Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 96/07728 ` 2 1 9 8 9 2 8 PCI'IUS95/11139
PRODUCTION AND SECRETION OF RECOMBINANT
FIBRINOGEN BY YEAST
GOVERNMENT RIGHTS
This work sulJpull~d in part by federal grant number HL37457. The
government has certain rights in this invention.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel expression system for
..,~inanL riL"ino~;~-" fibrinogen variants and subunits thereof, to yeasts
rullllr-cl with the ~A~ iO~ system, to the use of the e,.~ sio- system to
clone rib~ oy,~l~, fibrinogen variants and subunits thereof, and to the use of
.~.uu,bi.~a.~ fibrinogen, ri~ ûg~.. variants and subunits thereof as tools of
research or in medicine, e.g., in ~ nrStir assays or as therapeutics to treat
certain in~lir~tirnc
2. D~ lion of the Related Art
Fibrinogen is the soluble precursor of fibrin, which is the primary
~v..~ l of blood clots. The structure of fibrinogen has been extensively
~tudied. For a review of the structure, see M. Furlan, in Fibrinogen, Fibrin
W096/07721/ 2 1 9 8 9 2 8~ PCI~/US95/11139
Stabilization and Fibrinolysis, J. L. Francis ed., Chichester, Ellis Horwood, 1988,
pages 17-64; and R. F. Doolittle, 1984, Annu. Rev. Biochem., 53: 195.
Fibrinogen is composed of three different polypeptide chains (~ ci~nAt~d
Aa, B~ and ~), arranged as a dimer with each half-molecule cc.ntAinin~ a set of
each of the chains. The two half-molecules are linked together by three disulfide
bonds at the amino-terminal portions of the polypeptides. Two of the
symm~trirAl bonds are between adjacent r chains and one is between A chains.
In addition, a complex set of inter- and intrachain disulfide bonds (there are 29
disulfide bonds with no free sulfhydryl groups) are involved in maintaining
proper structure.
Fibrinogen has an essentially linear shape consisting of two terminal
bilobate domains tethered to a smaller central domain. The amino-termini of
poly~ d~s , r~ and y are contained and joined together in the central domain.
Fibrinogen is sensitive to thrombin. Thrombin cleaves peptides from the
ends of the and r~ chains of ri~lil,og~.~. The released chains are referred to as
the Ll,~ oræl)lides A and B, le~e~Liv~ly The removal of the ri~l;lw~ JIides A
and B from the amino-terminus of the fibrinogen and ~ chains gives rise to an
entity denoted "fibrin monomer," the ~,ol~neous polymerization of which
leads to fibrin.
Each fibrin monomer possesses spècific polym~ri7Ation sites (or "knobs")
in the central domain that in fihrinng~n are shielded by ril,l;nu~l,lides A and B.
Release of fiblil-o~plides A and B exposes these knobs, which are positively
charged and can link with ~ r~ y lle~l,liv~ly charged "holes" that lie on
the terminal domains of n.;gllbo~ing m~ C
WO 96/07728 2 1 9 ~ 9 2 ~ PCr/US95/11139
Initially, fibrin monomers polymerize to form double-stranded
protofibrils, wherein the central domain of one molecule ~c~cori~tPc with a
terminal domain from each of two n~i~hhnrin~ mnlPc~ c in a half-molecule
staggered overlap. These protofibrils cross-link in the presence of factor )(IIIa
(fibrin stabilizing factor) and Ca2~ to form fibrin. The cross-linking is also
catalyzed by thrombin, which converts the inactive enzyme precursor factor ~(IIIto the active form factor )~lIa
Fibrin clots are intended to be temporary sealants and, consequently, are
displaced as a part of the normal wound-healing process. Plasmin degrades
fibrin clots and the conversion of plasminogen to plasmin sets the pace of
~lics~ tion The most i~ ol ~dl~t plasminogen conversion process involves
tissue pla~ .og~l~ activator (t-PA), which is released from damaged endothelial
cells. t-PA on its own is not very effective in activating p!~...inn~,t.., but the
presence of fibrin and various fibrin breakdown products increase the activationprocess L-~ .cly,
In addition to its role in clot formation, fibrinogen is also involved in
platelet aggregation. High-affinity "platelet recognition sites" have been
localized to the hydrophilic carboxy terminal Ff~nt~ r~reptide at the opposite
ends of both y chains (residues 397~11). It appears that this segment forms a salt-
bridged y loop that fits the platelet ~;1 ;n~ receptor. After fibrinogen is bound
at either ~ terminal end by a platelet receptor, the molecule has sufficient "reach"
to form a bridge on its free end to a ~il,lillog~l~ receptor on an adjacent platelet.
