Note: Descriptions are shown in the official language in which they were submitted.
!
~`7~3~3
BIOSYNTHETIC CONSTR~E OF T~F-~
B~ckuround of the Invention
This invention relates to biosynthetic
peptide constructs o~ transforming growth
factor-beta, to synthetic genes encodi~g polypeptides
having transforming growth factor-beta-like
biological activity, to methods of producing such
synthetic genes using recombinant DNA technology, and
to the use of such biosynthetic peptide constructs as
regulators of cell proliferation and growth.
Transforming growth factor-beta (TGF-B) is a
multifunctional peptide regulator of activity
involving cellular or tissue response to injury or
stress. This factor has the ability to stimulate
cell proliferation in cells of mesenchymal origin,
while also being able to inhibit the growth of
epithelial cells, embryonic fibroblasts, endothelial
cells, and T and B lymphocytes. In addition, TGF-B
has a number of other regulatory activitites which
appear to be related uniquely to the specialized
function of a particular cell type. For example,
TGF-B stimulates the production of matri~ components,
e.g., inhibiting the synthesis and secretion of
proteolytic enzymes which act on tnese components,
thereby regulating the synthesis and degradation of
extracellular matriY ~for a reviow, see, e.g., Sporn
et al. (1987) ~. Cell Biol. lQ~:1039-104S).
TGF-B-type activities have been identified in many
normal fibronectin- and collagen-producing
, -2-- 2Q70393
fibroblasts, as well as tissues such as kidney
(Roberts et al. (1983) Biochem. 22:5692-5698),
placenta (Frolik et al. (1983) Proc. Natl. Acad. Sci.
(USA) 80:3676-3680), and platelets (Childs et al.
(1982) Proc. Natl. Acad. Sci. (USA) 79:5312-5316; and
Assoian et al. (1983) J. ~iol. Chem. 258:7155-7160),
as well as in tumor cells (see, e.g., Roberts et al.
(1980) Proc. Natl. Acad. Sci. (USA) 77:3494-3498).
There are four known molecular
configurations of TGF-B, each having an apparent
molecular weiqht of about 25,000 daltons. Three of
these species result from homodimeric (TGF-~l,
TGF-B2) and heterodimeric (TGF-Bl.2) combinations of
the monomeric subunits, Bl and B2. The fourth
species is a homodimer of a B3 subunit. Each subunit
is processed from a precursor of about 390 amino
acids, and the mature subunit protein includes
approximately 112 amino acids of its carbosy
terminus. The Bl and B2 subunits have about a 70%
amino acid sequence homology in their N-terminal
portions, and are highly conserved between species.
The deduced amino acid sequence of TGF-B3 shares
about 80~ homology with types Bl and B2, with many of
the differences being conservative substitutions.
TGF-Bl was originally isolated from hu~an platelets
and placenta ~EP 0128849), and bovine kidney (Roberts
et al. iki~-). TGF-B2 was originall~ identified as
cartilage-inducing factors tCIF) isolated from bovine
bone tUS 4,774,228). TGF-Bl.2 has been found in
porcine platelets and other cells which coexpress the
Bl and B2 chains (Cheifetz et al. (1988) J. ~iol.
Chem. ~~:10783-10789). TGF-B3 has been identifie~
--3--
2~7~393
in both human and chicken S~uke et al. (1985) Proc.
Natl. Acad. Sci~ (USA) 85:4715-4719).
The TGF-B's belong to a larger gene family,
the members of~which encode struct~rally similar
proteins that have similar regulatory activities
(reviwed in Massague (1987) Cell 4~:437-438).
Included in this family are: (1) Ygl, a protein
involved in mesoderm formation during XenoDus
development; (2) decapentaplegic complex (DPP), a
polypeptide encoded by a Drosophila gene responsible
for development of the dorsoventral pattern in the
embryo: (3) OPl, a region of a native osteogenic
protein sequence encoded by exons of a genomic DNA
sequence retrieved by applicants; (4) cartilage
inducing factors ~CIFs) isolated from bovine bone (US
4,774,228); (5) mammalian osteogenic bone matri~
proteins CBMP-2a, C~MP-2b, and CBM~-3, discovered by
applicants (see W089/01453); and (6) B-inhibin-a and
b, gonadal proteins that suppress pituitary secretion
of follicle stimulating hormone. All of these
proteins are believed to dimerize during refolding,
and are inactive when reduced to the monomeric form.
