
“amino acid residue” is broadly de?ned to include the
TrpR-TNS, TrpRTNl and TrpR in the presence (+) or 50 amino acids listed in the Table of Correspondence and
absence 6(—-) of lmg/ml SRRGDMS peptide as de
modi?ed an unusual amino acids, such as those listed in
scribed in Example 7.
37 CF. R. l.822(b)(4), and incorporated herein by refer
FIG. 4 illustrates peptide Inhibition of Cell Attach
ence. Furthermore, it should be noted that a dash at the
ment to Trp-TNS. Increasing concentrations of peptide
beginning or end of an amino acid residue sequence
SRRGDMS-NI-I; and GRGDSP-NHZ were present in 55 indicates a peptide bond to a further sequence of one or
cell attachment assays on TrpR-TN5 as described in
more amino acid residues or a covalent bond to an
Example 7. The control peptide MGSRSRD did not
amino-terminal group such as NH; or acetyl or to a
inhibit cell attachment.
carboxy-terminal group such as COOH.
Nucleotide: A monomeric unit of DNA or RNA
FIG. 5 illustrates a,- Bg-liposome binding to TrpR
TNS. Receptor liposomes were prepared by dialyses of
receptor and phosphatidylcholine (PC) plus 3H-PC as
described in Example 7. Shown is liposome binding to
?bronectin (FN) vitronectin (VN), TrpR-TNS, and
TrpR coated wells.
60
consisting of a sugar moiety (pentose), a phosphate, and
a nitrogenous heterocyclic base. The base is. linked to
the sugar moiety via the glycosidic carbon (1' carbon of
the pentose) and that combination of base and sugar is a
nucleoside. When the nucleoside contains a phosphate
FIG. 6 illustrates inhibition of Cell Attachment by 65 group bonded to the 3’ or 5' position of the pentose it is
referred to as a nucleotide. A sequence of operatively
LM609 Anti av [33. Shown is cell attachment in media
linked nucleotides is typically referred to herein as a
(or monoclonal antibody 81C6 1/200) and in presence
“base sequence” or “nucleotide sequence”, and their
of LM 609 1/200 as described in Example 7.
7
5,190,873
grammatical equivalents, and is represented herein by a
8
not having a common biological origin, i.e., evolution
arily different, are said to be “heterologous”.
formula whose left to right orientation is in the conven
tional direction of 5’-terminus to 3'-terminus.
Vector: a rDNA molecule capable of autonomous
replication in a cell and to which a DNA segment, e.g.,
gene or polynucleotide, can be operatively linked so as
Base Pair (bp): A partnership of adenine (A) with
thymine (T), or of cytosine (C) with guanine (G) in a
double stranded DNA molecule. In RNA, uracil (U) is
substituted for thymine.
Nucleic Acid: A polymer of nucleotides, either single
to bring about replication of the attached segment. Vec
tors capable of directing the expression of genes encod
ing for one or more proteins are referred to herein as
or double stranded.
“expression vectors”. Particularly important vectors
allow cloning of cDNA (complementary DNA) from
mRNAs produced using reverse transcriptase.
Polynucleotide: a polymer of single or double
stranded nucleotides. As used herein “polynucleotide”
and its grammatical equivalents will include the full
range of nucleic acids. A polynucleotide will typically
Receptor: A receptor is a molecule, such as a protein,
glycoprotein and the like, that can speci?cally (non-ran
refer to a nucleic acid molecule comprised of a linear
domly)
bind to another molecule.
strand of two or more deoxyribonucleotides and/or 15
Antibody:
The term antibody in its various grammati
ribonucleotides. The exact size will depend on many
factors, which in turn depends on the ultimate condi
tions of use, as is well known in the art. The polynucleo
tides of the present invention include primers, probes,
RNA/DNA segments, oligonucleotides or “oligos”
(relatively short polynucleotides), genes, vectors, plas
mids, and the like.
cal forms is used herein to refer to immunoglobulin
molecules and immunologically active portions of im
munoglobulin molecules, i.e., molecules that contain an
20 antibody combining site or paratope. Exemplary anti
body molecules are intact immunoglobulin molecules,
Gene: A nucleic acid whose nucleotide sequence
codes for an RNA or polypeptide. A gene can be either
RNA or DNA.
25
substantially intact immunoglobulin molecules and por
tions of an immunoglobulin molecule, including those
portions known in the art as Fab, Fab’, F(ab')z and F(v).
Antibody Combining Site: An antibody combining
cule comprising two strands of substantially comple
site is that structural portion of an antibody molecule
comprised of a heavy and light chain variable and
mentary polynucleotides held together by one or more
hypervariable regions that speci?cally binds (im
Duplex DNA: a double-stranded nucleic acid mole
munoreacts with) an antigen. The term immunoreact in
hydrogen bonds between each of the complementary
bases present in a base pair of the duplex. Because the 30 its various forms means specific binding between an
antigenic determinant-containing molecule and a mole
nucleotides that form a base pair can be either a ribonu
cleotide base or a deoxyribonucleotide base, the phrase
cule containing an antibody combining site such as a
“duplex DNA” refers to either a DNA-DNA duplex
whole antibody molecule or a portion thereof.
comprising two DNA strands (ds DNA), or an RNA
Monoclonal Antibody: The phrase monoclonal anti
DNA duplex comprising one DNA and one RNA 35 body in its various grammatical forms refers to a popu
strand.
lation of antibody molecules that contains only one
Complementary Bases: Nucleotides that normally
pair up when DNA or RNA adopts a double stranded
con?guration.
species of antibody combining site capable of im
munoreacting with a particular antigen. A monoclonal
antibody thus typically displays a single binding affinity
Complementary Nucleotide Sequence: A sequence of 40 for any antigen with which it immunoreacts. A mono
nucleotides in a single-stranded molecule of DNA or
clonal antibody may therefore contain an antibody mol
RNA that is sufficiently complementary to that on an
other single strand to speci?cally hybridize to it with
consequent hydrogen bonding.
ecule having a plurality of antibody combining sites,
each immunospeci?c for a different antigen, e.g., a bis
peci?c monoclonal antibody.
Conserved: A nucleotide sequence is conserved with 45
Fusion Protein: A protein comprised of at least two
respect to a preselected (reference) sequence if it non
polypeptides and a linking sequence to operatively link
randomly hybridizes to an exact complement of the
the two polypeptides into one continuous polypeptide.
preselected sequence.