Subse~lu~ ly, the receptors on both platelets migrate towards one another,
thereby l~il~l.illg the bridge. Other platelets are similarly engaged, thereby
leading to the formation of a lattice.
WO 961U7728 2 1 9 ~ 9 ~ ~ PCI~/US95/11139
Although the primary functions of fibrinogen are clot formation and
platelet ag~ gd~iu,~, fibrinogen also interacts with a wide variety of other
proteins and cells. For example, when provoked or damaged, endothelial cells
that line the vascular system also bind fibrinogen. The principal binding sites on
fibrinogen for endothelial cells appear to be the same as those involved in the
binding to platelets. See D. A. Cheresch et al., 1989, Cell, 58: 945. Moreover,
fibrinogen is unique among the plasma proteins in being able to "clump" certain
strains of Staphylococcus aureus. The principal "clumping" sites on fibrinogen
also appear to be the same as those involved in the binding to platelets. See J.Hawiger et al., 1982, Biochem., 21:1407.
The principal--and perhaps only--site of fibrinogen biosynthesis in
mammals is the liver. Hepatocytes are the principal site of synthesis and each of
the ~UlllpUllt~ chdins of fiL~lillo~ll is encoded by a separate gene. These genes
are expressed, the chains associate, the appropriate disulfide bonds are formed,and hexamers are released into the circulation and transported to the
endoplasmic reticulum, where they are glycosylated, phosphorylated, and
sulfated.
Hundreds of naturally occurring fibrinogen variants have already been
identified because of their clinical consequences or during routine screening.
These variants have ~rllli~ul~d much to the und~l~ldl dillg of ~ og~ fibrin
biO~llrllli~lly, in many instances providing important insights into structure-
function r~'~ti-7nchir~c As valuable as naturally occurring variants have been,
they will soon be overshadowed by site-directed ~ ;c r~yr-;lll~lll~ with
Ir. .~ r;l,. ;n~-~r .. ~ expressed in Ir~ulllbil~dl~ systems.
WO 9610M28 2 1 9 8 9 2 8 PCT/US95/11139
For example, in order to study the role in calcium binding of disulfide
bond ~Cys326-yCys339, one laboratory has already used site-directed mllt~gPnPCicto by.,llæsi~e a-polypeptide lacking Cys326 and Cys339 by expressing DNA
encoding the modified polypeptide in Escherichia coli. See M. G. Bolyard et al.,1990, Biochem. Biophys. Res. Comm., 174: 853.
InL~lPslil~gly, the same yCys326-~Cys339 bond has also been implicated in
clottability. R. Procyk et al., 1990, Biochem., 29: 1501-1507, showed that this bond
is one of the bonds that are cleaved during a mild reduction of fibrinogen with a
low concentration of diLlliu~ ul in the absence of calcium. A consequence of
the limited reduction is a loss of clottability, which was later determined to result
apparently from perturbation of carboxy-terminal polymerization sites on
fibrinogen. This perturbation of the carboxy-terminal poly...P~ i..,, sites was,in turn, appa~ lly a consequence of ~Cys326-~Cys339 bond cleavage. See R.
Procyk et al., 1992, Biochem., 31: 2273. Copending U.S. Patent Application Serial
No. 07/946,826, the entire contents of which are ill~ul~ulaL~d herein by reference,
teaches that ribl;l~og~l~ reduced in this manner has substantial biochemical andimmlln~ gical equivalency to fibrin and fibrin monomer and, therefore, is
useful as a sub~lilul~ for fibrin or fibrin monomer in assays requiring these
species. Such assays include, for example, conventional assays for the
quantitative det.ormin~ti~n of (i) fibrin monomers, (ii) plasmin activator
inhibitor activity, (iii) tissue plasminogen activator activity, and (iv)
immllnr~ ~CCayS.
It is possible to use site-directed mllt~Pnf~ciC to construct a DNA sequence
encoding such reduced ri~ o~ or some other fibrinogen variant and then
construct an ~ Pssiur, vector ~ such DNA, transform a suitable host
with such e~ ssiu,~ vector and then induce the host to express the DNA.
WO 96/07728 2 1 9 8 ~ 2 ~ PCI~/US95/11139
Our lab~lalulr and others have described systems in which transfected
animal cells produce recombinant r~ Og~.l. See S. N. Roy et al., 1991, 1. Biol.
Chem., 266: 4758 (expression in COS-l and Hep G2 cells); R. Hartwig et al., 1991, J.
Biol. Chem., 266: 6578 (expression in COS-1 cells); and D. H. Farrell et al., 1991,
Biochem., 30: 9414 (expression in baby hamster kidney (BHK) cells). These
systems, however, only produce small amounts of fibrinogen and they are
difficult to scale-up.