In addition, many include portions of a common
precursor peptide.
Identification of the regulatory activities
of the proteins in the TGF-B family, and the
elucidation of their amino acid sequences, have
resulted in research efforts directed to the
production o these proteins by recombinant means.
For e~ample, EP 0200341 discloses nucleic acid
sequences encoding native TGF-B and precursors
thereof which can ~e expressed in a host eukaryotic
2~3~3
cell transformed therewith. EP 0150572 discloses the
manufacture of structural genes coding for TGF-Bl and
analogs thereof and the expression of these genes in
microorganisms. ~owever, the desi~n and expression
of consensus protein constructs having considerable
sequence homology with a number of the proteins in
the TGF-B family, and displaying TGF-B-like activity,
has heretofore not been contemplated.
Accordingly, it is an object of this
invention to provide novel analogs of TGF-B having
TGF-B biological activities. Another object is to
provide an efficient method of producing novel,
active TGF~B analogs. Yet another object is to
provide genes encoding novel, non-native, TGF-B
species and methods for their production using
recombinant DNA techniques. Another object is to
provide novel truncated forms of T5F-B and structural
designs for proteins with TGF-B biological activity.
A further object is to provide methods of regulating
cell proliferation using TGF-B analogs.
These and other objects and features of the
invention will be apparent from the description,
drawings, and claims which follow.
--5--
s~m~aFy o~_thç_Inventinn 2 0 7 0 3 9 3
It has been discovered that forms of native
TGF-B which have been truncated at the N-terminus,
and which have fewer than the native number of
cysteine residues demonstrate TGF-~-like biological
activity, including the ability to induce an
anti-proliferative effect on mammalian epithelial
cells n vit~o. It has also been une~pectedly
discovered that truncated analogs ~f other
structurally similar proteins in the TGF-B family
known to have unrelated biological activities also
possess this TGF-B-like activity. These discoveries
enabled the design and construction of DNAs encoding
novel, non-native protein constructs which
individually and combined are capable of inhibiting
the proliferation of mammalian epi~helial cells in
culture.
Thus, in one aspect, the invention comprises
a truncated TGF-B analog produced by e~pression of
recombinant DNA in a host cell and capable of
inducing an antiproliferative effect in mammalian
epithelial cells Ln Vi~LQ- This protein construct
includes two polypeptide chains, each including a
biologically active domain, and each having ewer
than 9, and preferably 6 or ~, cysteine (Cys)
residues. It may further be characterized as being
unglycosylated.
In another aspect, the invention comprises a
protein produced by expression of recombinant DNA in
a prokaryotic host cell and including a pair of
polypeptide chains of fewer than about 112 amino
2~7~3~3
acids each. The sequence of amino acids in each
chain is sufficiently duplicative of the sequence of
TGF-B such that the protein is capable of inducing an
anti-proliferative effect on mammalian epithelial
cells in vi~o. Preferably, the polypeptide chain
contains fewer than 9, and more preferably 6 or 8,
cysteine residues, and further, may be
unglycosylated.
The cysteine residues are involved in the
formation of intra- and inter-chain disulfide bonds
(folding), the correct formation of which results in
an active construct having TGF-B-l~ke activity. In
eucaryotes, the synthesis and proper folding of the
protein can occur at least within those cells known
to express TGF-~; in prokaryotic calls, folding must
be performed Ln Vit~Q, a difficult feat in that any
number of combinations of disulfide linkages e~ist
between two polypeptide chains, each having less than
9, and prefera~ly 6 or 8 cysteine residues. An
important aspect of this inventior. is the discovery
that truncated constructs of the type described
herein may be post-translationally modified and
folded (by o~idation) in vit~Q to produce TGF-B-like
activity.