The two polypeptides linked in a fusion protein are
Hybridization: The pairing of substantially comple
mentary nucleotide sequences (strands of nucleic acid) 50 typically derived from two independent sources, and
therefore a fusion protein comprises two linked poly
to form a duplex or heteroduplex by the establishment
peptides
not normally found linked in nature.
of hydrogen bonds between complementary base pairs.
Upstream: In the direction opposite to the direction
It is a speci?c, i.e. non~random, interaction between two
of DNA transcription, and therefore going from 5' to 3’
complementary polynucleotides that ca be competi
tively inhibited.
Nucleotide Analog: A purine or pyrimidine nucleo
55 on the non-coding strand, or 3' to 5' on the mRNA.
Downstream: Further along a DNA sequence in the
direction of sequence transcription or read out, that is
tide that differs structurally from A, T, G, C, or U, but
is sufficiently similar to substitute for the normal nucle
traveling in a 3’- to 5'-direction along the non’coding
otide in a nucleic acid molecule.
DNA Homolog: Is a nucleic acid having a prese
lected conserved nucleotide sequence and a sequence
transcript.
strand of the DNA or 5'- to 3’-direction along the RNA
coding for a receptor capable of binding a preselected
Cistron: Sequence of nucleotides in a DNA molecule
coding for an amino acid residue sequence and includ
ligand.
ing upstream and downstream DNA expression control
elements.
cule produced by operatively linking two DNA seg 65 Stop Codon: Any of three codons that do not code
Recombinant DNA (rDNA) molecule: a DNA mole
ments. Thus, a recombinant DNA molecule is a hybrid
DNA molecule comprising at least two nucleotide se
for an amino acid, but instead cause termination of pro
tein synthesis. They are UAG, UAA and UGA and are
quences not normally found together in nature. rDNA’s
also referred to as a nonsense or termination codon.
5,190,873
The present invention concerns a method for prepar
ing hybrid proteins consisting of prokaryotic repressors
10
the formula, read from left to right and in the direction
of amino-terminus to carboxyterminus:
and receptor binding sites.
In the method of the invention, surface features of a
stable, prokaryotic repressor, such as Trp aporepressor,
are replaced with short peptide segments (oligopep
titles) that are active as ligand binding sites for receptors
produced by eukaryotic cells, such as antibodies, T-cell
receptor, hormone receptors, and the like. The prokary
In the formula, Z is an amino-terminal portion of the
tryptophan aporepressor, and U is a carboxyterminal
portion of the tryptophan aporepressor. The lengths of
Z and U are selected (adapted) such that B, the peptide
segment,
is expressed on the surface of the hybrid pro
otic repressor acts as a scaffold, or “context” protein, 10
tein.
Factors
considered when adapting the length of Z
for presentation of the peptide segment.
and
U
are
the
length in residues of B, whether B is
The peptide segment is typically at least 3, and prefer
substituted
or
inserted
without substitution into the
ably at least 5, and usually no more than about 20, and
aporepressor’s
native
sequence,
and in view of forming
preferably no more than about 10, amino acid residues
hybrid protein whose overall amino acid residue se
in length. Usually, the peptide segment will be in the
quence length is about 100 to about 120 residues, prefer
range of 6 to 9 (inclusive) residues in length. The length
ably about l05 to about 115 residues, and more prefera
must be suf?cient for the segment to form a binding site
bly
about 108 to 112 residues. Typically, when the E.
such that the hybrid protein mimics at least one biologi
coli tryptophan aporepressor is used as the context pro
cal activity, as evidenced by binding, of the native pro
20 tein, the hybrid protein length will be in the range of
tein ligand from which it is derived.
108 residues because that is the length of the native
An exemplary hybrid protein is a tryptophan apore
aporepressor.
For example, when a peptide segment
pressor containing within its amino acid residue se
that is 15 residues in length is to be inserted between
quence a peptide segment that is heterologous to (not
naturally occurring within) the aporepressor. The pep
?anking portions of the E.c0li tryptophan aporepressor
tide segment is located within the amino acid residue 25 protein, about 15 residues should be deleted from the
native aporepressor sequence to a hybrid protein length
sequence of the hybrid protein such that it is expressed
equivalent to that of the native aporepressor, i.e. about
on the protein’s solvent-accessible surface. Typically,
108 residues. The residues to be deleted are those adja
the peptide sequence is ?anked by amino-terminal and
cent
to the insertion site, i.e. interior residues, while the
carboxy-terminal sequences corresponding to respec
terminal portions, i.e. the amino- and carboxy- most
tive amino-and carboxy-terrninal sequences of the apo
(terminal) residues, are left intact. The dashes in the
repressor protein. However, the peptide segment can
formula represent peptide bonds that link the ?anking
also be located at the amino- or carboxyterminus of the
sequences to the peptide segment. Preferred hybrid
proteins are represented by the amino acid residue se
When the peptide segment is inserted or substituted
quences SEQ ID NO 1 through SEQ ID NO 19 listed
35
between two ?anking sequences, the amino acid residue
below.
sequence of the hybrid protein can be represented by
hybrid protein.