Recombinant fibrinogen will undoubtedly be useful as a research tool, but,
in addition, will also be useful in medicine, for example, in the preparation of"fibrin glue" for wound healing or, in some cases, for infusion into
l~y~orib~ o~ ic patients.
SUMMARY OF THE INVENTION
The principal object of the present invention was to provide a system for
expressing recombinant fibrinogen, recombinant fibrinogen variants and
~u~bi~a~l Lb.;l~oy~ subunits in relatively large amounts.
It was another object of the present invention to provide an expression
system for recombinant fibrinogen, recombinant fibrinogen variants and
~u~bi~a~l fibrinogen subunits, which would be easy to scale-up.
It was another object of the present invention to provide for re~omhin~n~
fibrinogen, .~.~...bi-~an~ fibrinogen variants and recombinant fibrinogen
subunits and the use of such ~ proteins in research and in medicine
wo 96/07728 2 19 8 9 ~ ~ rcr/usss/lll3s
~ 7
as ~ e--l;rc and in diagnostic assays.
These and other objects were met with the present invention, which
relates generally to an e~ iul~ vector for yeast comprising at least one cDNA
encoding a polypeptide chain of fibrinogen or a variant thereof. One
embodiment includes an expression vector for yeast containing a cDNA
encoding the A chain of fibrinogen or a variant thereof, a cDNA encoding the
B~ chain of fibrinogen or a variant thereof, and a cDNA encoding the ~ chain of
LL,.;.~o~ or a variant thereof.
A second embodiment of the present invention relates to yeast
r~""r~l with such a vector.
A third embodiment of the present invention relates to a method of
ssil~g ril,iino~;el- or a variant or subunit thereof in yeast, rr~mrricin~ the
steps of:
(a) constructing or obtaining an expression vector for yeast
at least one cDNA encoding a polypeptide chain or fiblil~o~èll or a
variant thereof;
(b) ~ cr.. ;.. ~ yêast with said exprêssion vector and selecting
for stable ~ c~ c,
(C) ~ inl~ the transformants in a culture medium under
r<-n~ nc wherein ril,lil.O~I or a variant or a subunit thereof is secreted into
said culture medium; and
WO 96/07728 2 1 ~ ~ ~ 2 8 PCI/US95/11139
(d) l~UVl~ g ri~.;.,OE;~., or the variant or subunit thereof from
the culture medium.
A fourth ~mho~lim~nt of the present invention relates to recombinant
fibrinogen or a variant or subunit thereof produced by the inventive process.
The inventive yeast system surprisingly produces at least 10 times mûre
recombinant fibrinogen than has been possible with other expression systems.
Also, the inventive yeast system is easy to adapt to produce large quantities ofrecombinant fibrinogen. Moreover, with the inventive yeast system,
bi~a~ll ril,l;l~O~II is the principal secretion protein and, thus, is easily
purified from the culture medium.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a map of plasmid pYES2.
Figure 2 shows SDS-PAGE and Western blot analyses of human plasma
and yeast l~....h;..~QI riL,lillo~;~ll.
Figure 3 shows SDS-PAGE and Western blot analyses of thrombin-induced
clotting and cross-linking of human plasma and yeast i~-ull~bil~dnl r;L,I;I,o~
DETAILED DESCRIPI'ION OF THE INVENTION
The present invention provides for recombinant human fibrinogen or
W096/07728 2 1 9 8 ~ ~ ~ PCIIUS95/11139
variants or subunits thereof, which then can be used in research or in medicine
as ~ a~ulics or as a reagent for ~liagnnsti~ assays. By ~ril,.;..ot;l~ variants" or
simply "variants" are meant polypeptides having essentially the amino acid
sequence of human fiblillo~rll, but wherein one or more insertions, deletions,
additions and/or substitutions int~ntinn~lly have been made by conventional
methods. By "fibrinogen subunits" or simply "subunits" are meant isolated
polypeptides having the amino acid sequence of the individual ,13 or ~ chains of
human fibrinogen or a fibrinogen variant or any combination of such chains
short of the complete molecule rnnt~inin~ two of each of the chains. Various
~u~ alio~ s of the chains are useful as research tools to study the b~laay~ ais
and assembly of the functional protein.
cDNAs for the , ~ and y chains of human fibrinogen have been isolated
and (~h~r~t~t~ri7~1 See, ~ta~e~livrly, M. W. Rixon et al., 1983, Biochem., 22: 3237;
D. W. Chung et al., 1983, Biochem., 22: 3244; and D. W. Chung et al., 1983,
Biochem., 22: 3250. However, due to code ~IPg~nf.r~y, there can be considerable
variation in nucleotide S~qllPn~.oc encoding the same amino acid sequence. For
the purposes of the present invention, such ~ r~-1lr variants" are also useful
and are , 1 ~ when reference is made to "cDNA encoding a polypeptide
chain of ri~iinogell". The term "degel~rlal~ variants", as used herein, refers to
any DNA sequence, which, owing to the degeneracy of the genetic code, encodes
the same amino acid sequence as another DNA sequence.