Several forms of TGF~ monomers are known in
nature, ~1, B~, and B3. Investigation of the
properties and structure of these native forms
enabled the development of a rational design for
non-native ( i . Q ., not known to be e~pressed in
nature), truncated protein constructs which also are
capable of differentially reyulating cell
proliferation in various cell types. Further, upon
examination of a number of unrelated proteins wi ~ ~ 7 ~ 3 9 3
some amino acid sequence homology, it was
une~pectedly discovered that they, too, possess
TGF-B-like activity.
Based on this knowledqe, a series of
consensus DNA sequences were designed with the goal
of producing active TGF-B analogs. The sequences
were based on partial amino acid sequence data
obtained from native TGF-B species and from observed
homologies with genes reported in the literature
encoding proteins of the TGF-B family (including Vql
and DPP), or on the amino acid seque~ces they encode,
having a presumed or demonstrated developmental
function. Several of the biosynthetic consensus
sequences have been expressed as fusion proteins in
prokaryotes, purified, cleaved, refolded, applied to
a mammalian L~ vitrQ assay system, and shown to have
TGF-B-like anti-proliferative activity.
In preferred aspects of tne invention, the
proteins encoded by these consensus sequences include
the generic amino acid sequences:
CXXXXLYXXFXXDXGWX~WXXXPXGYXAXXCXGXCPXXXX~X~3XXX~X~3XXX
100
XXLXXXXXPXXX~XXXCCVXK~K~JO~W UCL~XXXX~XXMXVXXCXCX
--8--
2~393
and
10 20 30 40 so
LYXXFXXnXGWX~3WX~PXGYiCAXXCXGXCPXXXX6~3X~lX~3XXX
100
~LXxxxxpx~axxxccw~ 3~vxxcxcx
wherein the letters indicate the amino acid residues
of standard single letter code, each ~X~
independently represents one of the naturally
occurring amino acid or a derivative thereof, and
each "~" independently represents an amino acid or a
peptide bond.
The currently preferred active peptide
constructs comprise amino acid sequences derived from
the three ~onomeric subunits of TGF-B (1, 2, and 3),
DPP, and OPl. The amino acid seguences of these
proteins are set forth in FIGURE 1 relative to the
sequence of TGF-Bl. Pre~erred amino acid sequences
within the foregoinq qeneric sequences are:
CCVRQLYIDFRKDLGWK-WIHEPKGYHANFCLGPCPYIWS--L-DTQ--Y-SKV
L P KR N A A S H R
Q V Y S R A T ~
KK H VE - V QN IA Q M Y Y E PLTEI NGSN AIL
RRHS S DD V L D Y H K F ADHF S V
100
LALYNQHNPGAS-AAPCCVPQALEPLPIVYYVGRKPKVEQL-SNMIVRSCKCS
S TI E S S D T L I KT I K
G L V
QT VHS E D-IPL TKMS ISM F DNNDNV LRHYE A DE G R
NN K V KA Q DSVA LNDQST KN QE T VG
2Q~03~3
and
2Q 30 40 50
LYIDFRKDLGWX~WIHEPKGYHANFCLGPCPYIWS--L-DTQ--Y-SKV
P KR N A A S H R
Q V Y S R A T T
VE - V QN IA Q M Y Y E PLT~I NGSN AIL
S DD V L D Y H K F ADHF S V
60 70 80 30 100
LALYNQHNPGAS-A~PCCVPQALEPLPIvYYVGRKPKVEQL-SNMIVRSCKCS
S TI E S S D T L I KT I K
G L V
QT VHS E D-IPL TKMS ISM F DNNDNV LRHYE A DE G R
NN K V KA Q DSVA LNDQST KN QE T VG
wherein, at each position where more than one amino
acid is shown vertically, any one of the amino acids
shown may be used alternatively in various
combinations, and "-" and n__n represent a peptide
bond. Note that the numbering of amino acids is
selected solely for purposes of facilitating
comparisons among alternative sequences. These
generic sequences have fewer than 9, and preferably
6-8 cysteine residues where inter- and/or
intramolecular disulfide bonds can form, and contain
other critical amino acids which influence the
tertiary structure of the proteins. Similar
structural features are found in the above named
known proteins of the TGF-~ family whose amino acid
sequences previously have been published. However,
of these only the TGF-B species (1, 2, and 3) have
been describad as capable of inducing an
anti-prolierative effect in mammalian epithelial
cells Ln vi~~Q.