SEQ ID NO
(1)
MAQQ-B
AAMAEQRHQEWLRFVDLLKNAYQNDLHLPLLNLMLTPDEREALGT
RVRIVEELLRGEMSQRELKNELGAGIATITRGSNSLKAAPVELRQW
LEEVLLKSD
(2)
MAQQSPYSAAMAEQRHQEWLRFVDLLKNAYQNDLHLPLLNLMLTPD
(3)
MAQQSPYSAAMAEQRHQEWLRFVDLLKNAYQNDLHLPLLNLMLTPD
EREALGTRVRIVEE-B
SQRELKNELGAGlATITRGSNSLKAAPVELRQWLEEVLLK SD
EREALGTRVRlVEELLRGEM-B
ELGAGIATITRGSNSLKAAPVELRQWLEEVLLKSD
(4)
MAQQSPYSAAMAEQRHQEWLRFVDLLKNAYQNDLHLPLLNLMLTPD
(5)
MAQQSPYSAAMAEQRHQEWLRFVDLLKNAYQNDLHLPLLNLMLTPD
(6)
EREALGTRVRIVEELLRGEMSQRELK-B
ATITRGSNSLKAAPVELRQWLEEVLLKSD
EREALGTRVRIVEELLRGEMSQRELKN-B
ITRGSNSLKAAPVELRQWLEEVLLKSD
MAQQSPYSAAMAEQRHQEWLRFVDLLKNAYQNDLHLPLLNLMLTPD
EREALGTRVRIVEELLRGEMSQRELKNELGAGIATITRGSNSLKAA
PVELRQWLEEVLLKSD-B
(7)
LHLPLLNLMLTPDEREA
LGTRVRIVEELLRGEMSQRELKNELGAGIATITRGSNSLKAAPVEL
RQWLEEVLLKSD
(s)
MAQQ-B
SPYSAAMAEQRHQEWLRFVDLLKNAYQNDLHLPLLNLMLTPDERE
ALGTRVRIVEELLRGEMSQRELKNELGAGIATITRGSNSLKAAPVE
LRQWLEEVLLKSD
(9)
LHLPLLNLMLTPDEREAL
GTRVRIVEELLRGEMSQRELKNELGAGIATITRGSNSLKAAPVELR
QWLEEVLLKSD
(10)
MAQQSPYSAAMAEQRHQEWLRFVDLLKNAYQNDLHLPLLNLMLTPD
EREALGTRVRIVEELL-B
QRELKNELGAGlATlTRGSNSLKAAPVELRQWLEEVLLK SD
(11)
MAQQSPYSAAMAEQRHQEWLRFVDLLKNAYQNDLHLPLLNLMLTPD
EREALGTRVRlVEELLRGEMSQ-B
5,190,873
11
-continued
SEQ ID NO
GIATITRGSNSLKAAPVELRQWLEEVLLKSD
(12) MAQQSPYSAAMAEQRHQEWLRFVDLLKNAYQNDLHLPLLNLMLTPD
EREALGTRVRIVEELLRGEMSQRE-B
ATITRGSNSLKAAPVELRQWLEEVLLKSD
(13) MAQQSPYSAAMAEQRHQEWLRFVDLLKNAYQNDLHLPLLNLMLTPD
EREALGTRVRIVEELLRGEMSQRELKNELGAGIATITRGSNSLKAA
PVELRQWLEEVLL-B
(14)
MAQQSPYSAAMAEQRHQEWLRFVDLLKNAYQNDLH LPLLNLML-B
TRVRl
VEELLRGEMSQRELKNELGAGIATITRGSNSLKAAPVELRQWLEEV
LLKSD
(15) MAQQSPYSAAMAEQRHQEWLRFVDLLKNAYQNDLHLPLLNLMLTPD
EREALGTRVRIVEELLRGEM SQR-B
IATITRGSNSLKAAPVELRQWLEEVLLKSD
(l6) MAQQS-B
AAMAEQRHQEWLRFVDLLKNAYQNDLHLPLLNLMLTPDEREALGTR
VRIVEELLRGEMSQRELKNELGAGIATITRGSNSLKAAPVELRQWL
EEVLLKSD
(17) MAQQSPYSAAMAEQRHQEWLRFVDLLKNAYQNDLHLPLLNLMLTPD
EREALGTRVRIVEEL-B
QRELKNELGAGIATITRGSNSLKAAPVELRQWLEEVLLK SD
(18)
MAQQSPYSAAMAEQRHQEWLRFVDLLKNAYQNDLHLPLLNLMLTPD
EREALGTRVRlVEELLRGEMS-B
ELGAGIATITRGSNSLKAAPVELRQWLEEVLLK SD
( 19)
MAQQSPYSAAMAEQRHQEWLRFVDLLKNAYQNDLHLPLLNLMLTPD
EREALGTRVRIVEELLRGEMSQRELKN-B
ATITRGSNSLKAAPVELRQWLEEVLLK SD
(1985)) and, typically, an additional restriction selection
step as described Wells, Philos. Trans. R. Soc. Lond. Ser.
A 317:415-423 (1986)). In this procedure, the gene en
When the peptide segment is located at the amino- or
carboxy-terminus of the hybrid protein, the hybrid pro
tein is represented by the formula B-U or Z-B, respec
tively, where Z and U are carboxy- or amino-terminal 30 coding the prokaryotic repressor, for example Trp apo
repressor, is prepared, for example by introducing
aporepressor sequences as previously described, and B
is the peptide segment.
unique restriction endonuclease sites, and is inserted
The phrases “surface accessible” and “surface expres
into an appropriate plasmid, such as pPYlSO, a rop't'
sion” indicate that the peptide segment is positioned
derivative of pBR322, which expresses trpR from the
within the primary structure of the hybrid protein so 35 lacUVS promoter (Bass, Science 242:240-245 (1988)) or
that it is available for speci?c binding by a receptor
pTACTERM, a top pBR322 derivative and site
when the hybrid is in a physiologically tolerable aque
directed mutagenesis to alter the sequence of the repres
ous solution. Windows or zones which are available for
sor to encode a desired peptide segment receptor bind
surface expression or which are surface accessible in the
ing site). This method can be used to make single
changes, or to “randomize" a codon by replacing it
with the mixed sequence NNS, in which N can be any
E. coli tryptophan aporepressor are de?ned by residues
5-, 44-52, 61-67, 67-77, 71-81 and 108 of the native
protein sequence as shown in SEQ ID NO 20.
of the four DNA nucleotides A, C, G or T and S can be
-C or G. This sequence represents all 20 amino acids
with 32 codons.
the proteins interferon alpha (IFN) and gp350 (EBV
coat protein) that interact with one major eukaryotic B 45
Substitutions are made in the prokaryotic repressor
lymphocyte surface receptor, CR2, are substituted or
using site-directed mutagenesis as described above to
inserted as peptide segments into the E. coli Trp apore
alter the DNA sequence to replace a stretch of contigu
pressor protein to form novel hybrid proteins.
ous DNA in the sequence of the repressor with the
In another embodiment, the hybrid proteins, at the
sequence of at least one selected binding site. Prefera
DNA level, are substrates for subsequent site-directed
bly, the selected binding site substituted into the prokar
In one embodiment of the invention, binding sites on
mutagenesis to generate mutant binding sites with al
tered biological properties, e.g. greater affinity for re
yotic repressor comprises at least three, and preferably
?ve amino acids. The gene encoding the repressor and
substituted receptor binding site is then cloned and
ceptor, to provide additional hybrid proteins with novel
activities.
expressed using standard molecular biology procedures.