The present invention also provides ~ ssi~l~ vectors for producing
useful quantities of purified r;l . ;I~Ogrll or variants or subuluts thereof in yeast.
The term "yeast", as used herein, is intended to ~ ....r~cc any yeast strain,
particularly strains of S~:l S~i. JC~ cerevisiae or Sa~ pombe, as well
as strains of other genera, for example, Pichia or Kluyveromyces, which have
WO 96/1~7728 2 1 9 ~ 9 2 8 PCI/~S95/11139
also been employed as ~lurlu~iull strains for l~-ulllbilral~ proteins.
In general, the vectors can comprise at least one cDNA encoding a
polypeptide chain (either a, ~ and/or ~) of fibrinogen or a variant thereof
operably linked to regulatory elements derived from yeast. Following
transformation of yeast cell lines with such vectors, the vectors can be induced to
express the encoded polypeptide chain or, where the vector comprises all three
chains, appropriately assembled fibrinogen or a variant thereof. Vectors for usein yeast are well known to those of ordinary skill in the art. Such vectors include
so-called "shuttle vectors," which replicate in both Escherichia coli and yeast A
general description of such vectors is given in J. D. Watson et al., RecombinantDNA, 2nd ed., New York, W. H. Freeman and Company, 1992, pages 235-253. A
more detailed description of such vectors, as well as of basic techniques of yeast
genetics, including preparation of yeast media, strain storage and revival, strain
growth and manipulation, mutagenesis, high-efficiency transformations,
selectable markers, expression cassettes, replicators, promoters, leaders,
Ir~ signal sequences for secretion, etc., is given in F. M. Ausubel et al.
(eds.)~ ', J~ cerevisiae." In F. M. Ausubel et al. (eds.), Short Protocols
in Molecular Biology, 2nd ed., New York, John Wiley & Sons, 1992, pages 13-1 to
13~9, the entire contents of which are hereby in.,J.yula~d by reference.
In general, yeast can be grown in either liquid media or on the surface of
(or embedded in) solid agar plates, but preference is given to liquid media. Yeast
are best grown on liquid media ~ont~ining dextrose (glucose), nitrogen,
phr~a~hulus, trace metals, and protein and yeast-cell-extract ~Iyrll~Jl~i,a~:s, which
provide amino acids, ....~I.oo~ Ul~UlD, vitamins, and other lll~abOli~s that
the cells would normally ~yl~ Si ~ de novo. Instead of dextrose, the yeast can
be grown on a variety of other carbon sources, for example, galactose, maltose,
WO 96/U7728 ~ 2 1 9 8 9 2 8 PCI/US95/11139
11
fructose, and raffinose. In a particularly preferred embodiment, which will be
described in more detail later, we have placed ~lal~S~ iull under the control of
the Gal-l promoter element, which is induced with galactose. The media should
be sterilized, for example, by autoclaving for 15 minutes at 15 Ib/in2, although
such times should be increased when large amounts of media are being prepared.
When cultured on such media, yeast cells divide approximately every 90
minutes.
Yeast vectors can be grouped into five general classes, based on their mode
of replication in yeast: YIp (yeast int~gr~tin~ plasmids), YRp (yeast replicating
plasmids), YCp (yeast centromeric plasmids), YEp (yeast episomal plasmids), and
YLp (yeast linear plasmids). With the exception of the YLp, all are shuttle
vectors. Ylp plasmids contain selectable yeast genes, but lack sequences that
allow autonomous replication of the plasmid in yeast. Instead, tr~ncfcrm~tinn
of yeast occurs by i~ gldliul~ of the YIp plasmid into the yeast genome. YRp
plasmids contain sequences from the yeast genome which confer the ability to
replicate dl~ ly YRp plasmids have high frequencies of tr~ncform~tinn
(103 to 104 tr~ncfnrm~ntc/~lg DNA), but transformants are ~c.. ~l;.. l~ unstable
during mitosis and meiosis. YCp plasmids contain DNA segments from yeast
IIUll.~ and this greatly increases stability during mitosis and meiosis. YLp
plasmids contain certain G-rich repeated sequences at their termini which
function as telomeres and allow the plasmid to replicate as a linear molecule.
However, for the purposes of the present invention, YEp plasmids are preferred.
These plasmids contain sequences from a naturally occurring yeast plasmid
called the "211m circle." These 211m sequences allow extrachromccnm~l
replication and confer high transformation frequencies (~104 to 105
tr Incform~ntc/llg DNA). These plasmids are relatively stable during mitosis and
meiosis and, ~ y, are commonly used for high-level gene ~AI~ sioll in
wo 96/07728 2 1 9 8 9 2 ~ PCI~/US95/11139
12
yeast.