--10--
Particularly useful sequences include analogs
having the following amino acid sequences:
TGF-~l
CCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLPTQYSKVLAL
100
YNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS;
TGF-~2
CCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLY
100
NTINPEASASPCCVSQDI.EPLTILYYIGKTPKIEQLSNMIVKSCKCS;
TGF-~3
CCVRPLYIDFRQDLGWKWVllEPKGYYANFCSGPCPYLRSAD'rTHSTVLGL
100
YNTLNPEASASPCCVPQDL,EPt.TIL.YYVGK'I`PKVF:QLSNMVVKSCKCS;
Vgl
CKKRHLYVEFKDVe,WQNWVIAPQGYMANYCYGEe'PYPL'rEILNGSNtlAIL
gO 100
QTI.VHSIEiPEDIPL,PCCVPTKMSPISMl.ti`Y[)NNDNVVLRHYENMAVDECGCR;
DPP
CRRHSLYVDFSDVGWDDWIVAPLGYDAYYCHGKCPFPLADHFNSTNHAVV
100
QTLVNNNNPGKVPKACCVPTQLDSVAMLYLNDQSTVVLKNYQEMT W GCGCR;
~, . . .
--ll--
and
CCVRQLYIDFKRDLGWKWVHEPKGYAANFCAGACPYLWSADTQHSRVLA
100
LYNTANPEASAAPCCVPQDLEPLTILYYVGRTPKVEQLSNM W KSCKCS.
or more truncated analogs such as:
TGF-~1
LYIDFRKDLGWKWIHEPKGYHANFCL.GPCPYIWSLDTQYSKVLAL
60 70 80 90 100
YNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS;
TGF-~2
LYIDFKRDLGWKWIllEPKGYNANFCAGACPYLWSSD'rQHSRVLSLY
100
NTINPEASASPCCVSQl)l,EPLTILYYIGKTPKIEQl.SNMIVKSCKCS;
TGF`-~3
LYIDF'RQl)l.GWKWVlll.PK(;YYANFC'SGPt~PYl.l~SAl)T'r~lS'rVt,GL
100
YNTI,NPE`ASASlCCVPQl)l,l`lLTII,YYVGRl`PKVEQl.SN~lVVKSCKCS;
V~31
~0 50
LYVEFKDVGWQNWVIAEQGYMANYCYGECPYPLlEIl,NGSNHAIL
100
QTLVHSIEPEDIPLPCCVYTKMSPISMLFYDNNDNVVLRtlYENMAVDECGCR;
;,~
-12-
DPP
LYVDFSDVGWDDWIVAPLGYDAYYCHGKCPFPLADHFNSTNHA W
60 70 80 90 100
QTLVNNNNPGKVPKACCVPTQLDSVAMLYLNDQSTVVLKNYQEMTVVGCGCR;
and
LYIDFKRDLGWKWVHEPKGYAANFCAGACPYLWSADTQHSRVLALYN
100
TANPEASAAPCCVYQDLEPI,TILYYVGRTPKVEQ~,SNM WKSCKCS.
The name given to each of these sequences designates
the natural source DNA sequence encoding the amino acid
sequence which, as far as applicants are aware,
exhibits the most homology with the recited TGF-
~analog.
The invention further includes DNA sequellces
encoding these constructs and a prokaryotic host cell
en~ineered to express these DN~ seq11ences. In a
preferred aspect of the invention, the p1okaryotic host
cell (~.g. E. coli) is tral1seGteci with a V~CtOI
., ~ ., ~ .~ ,,=
inC1Udin~ tl1C! TGF ~-enCOd illCJ I~N~ seq~1enc~. The
tral1sor1ned cell i* cl1ltr11tcd to express thc protc~
which .is then puliried ,~l1d ac~.ivated by oxidation ln
v.itro. T~1~? pl~o~eil1 so tl`~r~t~ )lcls t~1~ ahility to
induce an allt:i-ploli~etclt.ive effect on cultuled
mammalian epithelial cel1s.