_
Peptide segments forming binding sites can be substi
tuted for or inserted into regions of the aporepressor
55
The hybrid protein products are then puri?ed to pro
vide a hybrid repressor protein containing the substi
regions whose conformation is restricted by corepres
sor binding, without disturbing the corepressor binding
pocket. Corepressor analogs may be used to modify the
conformations of the peptide segments. Thus, several
different conformations of each peptide segment in the
hybrid Trp aporepressor may be made with different
whether the protein binds t cells containing the receptor
reactive with the binding site. Hybrid Trp repressor
resulting activities.
protein containing the tenascin receptor binding site
The method of the invention for preparing hybrid
tuted receptor binding site or sites.
Activity of the resulting hybrid protein is con?rmed
using a binding assay appropriate for the ligand being
mimicked. Typically, this involves determining
Bourdon et al., J. Cell. Biol, 10811149-1155 (1989) as
proteins is carried out as follows. Mutagenesis is per— 65 described in Example 7, infra, an assay to determine
formed on single or multiple codons of the prokaryotic
whether the hybrid protein binds to human tumor cells
repressor gene using a procedure such as that described
is carried out using the all attachment assay described
by Kunkel (Proc. Natl. Acaa’. Sci. USA 82:488-492
by Bourdon et al., supra.
5,190,873
13
- 14
The present invention also contemplates performing
further mutagenesis in the receptor binding site sequen
gens and immunogenic viral proteins. This favors the
induction of cellular immunity which plays a key role in
tumor rejection. A suitable virus, for example vaccinia
virus derived from a plaque-puri?ed virus of the Wyeth
smallpox, vaccine (New York City Board of Health
-
ces after prior mutagenesis of the repressor protein to
provide the binding site sequence in the repressor pro
tein to identify hybrid proteins with desired activities.
Changes are made in the flanking codons for amino acid
residues of the binding site in the hybrid protein to
optimize the presentation of the site to its corresponding
receptor. In addition, single amino acid changes are
made in the binding site to optimize the primary se
quence of the site. These will include mutations that
increase the speci?c activity of the site as determined by
standard binding assays.
strain), is used to construct a recombinant virus contain
ing the coding sequence of the hybrid protein under
control of the vaccinia virus “7.5K” promoter (Hue et
al., J. Virol. 62:176-180 (1988)). The recombinant virus
may then be administered intravenously as a vaccine to
protect against infection.
In addition to Trp repressor protein, any prokaryotic
repressor proteins may be used as the context protein
for expressing, in a conformationally restricted manner,
invention may be used as reagents for treatment of or 15 a receptor binding site. For example, the MET repres
sor, bacteriophage lambda repressor phage P22Arc
introduction into humans to combat infection or disease
repressor, and the like, can be used in place of the Trp
caused by defects in the interaction of the receptor and
repressor to carry out the invention.
its ligands. Thus, binding sites for receptors from prote
Hybrid proteins produced by the methods of the‘
Receptor binding sites for use in the present invention
ins involved in infection, for example from EBV _virus
are likewise not limited to those shown in the examples
herein, i.e. ?bronectin, tenascin, or IFN . Any binding
site that recognizes a cell surface receptor for which the
amino acid or DNA sequence is known may be used to
substitute or insert into the repressor protein to form a
proteins, may be engineered in hybrid proteins using the
methods of the invention to combat EBV infection.
Such proteins may mimic a compound such as inter
feron in vivo to block the binding or subsequent interac
tions of ligands to or with the receptors for EBV, pre
venting the effects of infection by this virus.
25
Therapeutic applications of the hybrid proteins of the
invention are carried out using pharmaceutical compo
sitions containing a pharmaceutically effective amount
hybrid.
The invention described herein further comprises
DNA sequences encoding the hybrid monomers previ
ously described. As used herein, the term “DNA se
quences” encompasses both double-stranded DNA and
of the hybrid protein and a pharmaceutically acceptable
carrier. The compositions may additionally include 30 single-stranded DNA containing information equiva
other reagents for treatment. Such compositions are
administered using conventional modes of administra
lent to that of the amino acid sequences as determined
by the genetic code; such single-stranded sequences can
tion including, but not limited to, topical, intravenous,
be either in the sense strand orientation or the antisense
intraperitoneal, oral and intralymphatic introduction.
strand orientation.
The hybrid proteins are used in a variety of dosage
forms which include, but are not limited to, liquid solu
monomer according to the present invention can be
tions or suspensions, tablets, pills, powders, supposito
ries, polymeric microcapsules or microvesicles, lipo
somes, and injectable or infusible solutions. The pre
ferred form depends upon the mode of administration 40
and the therapeutic application.
The compositions containing the hybrid proteins of
A double-stranded DNA sequence encoding a hybrid
operatively linked to a transcription-effecting DNA
sequence capable of effecting transcription of the DNA
sequence encoding the monomer. This transcription
effecting sequence is preferably one of Escherichia coli
Lac promoter, E. coli trp promoter, bacteriophage
lambda P1; promoter, and tac promoter, a hybrid trp-lac
the invention also preferably include conventional
promoter, but other promoters are known in the art and
pharmaceutically acceptable carriers and adjuvants
can be used.
known in the art such as human serum albumin, ion 45
exchangers, alumina, lecithin, buffer substances such as
phosphates, glycine, sorbic acid, potassium sorbate, and
-
This DNA sequence, comprising a DNA sequence
encoding a hybrid monomer operatively linked to a
transcription-effecting DNA sequence, can be incorpo
salts or electrolytes such as protamine sulfate.
The most effective mode of administration and dos
rated into a plasmid capable of stably transforming
matography, ion exchange chromatography electro
phoresis, and the like. Puri?ed hybrid proteins may be
ular Cloning Techniques” (Berger & Kimmel, eds.,
prokaryotic host cells to form a vector. The plasmid
age regimen for the compositions of the invention de 50 preferably has both a drug resistance marker and a
replication origin. Many suitable plasmids are known in
pends on the severity and course of the disease or dys
the art, including, but not limited to, the following:
functions, the patient’s health and response to treat
pBR322 and its derivatives; pUCl8, pUCl9, and their
ment, and the judgment of the treating physician. Ac
derivatives; bacteriophage u-derived plasmids, and bac
cordingly, the dosages of the compositions are titrated
55 teriophage M13-derived plasmids. Suitable host-vector
to the individual patient.
systems are described, for example, in B. Perbal, “A
Quantities of the hybrid proteins of the invention may
Practical Guide to Molecular Cloning” (2d ed., John
be readily made and puri?ed using additional standard
Wiley
& Sons, New York, 1988), and “Guide to Molec
procedures such as immunoaf?nity, gel exclusion chro
used as vaccines to immunize against certain disease.