In general, the heterologous structural sequence is assembled in
appropriate reading frame with trAncl~tinn initiation and t~rmin~tinn sequences,selectable markers and, preferably, a leader sequence capable of directing secretion
of translated protein into the extracellular medium. The selectable markers in
common use are wild-type genes such as URA3, LEU2, HI53 and TRP1 and,
preferably, use is made of all of them. These genes complement a particular
metabolic defect (nutritional auxotrophy) in the yeast host and, consequently,
successful tr~ncfnrm~ntc can be identified by their growth on selective media. In
the preferred embodiment, which is described in more detail below, use is made
of the MFal leader sequence, although other sequences will be similarly useful.
When the MF1 leader sequence is employed, the heterologous protein is
cleaved off by the yeast KEX protein. It may be helpful, in these cases, as
suggested by W. Fiers et al., "Secretion and Surface Expression in
Mi~luc~l~;al~ s of Heterologous Proteins Important for Medical Research and
Clinical Applications", in Harnessing Biotechnology for the 21st Century, M. R.
Ladisch et al. eds., American Chemical Society, 1992, pages 23-25, to place between
the pro-sequence and the heterologous gene sequence one or two Glu-Ala
dipeptides, which facilitate the cleavage of the pro-sequence.
All of the vectors described âbove carry strong promoters utilized by RNA
poly~ ldat II. The promoters can be either inducible (e.g., Gal-1, Gal-10, PH05) or
~ul~iluliv~ (e.g., ADHI, PGK or GPD). Transcription from these ~ ul~l~
depends on activator proteins bound to sites upstream of the llal S~ liùl start
site (in yeast, termed upstream activation sites or UAS). Preference is given tothe Gal ~JIUIII~ , particularly the Gal-1 (~ tnkin~ce) promoter. The Gal
~IUIIIUt~.s are regulated by the activator Gal~, which binds to UAS upstream of
WO 96107728 2 1 9 8 9 2 8 PCI/US95/11139
13
the lldl~s.l;l"iun start, and the negative regulator Gal-80, which su~p~sses
activation by the activator. Tlalls.l;~l;ull from the Gal-1 promoter, for example,
is massively induced when cells are grown in a medium that contains galactose
as the sole source of carbon; under these ~n~litions Gal-80 dissociates from Gal-4
and Gal-4 is bound to the Gal-1 UAS. Pl~r~ is, therefore, given to the use of
the Gal-1 promoter in conjunction with mediâ containing galactose as the sole
carbon source.
Suitable yeast transformation protocols are well known to those skilled in
the art; a yeast trAnsf~-rm~tinn kit may be purchased from BIO 101, Inc. (La Jolla,
CA) and the kit protocol was followed with slight mo~ifirAh~-ns in the examples
given below. D~ ;ol~ of the Gal-1 promoter occurs upon exhaustion of
medium galactose. Crude yeast ~ c are then harvested by filtration and
held at about 4C prior to further purifi~Atinn.
In a preferred embodiment, we have achieved superior yields utilizing the
e~ D~;ul~ vector pYES2, the details of the construction of which are described in
the examples below. Vector pYES2 was constructed with all three fibrinogen
cDNAs in tandem, each under the control of the Gal-1-promoter element fused
with the MFal prepro secretion signal cascade. As will be discussed in greater
detail below, I~ulllbil~ànl fibrinogen secreted from yeast is similar to plasma
r;~ u~ when analyzed on polyacrylamide gels and, moreover, like naturally
occurring plasma fibrinogen, recombinant fibrinogen secreted from yeast is
capable of forming a thrombin-induced clot.
In the yeast system, fihrinog~n is the principal secretion protein in the
culture medium and, thus, is easily purified by ~:ullv~l~liul~al purifying methods
for proteins, for example, by ~ulllb;llaliul~s of salhing out, ultrsfiltrstil-n, dialysis,
WO 96/07728 2 1 9 8 9 ~ 8 PCI/US95/11139
14
ion exchange chromatography, gel filtration, electrophoresis, affinity
~hrc)m~ , etc.
It is also possible to achieve appropriately assembled fil,lil.o~;~l, by co-
l,.".~r~ the yeast cells with each Lblino~ cDNA in a separate expression
vector, rather than in tandem in a single vector. If use is made of this
embodiment, then each vector should contain a different selection vector in
order to facilitate selection of stable transformants containing all three cDNAs.