The biosynthetic constructs disclosed herein may be
used to regulate cellular activities such as
proliferation and growth. In this regard, these
.
2~703~3
constructs have wide potential clinical applications,
for example, by controlling the proliferation of
various tumor cell lines, or by enhancing the growth
rate of T and B lymphocytes in immunosuppres~ed (e.g.
Acquired Immunodefficiency Syndrome (AIDS) patients.
The constructs also may be used in cell cultures to
modulate growth of various types of eucaryotic cells.
Brief ~es~iption of th Q rawin~ 2 Q 7 0 3 9 3
The foregoing and other objects of this
invention, the various features thereof, as well as
the invention itself, may be more ully understood
from the following description, when read together
with the accompanying drawings, in which:
FIGURE 1 is a comparison of the amino acid
sequence of various proteins in the TGF-B family to
TGF-Bl; and
FIGUREs 2A and 2B are rep--esentations of a
DNA sequence and corresponding amino acid sequence of
a modified trp-LE leader sequence, two FB domains of
protein A, an Asp-Pro cleavage sit~, and (A) the 6
Cys TGF-Bl sequence, and (B) the 8 Cys TGF-Bl
sequence.
DescriP~ion 2070~93
Nucleic acid sequences encoding truncated
TGF-B analogs were designed based on sequence data
reported in the literature, codons inferred from
known amino acid sequences, and observations of
partial homology with known genes of the TGF-B
family. These sequences have been refined by
comparison with the sequences present in certain
regulatory genes from the TGF-B family.
The naturally occurring proteins of the
TGF-B family are made as precursors~ with a large and
poorly conserved N-terminal domain and a
characteristic C-terminal domain in which a pattern
of 7 cysteines (Cys) residues is highly conserved.
In addition to these Cys residues, certain other
amino acids are found in members of the family very
nearly in the same relative positions in sequence as
set forth below:
CXXXXLYXXFXXDXGWX6WXXXPXGYXAXXCXGXCPXXXX6~X~XXX6~X~XXX
~0 90 100
XXLXXXXXP~XX0XXXCC ~ ~XXMXVXXCXCX
wherein each X independently repre3ents a naturally
occurring amino acid, and ~ or ~ represents an amino
acid or peptide bond.
The N-~erminal sequence o~ mature ~GF-B and
other related proteins contains a variable number of
Cys residues which appear to be crosslinked among
each other or with a residue of another amino acid
chain, but not to Cys residues in :he C-terminal
-16-
2~!7~393
dom~in. Maturation of the precursor to the mature
form of these proteins occurs by trypsin-like
cleavages between the precursor and the mature
protein, and possibly also within the precursor form
as other similar cleavage sites ar~ presen~ therein.
All members of the Vgl-related subyroup of
the TGF-B ~amily (includinq Vgl, DPP, OPl, CBMP-2a,
CBMP-2b, and CBMP-3) share the feature of two basic
residues (i.e., Lys-Lys, Arg-Arg, ~rg-Lys) following
the first Cys in the conserved C-terminal domain.
The conserved double basic residues may represent
another secondary maturation site. Cleavage by
trypsin or related protease releases a C-terminal
domain containing only 6 Cys residues. Since the
precursor region of TGF-B contains up to S Cys
residues which are not crosslinked to the C-terminal
domain, the first of the 7 Cys residues may not be
crosslinked to the C-terminal domain either.
Therefore 6 Cys residues appear to be sufficient for
a properly folded C-terminal domain.
In view of this disclosure, skilled genetic
engineers can design and synthesize genes which
encode a number of appropriate amino acid sequences.
These genes can be e~pressed in various types of
eucaryotic cells but, or reasons of efficiency are
preferably produced in prokaryotic host cells,
thereby providing large ~uantities of active
synthetic proteins such as truncated analogs,
muteins, fusion proteins, and other constructs, all
mimicking the biological activity af native TGF-B,
including the ability to induce an anti-proliferative
e~fect on cultured mammalian epithelial cells.