Procedures for preparing such vaccines are known in
Academic Press, New York, 1987; Volume 152 of Meth
ads in Enzymologv). Preferably, the prokaryotic host
cells are E. coli. Prokaryotic, eukaryotic and archaebac
terial host cells stably transformed with such a vector
the art (see, eg Estin et al., Proc. Natl. Acad. Sci. (USA)
are also within the present invention.
85:1052 (1988)). Brie?y, recombinant viruses are con
structed for expression of the cloned gene encoding the 65
EXAMPLES
hybrid protein. Cells infected with the recombinant
The
following
description
provides details of the
viruses will express the hybrid protein at the surface of
manner in which particular embodiments of the present
the cells together with the host’s incompatibility anti
15
5,190,873
invention may be made and used. This description,
while exemplary of the present invention, is not to be
construed as speci?cally limiting the invention. Varia
tions and equivalents, now known or later developed,
which would be within the understanding and technical
16
complex. The aporepressor has been crystallized in
more than eleven different forms, most of which dif
fract clearly to a resolution of less than 2 A. The crystal
structure of aporepressor reveals that the protein is
comprised of six alpha helices joined by short surface
competence of one skilled in this art are to be consid
turns. Four of these alpha helices, A,B,C, and F, are
involved in the formation of an extraordinary stable
hydrophobic core; the aporepressor dimer denatures
at>90° C. The ?rst three helices are intertwined such
ered as falling within the scope of this invention.
1. Oligonucleotide Design for Producing Trp
Aporepressor Hybrid Recombinant DNA
that many of the van der Waals interactions that stabi
lize the core of the aporepressor dimer are intermolecu
lar. In addition the alpha-helix F from one monomer
In this invention, surface features of a stable procary
otic aporepressor, in this instance E. coli tryptophan
aporepressor (TrpR), are replaced with short oligopep
makes contacts with both the A and C helices from its
tides that are active as ligands or binding sites for recep
partner monomer. The extraordinary stability of the
tors on eucaryotic cells. The procaryotic aporepressor - 5 core is emphasized by the fact that the coordinates of
then acts as a scaffold or context protein to allow pre
atoms in these four-helices vary little from crystal struc
sentation of the oligopeptide binding site. The nature of
ture to crystal structure.
the conformation of a receptor binding site can then be
In contrast, the coordinates of helices D and E vary
evaluated with respect to the speci?city of its interac
greatly from structure to structure. When aporepressor
tions with its receptor.
20 and repressor are crystallized in the same space group,
To achieve this invention, different regions of the
the major difference between the two structures is
gene encoding TrpR were separately subjected to
Oligonucleotide-mediated site-directed mutagenesis as
described below to produce receptor binding sites in
found in the coordinates of helices D and E, leading to
a postulate that these two secondary substructures com
prise a “flexible reading head” that can interact with
those locations. The nucleotide sequence for a potential 25 DNA when its structure is altered by tryptophan (core
integrin binding site derived from human tenascin was
pressor) binding. This idea is also a supported by results
engineered into eight different regions on the surface of
showing that changes at several critical residues in D
TrpR. The regions were mutagenized such that a fusion
(Lys72) and E (Thr8l) either abolish DNA-binding, or
protein having the amino acid residue sequence
SRRGDMS (SEQ ID NO 21) flanked by TrpR amino
alter the speci?city of the Trp repressor/DNA binding
interaction as described by Bass et al., Science,
acid residues was produced.
Multiple sites on the TrpR gene which is 324 base
pairs in length were chosen to provide a reasonable
242:240-245 (1988). Two-dimensional NMR analyses of
both aporepressor and repressor indicate that "alpha
opportunity to place the engineered recombinant bind
X-ray diffraction analysis indicates that D and E form
helix D” is not ordered as a helix in solution although
ing site in a variety of structural contexts, one or more 35 helix-turn-helix conformation. Both structural models
of which may be appropriate for receptor binding in the
sites for site-directed mutagenesis was based on the
predict that substitutions of a SRRGDMS peptide in
between D and E helices will be solvent accessible and
unlikely to disrupt the structure of the rest of the TrpR
extensive nuclear magnetic resonance (NMR) and
protein.
fusion protein. The rationale for selecting particular
X-ray crystallographic characterization of Trp apore
All synthetic oligonucleotides for use in accomplish
pressor protein. See Joachimiak et al., J. Biol. Chem,
258:1264l-l2643 (1983); Luisi et al., Biochim. Biophys.
Acza., 1048:113-126 (1990); and Arrowsmith et al., Bio
ing this invention are listed in Table 1 with the corre
sponding SEQ ID NO. The oligonucleotides were syn
thesized on an Applied Biosystems DNA synthesizer,
model 381A, following the manufacturer’s instructions.
chem, 29:6332 (1990).
Brie?y, E.c0li Trp aporepressor is a small dimeric 45 The use of these oligonucleotides in the construction of
regulatory protein that binds two molecules of the core
TrpR binding site recombinants by site-directed muta
pressor, L-tryptophan, to form the active repressor
genesis is described in Example 2.
55
65
5,190,873
17
18
on
:
0
.
on
ac
I
00
mo
POP
00
0<0
(CL.
E.
00
mm
F0
on
00
em
5.
P0
F0
no
00
00
560mi0.3uAN:no.25w3cm-0u:.»av5
mZP
an
<0
00
2.
0P0
3.
<0
Na
g
21
5,190,873
2. Preparation of a Recombinant TrpR Plasmid
TERM was performed following the site-directed mu
Expression Vector
tagenesis procedure for the construction of hybrid
TrpR containing receptor binding sites as described
a. Puri?cation of TrpR Gene
A 1.3 kilobase insert from E. coli containing the TrpR
gene was engineered into a plasmid RPGS, a derivative
of pBR322, following procedures described by Gun
salus et al., J. BacterioL, 140:106-113 (1979) and Gun
salus et al., Proc., Natl. Acad. Sci, 77:7ll7-712l (1980).