For the pl~,al~Livl~ of ril,lino~,. variants or variant subunits, advantage
is taken of ~vl~v~l~Livl~al mlltAgPnPcic techniques to alter the DNA sequence ofthe native cDNA(s). For example, oligonucleotide-directed site-specific
mllt~gPnPcic procedures can be employed to provide an altered "gene" having
particular codons altered according to the substitution, deletion or insertion
required. Where the fibrinogen variant is intended for research, cassette
mutagenesis with degenerate /71igonllrlPctides can be used to create a large
collection of random mutations in a single experiment. Details of these
techniques are well known to those of ordinary skill in the art and are not
repeated here. However, references are made to J. D. Watson et al., supra, pages191-211; F. M. Ausubel et al., supra, pages 8-1 to 8-25; and J. Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Plainview, New York, Cold
Spring Harbor Laboratory Press, 1989, pages 15-1 to 15-113, the entire contents of
each of which are hereby ill~Vl~Ul~ li by reference.
Thus, as mPnticnPcl previously, the yCys326-~Cys339 bond has been
r 1 ~ I in clottability. For a variety of diagnostic assays, it is desirable to use a
reagent that will not clot during the assay, but which is, nevertheless,
bio-~hPmicAlly and immunologically indistinguishable from fibrin or fibrin
W096/07728 2 1 9 ~ 9 2 ~ PCr/US95/11139
monomer. According to U.S. Patent Application Serial No. 07/946,826, supra, a
reagent suitable for this purpose is achieved by subjecting r;l., ;r,r~,~ . to a limited
reduction, which, according to R. Procyk et al. 1992, supra, resul~n the cleavage
of certain Cys-Cys bonds. It may be possible to prepare such ~reduced r;l..; ,~r.~
by recombinant methods. For example, it is possible, using oligonucleotide-
directed site-specific mlltA~rnrcic procedures to modify the cDNAs encoding the
various chains by replacing the codons for cysteines at the positions that are
broken during the limited reduction with codons for other amino acids, for
example, glycine or, perhaps, methionine, which, like cysteine is hydrophobic
and contains sulfur in the side-chain.
In addition, l~.u-l-blndl~l ri~ og~l~ and variants thereof will have use in
medicine as lI~lalJ~Ulic agents. Fibrinogen is one of the "acute phase proteins,"
the biosynthesis of which is markedly increased in times of trauma and insult.
See, R. F. Doolittle: "The Molecular Biology of Fibrin". In G.
Stamdl~,yanl,~oulos et al. (eds.), The Molecular Basis of Blood Diseases, 2nd ed.,
Philadelphia, W. B. Saunders Company, 1994, pag 712. Fibrinogen re~ rmr-nt
may be required in patients with liver failure or fliccr-rnin~tr-cl illLlavas~ular
coagulation (DIC) or in patients with r/~n~nit~l rib.i~o~ deficiency. Currently,ad.. i..i~l.ation of .l~ap.~ dl~d antihemophilic factor (AHF), which is also
called factor VIII, is the preferred treatment for fibrinogen rr-p1ar~mr-nt Each bag
of .I ~ .ildl~d AHF contains about 250 mg of fibrinogen. A plasma level of
at least 50 mg/dl of ri~ is required for adequate hPrn~stacic with surgery
or trauma. See, M. S. Kennedy: "Transfusion Therapy". In D. ~rmr-nin~-
Pittiglio et al. (eds.), Modern Blood Banking and Transfusion Practices, 2nd ed.,
Philadelphia, F. A. Davis Company, 1989, page 268. Administration of
recombinant ril,lino~ or variants thereof to such patients will be by the
il~lla~nous route and the typical daily dosage will be such as is necessary to
wo 96/07728 219 ~ 9 ~ ~ PCINS95/1113g
maintain the ~ lplasma level of 50 mg/dl of fibrinogen. For such
purposes, le..... bi. anl riL,I;I-Ogèl~ or variants thereof can be added to whole
blood or blood products, e.g., plasma, ~ è~ lè~ etc., or to the .ul~ve- ~ al
ph~rm~uti~Al vehicles.
The invention will now be described in greater detail by reference to the
following non-limiting examples:
The è,~.e~iOI- vector (pYES2) and the yeast strain (INSVC1, MAT~
his3-A1 leu2 trpl-289 ura3-52) were obtained from Il~vi~l~Jgell, Inc. Medium to
grow the yeast in selective conditions was purchased from BiolO1, Inc. Galactose,
raffinose, tunicamycin were obtained from Sigma. Antibodies to human
riblillogèl- was from Dako Cul~Jlatiul~, restriction enzymes, Klenow fragment,
calf intestinal phl~ h~l~ce (CIP) were from Boehringer, M~nnh~im, endoglycosidase-
H from Genzyme, T4 DNA ligase from New England Biolab, L-[3sS] methionine
(1100 Ci/mmol) was from New England Nuclear Corporation-Du Pont. Other
reagents used have been described previously (see S.N. Roy et al., J. Biol. Chem.,
267: 23151 (1992); S.N. Roy et al., J. Biol. Chem., 269: 691 (1994); and S.N. Roy et
al., J. Biol. Chem., 266: 4758 (1991)).