-17-
2~393
More specifically, the DNA sequences
designed according to the above criteria and logic
were constructed using known techniques involving
assembly of oligonucleotides manufactured in a DNA
synthesizer. The sequences may be expressed using
well established recombinant DNA tachnologies in
various prokaryotic host cells, and the e~pressed
proteins may be cleaved from precursors, osidized,
and refolded in vitro for biological activity. This
approach has been successful in producing a number of
novel protein constructs not found in nature (as far
as applicants are aware) which hava TGF-B-like
activity, i.e., the ability to induce an
anti-proliferative effect on cultu~ed mammalian
epidermal cells.
The design and production of such
biosynthetic proteins, and other m3terial aspects
concerning the nature, utility, how to make, and how
to use the subject matter claimed herein will be
further understood from the following non-limiting
e~amples, which constitute the best method currently
known for practicing the various aspects of the
invention.
~,~
1. Consensus Sequence Design
Published amino acid sequences for TGF-B2,
TGF-B3, Vgl, and DPP-C were used to determine which
amino acids showed strong homology with the TGF-Bl
sequence. FIGURE 1 compares the amino acid sequences
~ 8-
~7~3~3
of these proteins with the sequence of TGF-
~denotes a match), and TABLE 1 summaries the e~tent of
homology.
TABLE 1
comDarisonno. of matches % homology
TGF-B2/TGF-B1 78/115 67.8
TGF-B3/TGF-B1 85/115 73.9
DPP/TGF-Bl 34/115 29.6
Vgl/TGF-Bl 35/115 29.6
-
In determining an appropriate consensus
amino acid sequence for TGF-B analogs, from which
encoding nucleic acid sequences can be determined,
the following points were considered: ~1) the known
amino acid sequence of natural source TGF-~l, 2, and
3 are ranked highest; (2) where an amino acid in the
sequence matches for all three proteins, it is used
in the synthetic gene sequence; (3) matching amino
acids in DPP and Vgl are used; (4) if Vgl or DPP
diverge, but either one were matched by TGF-Bl, 2, or
3, this matched amino acid was chosen; and (5) where
all sequences diverge, the amino acid residue alanine
was chosen, provided that the secondary structure is
maintained.
--19--
2Q~3~3
Using these criteria, the preferred sequence
i s :
3~ 40 50
CCVRQLYIDFKRDLGWKWVHEPKGYAANFCAGACPYLWSADTQHSRVLA
6~ 70 80 90
LYNTANPEASAAPCCVPQDLEPLTILYYVGRTPK~EQLSNMVVXSCKCS.
In addition, the first consensus sequence
was designed to preserve 8 of the disulfide
crosslinks and the apparent structural homology among
the related proteins, while the secoffd more highly
truncated consensus sequence was d~signed to preserve
6 disulfide bonds. That sequence is:
LYIDFKRDLGWKW~HEPKGYAANFCAGACPYLW.CADTQHSRVLALYN
100
TANPEASM PCCVPQDLEPLTILYYVGRTPKVE~SNMVVKSCKCS.
2. Gene Preparation and Expression
The synthetic genes designed using the
criteria set forth above are produced by assembly of
chemically synthesized oligonucleotides. 15-lOOmer
oligonucleotides are synthesized on a Biosearch DNA
~odel 8600 Synthesizer, and are p~rified by
pol~acrylamide gel electrophoresis ~PAGE) in
Tris-Borate-EDTA buffer (TBE). T~.e DNA is then
electroeluted from the gel. Overlapping oligomers
are phosphorylated by T4 polynucl~otide kinase, and
then ligated into larger blocks which may also be
-2~-
2Q7~393
purifed by PAGE. Alternatively, n~tural gene
sequences and cDNAs may be used for e~pression. The
two resulting genes are shown as tne latter portion
of the fusion sequences in FIGUREs 2A and 2B. The
sequence shown in 2A is a truncated form of 2B; five
amino acids at the N-terminus have been eliminated.