Brie?y, MC4lOO pheA905 thrzMu c(Ts) [lambdapl(209)
22
In the instant invention, subcloning into pTAC
below. For the tenascin construct, a 409 bp Barn HI
fragment containing the entire TrpR gene and the re
ceptor binding site prepared in Example 3a was sub
cloned into a Barn HI linearized pTACTERM vector to
form a TrpR expression vector as described in Example
O 6.
was constructed and a lysate of this lysogen was pre
3. Preparation of TrpR-Heterologous Receptor Binding
pared by UV light induction to infect MC4lO0 aroF922
fal thr-90O trpR (lambdacI857S7). Lambda thr+trans
ducing phage were recovered and the thr+transduc
Site Recombinant pPYlSO Plasmid Vectors
a. TrpR Recombinant Hybrids Having an Integrin
Receptor Binding Site
1) Tenascin
The tenascin-derived peptide sequence SRRGDMS
has been shown to be important in mediating cell bind
ing to the extracellular matrix protein tenascin Bourdon
tants were assayed for anthranilate synthetase activity
for measurement of those carrying the trpR gene. Eco
RI restriction digestion fragments from the trpR con
taining lysogens were ligated into the single Eco RI site
of the CM Tc plasmid vector pACYCl 84. The recipient
strain for the transformation was W31 l0SRT4 and was
0
et al., J. Cell Biol, 109:317-330 (1989). The peptide is a
mutant in trpR. Transformants containing the trpR gene
were selected and plasmid DNA was purified following
competitive inhibitor of speci?c RGD-dependent re
ceptor binding to tenascin and ?bronectin. In addition,
antisera speci?c for SRRGDMS block cell binding to
tenascin. Oligonucleotide mediated site-directed muta
techniques well known to those skilled in the art.
Maniatis, Molecular Cloning: A Laboratory Manual, 2
ed, Cold Spring Harbor Laboratory Press, NY, NY.
genesis was used to introduce nucleotide sequences
(1989). This plasmid was designated pRPGS and con
tained the entire TrpR gene. For construction of
pPYl5O as described below in which the TrpR gene
was mutagenized, pRPGS was first digested with HpaII
to form a smaller TrpR gene fragment consisting of 424
coding for the SRRGDMS binding site into the TrpR
gene in the pPYlSO expression vector prepared in Ex
ample 2b.
The SRRGDMS sequence was introduced at 8 sites
within the TrpR gene. These sites included the amino
terminus, a site between alpha-helix A and alpha-helix
B, two sites between alpha-C and alpha-D, three sites in
alpha-D, or the alpha-D/alpha-E transition, and at the
bp. Bam I-II linkers were constructed on this fragment
and then was ligated into a Bam HI linearized pBR322
plasmid. The ligated plasmid containing the smaller
TrpR gene was designated pRPG47 and was subse
quently digested with Eco RI and Pvu II to form ‘a
carboxyl-terminus of TrpR. The oligonucleotides used
to encode the binding peptide sequence at each site are
shown in Table 1. Three substitutions of the
TrpR gene containing fragment. This fragment was
ligated to a similarly digested fragment from plasmid
pZ152 described Zagursky et al., Gene, 27:183-191
SRRGDMS sequence in the D/E transition designated
TN4, 5 and 6 were made. Substitution of residues for the
region between Lys72 and Thr8l was performed to
(1984) which contains an M13 origin to form a ligated
plasmid which contains the TrpR gene and an M13
replace this ?exible portion of aporepressor without
origin necessary for subsequent site-directed mutagene
disrupting the core structure of TrpR. The amino termi
nal and carboxy terminal sites were chosen because they
were solvent exposed, disordered structures as revealed
sis and helper phage rescue.
b. Insertion of the TrpR Gene-Containing Fragment
into an Expression Vector
l) pPYlSO Expression Vector
The plasmid expression vector pPYl50, a rop-l"
derivative of pBR322, was used as a recipient cloning
45
by both NMR and X-ray diffraction data which would
likely mimic the active linear synthetic peptides.
The alpha-B helix is between the highly stable alpha
A and alpha-C helices of the hydrophobic core. Substi
tution of the SRRGDMS sequence at the beginning of
vector for insertion of the 424 bp TrpR gene fragment 50 alpha-B was determined to not be likely to disrupt core
structure but its location between the highly stable
as described in Example 2a. The resulting ligated plas
structures of the alpha-A and alpha-C would define a
mid carried the M13 origin from pZ152 and a fusion of
' the PlacUVS promoter to the TrpR structural gene
particular set of constrained conformations as a result.
The site in alpha-C/alpha-D transition is solvent ex
from pRPG47. The expression of the TrpR was driven
55 posed, but due to its proximity to the hydrophobic core,
from the lacUV5 promoter.
would provide a unique conformational environment
2) pTACTERM Expression Vector
for the SRRGDMS peptide. This SRRGDMS substitu
tion site would be unlikely to disrupt the core structure
In an alternative to expressing TrpR protein in
and limit the ability to obtain NMR data.
pPYl50, the aporepressor alone or containing a hybrid
binding site can be expressed from a rop pBR322 deriv 60 Oligonucleotide-mediated site-directed mutagenesis
of TrpR pPYlSO expression vector was performed by
ative designated pTACTERM 62 which has a stronger
the method described by Kunkel, Proc. Natl Acad. Sci,
tac promoter. The advantage of using pTACTERM
USA,_ 82:488-492 (1985) using a Muta-Gcne In Vitro
over pPYlSO is that plasmids replicate to a higher plas
mid copy number due to the absence of the rop gene.
When expressed from pTACTERM, wild-type TrpR
and mutant aporepressors comprise l to 5% of the total
cell protein. Paluh et al., Nuc. Acids Res, 14:7851-7860
(1986).
Mutagenesis Kit (Catalog Number 170-3576, Bio-Rad
Laboratories, Richmond, Calif). The pPYlSO plasmid
containing the TrpR gene prepared in Example 2bl) was
transformed using standard procedures well known to
one skilled in the art in the bacterial strain C1236 (Bio
23
5,190,873
this bacterial host strain was de?cient for dUTPase and
uracil~N-glycosylase which results in an occasional
substitution of uracil for thymine in newly synthesized
DNA. The resulting DNA, when transformed into a
wild-type host with an active uracil-N-glycosylase is
inactivated. The uracil-containing DNA was then used
as a template for oligonucleotide-mediated site-directed
mutagenesis wherein the complementary strand was
synthesized using the mutagenic oligonucleotide to
24
ammonium acetate at pH 7.5 and 20% polyethylene
glycol (PEG). The resulting admixture was maintained
at 25° C. for 15 minutes. The admixture was centrifuged
at 11,000X g for 20 minutes to pellet the single-stranded
phage present. The resulting supernatant was removed
Rad Laboratories). In an overview of the procedure,
10
from the phage pellet.
The phage pellet was resuspended in 300 pl of TE
buffer consisting of 10 mM Tris-HCl (Tris [hydroxyll
aminomethane hydrochloride) pH 8.0 and 1 mM
EDTA (ethylene-diamine tetraacetic acid). The result
prime DNA synthesis. The resultant double-stranded
wild-type/mutant heteroduplex molecule was then
' ing solution was admixed with an equal volume of phe
transformed into a host with an active uracil-N-glycosy
ferred to a fresh tube. The aqueous phase was reex
lase resulting in the inactivation of the uracil-containing
wild-type parental strand and the enrichment of the
tracted with phenol/chloroform until no signi?cant
interface between the organic and aqueous phase was
observed. The aqueous phase was then admixed with an
nol/chloroform and the resulting aqueous phase trans
mutant strand.
Brie?y, the uracil-containing single-stranded tem
plate required for the site-directed mutagenesis proce
dure was prepared ?rst by maintaining a 5 milliliter (ml)
culture of XLl—blue cells (Stratagene, La Jolla, Calif.)
containing the pPYlSO plasmid at 37° C. for 3 hours.
One pl of a solution containing 1X 1011 plaque forming
units (pfu) per ml of M13K07 helper phage provided
20
with the kit was then added to the 5 ml culture and the
culture was further maintained at 37° C. for 3 hours. 25
equal volume of chloroform and the single-stranded
DNA isolated from the aqueous phase by ethanol pre
cipitation using ammonium acetate.
The amount of single-stranded pPYlSO DNA isolated
was determined by gel electrophoresis. This single
stranded pPYlSO DNA was then used as a template in
the site-directed mutagenesis procedure below.
The nucleic acid segment coding for the tenascin
receptor binding site, SRRGDMS, (SEQ ID NO 45)
During this time, single-stranded M13 phage containing
was then inserted into the plasmide pPY150 using the
the single-stranded pPY15O DNA were formed and
released from the XLl-blue bacteria.
The bacteria in this culture were killed by heating the
pPY150 single-stranded DNA template prepared above
culture to 68° C. for 15 minutes. The killed bacteria 30
were removed from culture, by centrifuging the culture
at 10,000Xg to produce a supernatant containing the
pPYlSO single-stranded phage. One ml of this superna
tant was added to a previously established 5 ml culture
and the oligonucleotides, TNl through TN8, listed in
Table I in the site directed mutagenesis protocol de
scribed in the manufacturer’s instructions provided with
the Mutagenesis Kit (Bio-Rad). The nucleic acid seg
ment coding for the SRRGDMS was placed at eight
different locations within the pPYlSO template DNA.
Five ug of the pPYlSO single-stranded template pre
of C1236 bacteria that was in the late log phase of 35 pared above were admixed with 50 ng of each of the
growth. The culture was maintained at 37° C. for 10
oligonucleotides, TNl through TN8, to form a 10 ul
minutes and then 10 rnicroliters (pl) of the culture was
admixture. The admixture was maintained for 10 min
plated onto LB bacterial plates (LB media contains 10
utes at 68° C. followed by 5 minutes at 25° C. to allow
grams/Liter (g/L) of bactotryptone, 5 g/L of yeast
the oligonucleotide to anneal to the single-stranded
extract, 5 g/L of NaCL containing both 50 pg/ml of 40 template. The complementary strand was then synthe
ampicillin and 30 pg/ml of chloramphenicol. The re
sized by admixing 4 ul of a solution containing 10 mM of
sulting bacterial plates were maintained at 37° C. for
adenosine triphosphate (ATP), 4 ul of a buffer contain
12-18 hours to allow individual colonies of CJ236 bac
‘ing 0.66 M Tris-HCl at pH 7.6, 50 mM MgClz, 50 mM
teria containing the pPYlSO single-stranded M13 phage
dithiothreitol (DTI‘), 2 ul of a solution containing 2.5
to form.
45 mM each of dCTP, dATP, dGTP, and dTTP, 1 ul of T4
One of the resulting colonies was selected and used to
DNA ligase (Stratagene) and 1 ul of the Klenow frag
initiate a 5 ml bacterial culture in superbroth media
ment of DNA Polymerase I (Stratagene) to form a
consisting of 35 g/L bactotryptone, 20 g/L yeast ex
mutagenesis reaction admixture. The mutagenesis reac
tract, 5 g/L sodium chloride at pH 7.5. This 5 ml cul
tion was terminated by admixing 1 ul of 50 inM EDTA.
ture was maintained at 37° C. for 6 hours and then trans 50 The resultant mutagenized closed circular cDNA was
ferred to 150 mls of superbroth in a 250 ml ?ask and
then transformed into competent CG103F’ Fan bacteria
further maintained at 37° C. for one hour with constant
(Bio-Rad) and kanamycin resistant clones containing
shaking. Then 100 pl of a solution containing 1X10ll
the mutagenized DNA were selected. DNA was then
PFU/ml of M13K07 helper phage (Stratagene) was
isolated from the resulting transformants using the
added to the culture and the culture was maintained at 55 DNA isolation procedures of Sambrook et al., Molecu
37° C. for 12-18 hours with constant shaking. During
this time, a culture of C1236 bacteria containing the
Iar Cloning: A Laboratory Manual, Second Edition, Cold
Spring Harbor Laboratory, Cold Spring Harbor, New
pPYlSO containing TrpK gene single-stranded DNA
York, N.Y. (1989). The presence of mutations were
template was produced.
con?rmed by dideoxynucleotide sequencing of the iso
This culture was then used to isolate the pPY150 60 lated double stranded DNA using the manufacturer’s
single-stranded template using the single-stranded
phage DNA isolation procedures described in the p
Bluescript II manual (Stratagene). Brie?y, 50 pl of the
culture containing pPY150 single-stranded phage pro
instructions in the AMV Reverse Transcriptase 35S
dATP Sequencing Kit (Stratagene).
Following site-directed mutagenesis and veri?cation
of the construction of the hybrid TrpR-SRRGDMS
duced above was centrifuged at 17,000X g to produce a 65 containing plasmid, the region containing the entire
clari?ed supernatant. Approximately 1.2 ml of this su
mutagenized TrpR gene was subcloned into a pTAC
pernatant was transferred to an eppendorf centrifuge
TERM expression vector for expression of the hybrid
tube containing 300 pl of a solution consisting of 3.5 M
protein as described in Example 6.
5,190,873
25
26
for binding to ?bronectin receptors as described in Ex
2) Fibronectin
ample 8.
A peptide, GRGDSP (SEQ ID NO 46), derived from
4. Preparation of TrpR-Heterologous Receptor Binding
the extracellular matrix protein ?bronectin containing
Site Recombinants to Produce Cysteine Disulfide
the integrin receptor binding site RGD has been de 5
scribed. Pierschbacher et al., J. Cell Biol, 28:115-126
Loops
(1985). The receptors alpha 5 betal, alpha,beta1, alphav
betas, for example recognize an RGD-binding site in
a. TrpR Recombinant Hybrids Having an lntegrin
Receptor Binding Site Containing Cysteine Disul?de
Loops
matrix ligands. The receptors have overlapping but
distinct ligand speci?cities. Peptides from ?bronectin
The conformational context of the SRRGDMS (SEQ
ID NO 21) or GRGDSP (SEQ ID NO 46) sequence
(GRGDSP) or tenascin (SRRGDMS) act as competi
tive inhibitors of receptors binding ?bronectin, tenascin
and vitronectin and GRGDS (SEQ ID NO 46) peptides
within TrpR is further altered by creating cysteine di
sul?de bridges to create a loop containing the binding
can inhibit tumor metastasis, tumor invasiveness and in
site sequence. The rationale for this is several fold. First,
vitro angiogenesis. The peptides do not have, however, 15 modeling predictions (Cachau et al., J. Mol. Res,
the receptor speci?city of the natural matrix ligand.
22179-186 (1989)) and results with cyclic peptides
Conformation must play an integral role in receptor
(Pierschbacher et al., J. Biol. Chem, 262:17294-17298
recognition of the RGD binding sites. This has impor
(1987))
and a helical dimers (Engel et al., Bic/rem,
tant implications since this conformational recognition
30:3161-3169 (1991)) indicate that the most active con
can result in very distinct cellular responses. Fibronec
tin is a strong cell adhesion and spreading factor, while
tenascin prompts reduces cell adhesion and increased
motility but not spreading. The two proteins contrast
and complement each other in modulating adhesion and
cell motility.
The GRGDSP sequence is placed at the same sites as
formations are likely to be contained in a turn structures
or loops. Second, the snake venom disintegrin proteins
have the RGD site ?anked by cysteines (Gould et al.,
P.S.E.B.M., 195:168-171 (1990)). Third, the active
TrpR-TNS hybrid protein produced in Example 6 is
25 likely to reside within an extended or loop structure.
Finally, cysteine disul?de bridges constrain the binding
with the tenascin peptide in TrpR as described in Exam
sequence in ways not possible through the TrpR con
text alone. By selecting the ?anking distance from the
ple 3al). Oligonucleotide-mediated site-directed muta
genesis is performed as described using the ?bronectin
binding-site encoding oligonucleotides listed in Table l.
The resulting TrpR-GRGDSP hybrid constructs are
binding sequence, loops of various sizes and varying
then subcloned as described in Example 6 into a pTAC
ial cyclic peptides. However, unlike cyclic peptides the
conformation contexts are generated. The formation of
disul?de bridges is analogous to the formation of artific
TERM expression vector for expression of the hybrid
NH; and COOH protein context is not lost.
This strategy is most applicable to TrpR-hybrids at
for binding to ?bronectin receptors as described for 35 the NH; and COOH-termini (TrpR-TNl, TrpR-TN7)
protein. The expressed hybrid protein is then assayed
tenascin receptors in Example 7.
and D/E transition (TrpR-TN4, TNS, and TN6), be
b. TrpR Recombinant Hybrids Having a Lymphocyte
cause these sites are ?exible regions of Trp aporepressor
and changes in these regions are least likely to disrupt
the hydrophobic core. Core disruption would make
Receptor Binding Site, CR2
1) Alpha Interferon (IFNa)
NMR spectroscopy and X-ray crystallographic analysis
exceedingly difficult. Because both TrpR-Tn] (NH;
The peptide QLNDLEACV (SEQ ID NO 47) is the
receptor binding site on IFNa which is recognized by
the B lymphocyte receptor called CR2. Oligonucleo
terminal) and TrpR-TNS (D/E) are both active recep
tor binding proteins, cysteine bridges in these mutants
tide-mediated site-directed mutagenesis was performed
are generated ?rst. Cysteine pairs ?anking the
as described using the interferon binding-site encoding 45 SRRGDMS sequence are introduced into the TrpR
oligonucleotides listed in Table 1. The resulting TrpR
TNl and TNS by a single round of site-directed muta
genesis as described in Example 3 using paired oligonu
cleotides coding for a cysteines ?anking the
GRGDSP hybrid constructs were then subcloned as
described into a pTACTERM expression vector as
described in Example 6 for expression of the hybrid
SRRGDMS substitution site. The oligonucleotides used
protein. The expressed hybrid protein was then assayed 50 for generating hybrid TrpR-SRRGDMS fusion prote
ins having disul?de loops are listed in Table 2 below.
TABLE 2
CYS-CYS LOOPS:
SSL 1A(TN1) (SEQ ID NO 48)
l
2
3
M
A
C
ATT ATG GCC
CAA
TGC
_
S
TCA
R
CGC
R
CGC
G
GGA
C
TGC
l0
A
GCA
D
GGC
M
ATG
S
TCA
D
GAC
M
ATG
81
S
C
T
TCC TGC ACG
l1
M
ATG
12
A
GCA
SSL IBaCI'Nl) (SEQ ID NO 49)
1
CGA CAT
A'I'T
3
4
M
C
Q
Q
S
R
ATG
TGC
CAA
CAA
TCA
CGC
C
TGC
l2
A
GCA
l3
E
GAA
l4
Q
CAG
15
R
CGT
C
TGC
S
TCA
R
CGC
R
CGC
G
GGA
SSL] BbCI‘Nl) (SEQ ID NO 50)
8
M
S
ATG TCA
9
A
GCA
l0
A
GCG
SSL 5A(TN5) (SEQ ID NO 51)
68
69
Q
R
CAG CGT
70
E
GAG
71
L
TTA
SSLS Bb(TN5) (SEQ ID NO 52)
82
83
l
T
ATI' ACG
8
CG