Example 1~ of E~,.. Vector
Expression vectors l ~ fibrinogen cDNAs for single chains, 2 in
~.""l,;n~ and all 3 chains are inserted to the multiple cloning sites at the 3'
end of the Gal-1 promoter fused with the MF1 prepro secretion signal (SS)
cascade in pYES2 plasmid, which is depicted in Figure 1. To prepare pYES2A,
pYES2BB and pYES2~, full-length cDNAs were released by appropriate restriction
enzymes se~ ély from pleviOu~l~y described constructs (see S.N. Roy et al., J.
-
wo 96107728 2 1 9 ~ 9 2 ~ PCr/usgs/1113s
Biol. Chem., 269: 691(1994); and S.N. Roy et al., l. Biol. Chem., 266: 4758 (1991)).
Other Co~ u.l~, pYES2AaBB, pYES2A~, pYES2BB~ and pYES2AaBB y were
made by ligating ril,l;.,O~ chain cDNAs in tandem, each under the control of
the Gal-l-SS promoter. The procedures for elution of DNA fragments from
agarose gel, d~,ho~h~.~lation of plasmids by CIP, the fill-in reaction by Klenowfragment and ligation were performed as described elsewhere (see S.N. Roy et al.,
1. Biol. Chem., 267: 23151 (1992); S.N. Roy et al., l. Biol. Chem., 269: 691 (1994); and
S.N. Roy et al., 1. Biol. Chem., 266: 4758 (1991)).
Example 2: T ~ of Yeast
Transformation of S. cerevisiae (INVSCl) with pYES2 vectors containing
fibrinogen cDNAs were performed by the alkali-cation method and the cells were
plated on SC-ura plates (see L.D. Schultz et al., Gene, 54: 113 (1987)). Single
colonies from each plate were grown in SC-ura medium . . "~ 4% raffinose
at 30~C with vigorous shaking overnight and kept as stock culture. Transformed
yeast cells with the above described constructs were named INVSClA,
INVSClBB and INVSC1~, INVSClABB, INVSClA~, INVSClBB~ and
INVSClABBy.
S. cerevisiae cells (INVSCl) stably transformed with vector pYES2ABB~
were prepared in the foregoing manner and deposited with the American Type
Culture C~ ction, Rockville, MD, on August 12,1994, under accession number
ATCC 74296. The deposit was made pursuant to the Budapest Treaty.
Example3: E..~ and Treatmentwith T~
Stock culture was grown in 5 ml of SC-ura medium overnight at a density
WO 96/07728 2 1 9 ~ 9 2 ~ PCI/US95/11139
18
of lxlO8/ml. The cells were harvested at 500 xg, ~ 1 in SC-ura medium
in;,~ 2% galactose and grown for an ~r~itir)n~l 16 hr for induction of
r;l..;..O~2,.,,~ chain synthesis. The cells were harvested at 500 xg, resuspended in
SC-ura-met medium ~ ini~ 50 IlCi/ml of L-[3sS]methionine and incubated
for 1 hr at 30C. In some cases, the cells were preincubated with medium
cc-nt~ining 10 llg/ml of tunicamycin for 1 hr and L-[3sS]methionine as usual.
When dr~t~rminin~ intracellular fibrinogen, the cells were harvested, washed
with phosphate buffered saline (PBS), Iysed with 0.5 ml of IP buffer (50 mM
Tris.HCI, pH 7.4, 1% Triton X-100, 0.2% SDS, 150 mM NaCI, 5 mM EDTA, 10
U/ml Trasylol, 1 mM PMSF, 0.1 mM TPCK, 1 ~g/ml Soyabean-trypsin inhibitor)
and 200 mg (0.5 mm dia) of acid-washed glass beads/108 cells by vortexing twice
for 45 sec (see J.R. Flal~uSOrr et al., Methods in Enzymology, 194: 662 (1991)). The
cell Iysate was diluted to 1 ml with water and centrifuged at 15000 xg for 15 min at
4C. Fibrinogen was isolated by imll-u.w~ d~illg the cytosol with a human
polyclonal Ll,~ og~.~ antibody as described elsewhere (see S.N. Roy et al., 1. Biol.
Chem., 267: 23151 (1992); S.N. Roy et al., l. Biol. Chem., 269: 691 (1994); and S.N.
Roy et al., 1. Biol. Chem., 266: 4758 (1991)).
Example 4: Secretion of Fibrinogen
Yeast cells 1-.. -- r .. ~cl with pYES2AB~y and grown from single colony
were inr~~ tr~rl in 50 ml of SC-ura medium .~ .i..g 4% raffinose and grown
overnight at 30C. The cells were then induced with 2% galactose and incubated
for an ~r~l~itir n /1 16 hr. The culture medium was centrifuged at room
l~ul~la~ul~: for 5 min at 500 xg. The pH of the medium was adjusted to 7.0 with
1 M Tris-HCI buffer pH 8.0 and a cocktail of protease inhibitors (10 U/ml Trasylol,
1 mM PMSF, 0.1 mM TPCK, 1 llg/ml Soyabean-trypsin inhibitor, 1 mg/ml
papstatin) was added. ri~l;nog~l~ was isolated from the medium by absorption
W096/07728 2 1 9 ~ 9 2 8 PCI/US95/11139
19
on a p~vL~ e sulfatc S~,ha.vs~ 6B column (10 ml) calibrated with buffer A (50
mM Tris.HCl, pH 7.4, 5 mM EDTA). The column was washed with buffer A
g 0.8 M NaCl and bound r;l,.;..~g.~.~ was eluted with 0.1 M N~ . c~t~t~,
pH 4.5. The pH was adjusted to 7.0 with 1 M Tris.HCl, pH 8.0 (see C.E. Dempfle et
al., Thromb. Res., 46: 19 (1987)).
Example 5: Q ~ A of Secreted Fibrinogen
The amount of fibrinogen secreted in the medium was measured by
competition ELISA using two different fibrinogen chain-specific monoclonal
antibodies [1-8C6(anti BB 1-21) and Fd4-7B3 (anti ri~li.,Oy,~l~ fragment D)]. Details
of the assay using Fd4-7B3 were reported previously (see S.N. Roy et al., J. Biol.
Chem., 266: 4758 (1991)). A new assay has recently been developed with antibody
1-8C6 whose specificity has been described (see B. Kudryk et al., Molec. Immun.,20:1191 (1983)). A horseradish peroxidase-labeled form of antibody 1-8C6 is
utilized in this assay and fibrinogen concentrations as low as 0.05 llg/ml can
readily be measured.
xample 6: Ct ~ of the Properties of Human Plasma and Yeast
Recombinant Fibrinogen
Secreted recombinant fibrinogen was treated with thrombin (6.8 NIH
U/ml) with or without factor XIII (1.0 U/ml) to ~ t~rmin~ its ability to form a
thrombin-induced clot and to crosslink. The fibrin complexes were separated by
SDS-PAGE and detected by staining with rnnm~cci.o blue and by Western
using chain-specific ~ntiho~ oc 1C2-2 (anti fibrinogen A~/fibrin c~)
(R. Procyk et al., Thromb. Res., 71:127 (1993)); Ea3 (anti ri~ 0~ BB/fibrin B) (B.
Kudryk et al., "M~ rl-m~l Antibodies as Probes for Fibrin(ogen) Proteolysis."
WO 96107728 2 1 9 8 9 ~ ~ PCr/US95/11139
In~ ' Antibodies in I . '~u~ (J.F. Chatal, ed.), CRC Press,
Boca Raton, FL, pp. 365-398); T2G1 (anti fibrin B) (B. Kudryk et al., Molec.
Immun., 21: 89 (1984)); and 4-2 (anti ri~ og~l~ r/fibrin ~-dimer) (R. Procyk et al.,
Blood, 77:1469 (1991)).
In order to assess the similarity of yeast recombinant fibrinogen to human
plasma fibrinogen, comparisons of the two products were made on
polyacrylamide gels.
Figure 2 shows the immunoreactivity of recombinant fibrinogen with
chain specific antibodies. Recombinant fibrinogen was separated on 4-10%
g-radient SDS-PAGE under reducing or non-reducing conditions and analyzed by
Western ill.ll.ul~lc,~s using different chain specific antibodies.
Panel A: Reduced gel stained with coomassie blue.
Panel B: lI1~ UI~UIJIV~ of reduced samples reacted with MAb to A/~ chain.
Panel C: Same as B reacted with MAb to BB/B chain.
Panel D: Same as B reacted with MAb to r chain.
Panel E: Tmmlln:' !ol of non-reduced samples reacted with MAb to -y chain.
Lane 1, molecular size markers; lane 2, plasma Fbg; Lane 3, ie-vll~ dl~l yeast
Fhg.
The data shows that recombinant fibrinogen secreted by trAncform~d yeast
cells has similar electrophoretic and immunoreactive properties as plasma
ri~ o~
Figure 3 shows the clotting ~.vp~Llie~ of ~u--lbina-ll yeast fibrinogen.
Purified recombinant rib.;.~og~l~ secreted by yeast cells was inrllhAt~d with