To enable the expression ~f the synthetic
gene shown in FIGURE 2A in an E. Ç:~li host, the qene
is modified by cassette mutagenesis, The N-terminus
is replaced up to the ClaI site wi.h a hinge region
that provides for release of the TGF-B protein from
the leader, preceded by a BamHI sïte for attachment
to leader peptides. The modified ~ene is then
attached to an FB-dimer leader at :he BamHI site.
The complete fusion gene is shown in FIGURE 2A.
The fusion gene is then inserted as an EcoRI
to PstI f raqment into an e~pression vector based on
pBR~22 and containing a synthetic tryptophan (trp)
promoter/operator and a modified tIp-LE (MLE) leader
(which is similar to the one described by Huston et
al. in Proc. Natl. Acad. Sci. (USA) (1988)
85:5~79-5883, but having only a single Eco~I site at
the hinge of the MLE leader). The vector is opened
at the EcoRI and PSTI restriction iites, and a
F~-F~TGF-B gene fragment is then inserted
therebetween, where FB is f ragment B of
Staphylococcal Protein A. The resulting e~pression
vector includes the TGF-B gene to a ragment encoding
FB.
~21-
~Q~93
3. Production of Active Analogs
The protein constructs are expressed in ~
ÇQli host strain JM101 (e.g.) grown in minimal medium
(M9) after starvation for trp and induction by
indoacrylic acid (I~A). The cells are lysed and the
inclusion bodies collected by differential
centrifugation. The fusion proteins are purified
from the inclusion bodies by urea or quanidine
solubilization. The FB sequence is then chemically
cleaved from the TGF-B protein construct at the hinge
region of the fusion protein. The hinge region has
the sequence Asp-Pro-Asn-Gly which can be cleaved at
the Asp-Pro site with dilute acid, or at the Asn-Gly
site with hydrosylamine. The resulting cleavage
products are passed through a Sephacryl-200HR column
which separates most of the uncleaved fusion products
from the TGF-~ analogs.
Protein refolding is performed under the
conditions of 50 mM Tris-HCl, pH 8.0, 3 M guanidine
hydrochloride (GuHCl), 10 mM dithiothreitol ~DTT),
and 1-10 mM o~idized glutathione.
4. TGF-B Activity Assay
This assay is based on the ability of TGF-
~to inhibit DNA synthesis in the mink lung epithelial
cell line, ATCC no. CCL 64. A confluent culture o
CCL-64 maintained in Eagle's minimum essential medium
(EM~M) supplemented with 10% fetal bovine serum
(F~S), 200 units/ml penicillin, and 200 ~g/ml
streptomycin, is used to seed a 48-well cell culture
plate at a cell density of 200,000 cells per well.
--22--
2~393
When the culture becomes confluent, the media is
replaced with 0.5 ml of EMEM containing 1% FBS and
penicillin/streptomycin. The culture is incubated
for 2~ hours at 37C. The TGF-B test samples in EMEM
containing 0.5% FBS are then zdded to the wells, and
the cells are then incubated for another 18 hours.
After incubation, 1.0 ~Ci of 3H-thymidine in 10 ~1 is
added to each well. The cells are incubated for 4
hours at 37C. The media is then removed and the
cells washed once with ice-cold phosphate-buffer
saline. The DNA is precipitated by adding 0.5 ml of
10% TCA to each well and incubating at room
temperature for 15 min. The cells are then washed
three times with ice-cold distilled water and lysed
with 0.5 ml 0.4 M NaOH. The lysate from each well is
then transferred to a scintillation vial and the
radioactivity recorded using a scintillation counter
(Smith-Kline Beckman).
Each test sample is assay~d in triplicate.
A TGF-~ control is included in each assay. The
inhibition activity of each sample is espressed as
the 50% effective dose ~ED50), which is defined as
the amount of material in ng/ml required to induce
50% reduction in ma2imal incorporation o
3H-thymidine.
The invention may be embodied in other
specific forms without departing from the spirit or
essential characteristics thereof. The present
embodiments are therefore to be considered in all
respects as illustrative and not restrictive, the
scope of the invention being indicated by the
appended claims rather than by the oregoing
-23-
207~93
description, and all changes which come within the
meaning and range of eguivalency of the claims are
therefore intended to be embraced therein.
What is claimed is:
