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had no appreciable effect on the Stypven-activated
HOOKWORM ANTICOAGULANT factor X clotting time; the anticoagulant was characterized as a protein with a molecular weight between
TECHNICAL FIELD OF THE INVENTION 20,000 and 50,000 daltons (Eiff, J. A., ParasitoL 52:
This invention relates to an anticoagulant isolated 5 833-843 (1966)). Other investigators, on the other hand,
from hookworms. demonstrated that extracts of the whole worms did, in
fact, prolong the Stypven time, arguing in favor of the
BACKGROUND OF THE INVENTION presence of an inhibitor of factor Xa (Spellman and
Hookworms are intestinal nematodes that infect over Nossel, cited above).
1 billion persons worldwide, with a higher prevalence 10 Blood coagulation, initiated by substances in injured
in children than in adults (briefly reviewed in CeciFs tissues, is propagated by an interlocking network of
Textbook of Medicine, 19th ed., W. B. Saunders Co., enzymatic activation, propagation, and control events,
1992, page 2010). These individuals suffer from intesti- the so-called coagulation cascade. These complex reac
nal hemorrhage as a direct consequence of blood loss tions ensure that blood coagulation happens quickly and
caused by the adult hookworms attached to the mucosa. 15 yet remains localized. Blood coagulation results in the
Hookworm disease is most common in tropical and less formation of a protein scaffolding, the fibrin clot, that
developed countries, where environmental and socio- controls bleeding and serves as a nidus for subsequent economic conditions including warm, moist soil, lack of cellular ingrowth and tissue repair. After several days,
public sewage disposal systems and the habit of walking the fibrin clot is lysed and replaced with a more perma
barefoot especially favor transmission. Although other 20 nent scaffolding of connective tissue matrix molecules,
routes of infection are known, such as lactogenic trans- Abnormalities that result in delay of clot formation or
fer of larvae to infants and use of soiled bedding and premature lysis of clots are associated with a bleeding
clothing (Hotez, P. J., Pediatr. Infect Dis. J., 8: 516-520 tendency.
(1989)), infection often occurs when exposed skin main- Coagulation and fibrinolysis involve many blood
tains contact for several minutes with soil contaminated 25 ... proteins (see> for examplej Tabie i55.i m Cecil)
with parasite eggs containing viable larvae. These pene- cited above> page im)> ^ the list ... ionger as
trate the skin and journey to the lungs to develop into blood coagulation mechanisms are studied in greater
adults that eventually make their way to the upper small detail Structural md f^^^ similarities can be em
mtestme, where they attach to the mucosa ployed to group the proteins. For example, one group
Hookworm disease is due pnmarly to gastromtestmal 30 ^ of serine t ^ hence members of
blood loss and attendant iron deficiency anemia. Adult „ • ° „,„^„„„ e„Z;u, „r „,u;„u :„„i„,i„,
j. ■ j the serine protease family ot proteins which mcludes
worms attached to the mucosa digest mgested blood as . . , ^ . . l i t J *t ■
well as cause focal bleedine Each hookworm can suck trypsm' ... elastase, plasmin and cathepsin
as much as 0.2 ml of blood per day (Spellman, G. G., £ ^ ^agulation cascade Factors II, VII, IX X,
and Nossel, H. L., Amer. J. Phys. 220: 922-927 (1971)). 35 £ ^ pTMtemuC 316 m ^ lerfe pr°teaSe famly
This dramatic blood loss can reduce peripheral hemo- These are modified by a vitamin K-dependent posttran
globin concentrations to as low as 3 g/100 ml. More slational carboxylation of glutamic acid residues which
commonly, however, blood loss is insidious, and results ^ Protems to bmf .call;lum <fd Phospholipids
in chronic iron-deficiency anemia. Thus, in its human !md &eTeby participate efficiently m blood coagulation,
host, the adult hookworm functions as a conduit that 40 Tissue plasminogen activator m the coagulation cascade
empties blood into the intestinal tract, producing blood K ^so a serme protease. Other proteins are Serine prote
loss on a global scale equivalent to the exsanguination of ase inhibitors and hence members of the "serpin" family
1.5 million people per day (Hotez, cited above). Nutri- of proteins, which includes antithrombin III, heparin
tional deficiencies secondary to coexisting conditions cofactor II, and plasminogen activator,
that result in low iron stores contribute to morbidity. 45 Blood coagulation can be initiated by exposure of
The remarkable ability of a single small parasite to blood to tissue factor, the so-called "extrinsic system", cause so much blood loss raises the question of an effec- or by activation of contact factors of plasma, the sotive anticoagulating mechanism. Loss of blood from the called "intrinsic system". Both of these initiation pathgastrointestinal tract would be facilitated if the ability of ways lead to a common pathway, which results in the blood to clot were impaired in persons infected with 50 elaboration of thrombin, the master coagulation enthis parasite. Early in this century, researchers observed zyme. Two major coagulation tests mentioned above that extracts of the dog hookworm contained a sub- differentiate these pathways. In the prothrombin time stance that delayed coagulation of human blood (Loeb, (herein denoted FT) test, tissue factor is added to L., and Fleisher, M. S., J. Infect Dis. 7:625-631 (1910)). plasma so that activation proceeds by the extrinsic pathSome fifty years later, it was subsequently noted that 55 way. In the partial thromoplastin time (herein denoted hookworm protein, when added to mammalian plasma, PTT) test, blood plasma is activated by the intrinsic markedly prolongs both the prothrombin and partial pathway. The pathways are related somewhat because thromboplastin times (Spellman and Nossel, cited deficiencies of Factor IX, an intrinsic factor, as well as above, and Carroll, S. M., et al., Thromb. Haemostas. 51: the factors that follow Factor IX in the intrinsic and 222-227 (1984)). 60 common pathways and Factor VII, an extrinsic factor
Although some of this effect has been attributed to a that activates LX and X, are all associated with a bleed
fibrinogenolytic and fibrinolytic protease that degrades ing tendency. In contrast, deficiency of Factor XII and
fibrinogen (Hotez, P. J., et al., J. Biol Chem. 260: prekallikrein, which activates XII, does not cause a
7343-7348 (1985)), the exact location in the clotting bleeding problem.
cascade at which the predominant anticoagulant effect 65 „,„„. . DV TMTM -nenrc^-mr^s is exerted has not been determined. One investigator SUMMARY OF THE INVENTION reported that extracts of hookworm cephalic glands, It is an object of the invention to provide a new antiwhile significantly prolonging the prothrombin time, coagulant.
DESCRIPTION OF THE FIGURERS
It is a further and more specific object of the inven- toma duodenale, Necator americanus, and, less com
tion to provide an anticoagulant that can be used as a monly, A. ceylonicum, as well as hookworms that infect
therapeutic agent for the treatment of numerous vascu- other animals such as Ancylostoma caninum, Bunos
lar disorders, as well as for the development for vac- tomum phlebotomum, Agriostomum vryburgi, B. trigono
cines for hookworm infection and strategies for lessen- 5 cephalum, Gaigeria pachyscelis. Other blood-sucking
ing the sequelae of chronic infection. nematodes such as Haemonchus species, e.g., H. contor
These and other objects are accomplished by the tus, are also encompassed by this invention. Ancylostoma
present invention which provides a soluble protein anti- caninum is preferred in one embodiment,
coagulant isolated and purified from Ancylostoma, In the practice of this invention, a soluble protein
particularly Ancylostoma caninum, hookworms. In 10 anticoagulant is isolated and purified from hookworms,
clotting assays, the protein prolongs the prothrombin By "purified" is meant essentially homogenous, yielding
time and partial thromboplastin time. It inhibits clotting one polypeptide band on electrophoresis in a system
factors Vila, the enzyme responsible for initiating the that separates proteins; purified anticoagulant is thus
human clotting cascade, and Xa, thus exhibiting com- substantially free of other hookworm constituents, in
mon pathway inhibitory activity as well. The protein 15 eluding associated proteins. Generally, the preparation
does not inhibit thrombin or clotting factors II and V. is carried out by homogenizing or lysing the nematodes
The protein has an apparent molecular weight of to obtain soluble extracts, and purifying the protein
about 6500 daltons. It contains the amino acid sequence from the extracts. Any type of protein purification
Tyr-Pro-Glu-Cys-Gly-Glu-Asn-Cys-Gly-Leu, thus ex- scheme familiar to the skilled artesan can be employed,
hibiting sequence homology with polypeptides belong- 20 such as, for example, affinity, ion-exchange, exclusion,
ing to the Kunitz-type family of serine protease inhibi- partition, liquid and/or gas-liquid chromatography;
tors. zone, paper, thin layer, cellulose acetate membrane,
The invention also provides DNA encoding the agar gel, starch gel, and/or acrylamide gel electropho
hookworm anticoagulant, biologically functional circu- resis; immunochemical methods; combinations of these
lar plasmid or viral DNA vectors comprising the DNA, 25 with each other and with other separation techniques
and procaryotic or eucaryotic host cells such as E. coli. such as dialysis; and the like.
transformed or transfected with the vectors in a manner In one embodiment, protein is obtained by separating
allowing the host cell to express the protein. proteins in a hookworm extract using a Sepharose ion
exchange column, followed by purification on an affin
30 ity column consisting of purified human factor Xa
FIG. 1 shows selective inhibition by hookworm ex- bound to agrose resin and gel filtration through a Se
tract of the chromogenic hydrolysis of substrate (40 ug pharose gel column. Experimental details are given
Chromozym X) by 0.01 units of purified factor Xa hereinafter.
(open bars). Equal amounts of hookworm protein do Hookworm anticoagulant protein so obtained pro
not inhibit the activity of purified thrombin versus its 35 longs the prothrombin time and partial thromboplastin
substrate (Chromozym TH, hatched bars). time in clotting assays, as well as the factor X (Stypven)
FIG. 2 shows partial purification of hookworm inhib- clotting time. It inhibits extrinsic factor Vila, the en
itor by Q Sepharose column chromatography. Hook- zyme responsible for initiating the human coagulation
worm extract from 100 adult worms was added to a cascade. In addition, it is also capable of binding to
1.7X9 cm Q Sepharose column using gravity flow. A 40 factor Xa in the common pathway of the coagulation
major protein peak (denoted—) was eluted .from the cascade. As such, it bears a striking similarity to the
column with a 2.0M NaCl gradient. Individual column mammalian Extrinsic Pathway Inhibitor (EPI), a major
fractions (approximately 1 ml) were collected and as- endogenous anticoagulant produced by human tissues
sayed for factor Xa inhibitory activity by chromogenic to regulate the coagulation cascade. Hookworm antico
assay (denoted—). 45 agulant does not inhibit the hydrolytic activity of puri
FIG. 3 shows the effect of Q Sepharose column frac- fied thrombin and does not inhibit clotting factors II
tions on prothrombin (open circles) and partial throm- and V.
boplastin times (closed circles; control clotting times: The hookworm anticoagulant of this invention exhibPT= 1.0 seconds, PTT=28.3 seconds). The peak of its a molecular weight of about 6500 daltons. It contains anticoagulant activity, i.e., prolongation of PT/PTT, 50 the amino acid sequence Tyr-Pro-Glu-Cys-Gly-Glucorresponds to the peak of inhibition observed with the Asn-Cys-Gly-Leu, thus exhibiting sequence homology chromogenic assay of factor Xa activity. with polypeptides belonging to the pancreatic trypsin FIG. 4 shows the effect of purified hookworm antico- or Kunitz-type family of serine protease inhibitors agulant on factors Vila (open bars) and Xa (hatched (named after the first inhibitor to be isolated in crystalbars ) using an in vitro chromogenic assay analogous to 55 line form, the first for which typical 1:1 enzyme-inhibithat described for FIG. 1 above. tor stoichiometry was determined, the first for which
reversibility was demonstrated, the first to be sequenced, and the first to have the three-dimensional structure determined; reviewed by Laskowski, M., and This invention is based upon the finding that a low 60 Kato, I., Ann. Rev. Biochem. 49: 593-626 (1980)). Memmolecular weight protein isolated and purified from bers of this polypeptide family have been found in many hookworms binds to and inhibits both extrinsic factor species including other mammals, snails, and sea anemoVlla, the enzyme responsible for initiation of the human nes, as well as in soybeans and snake venoms. The gene coagulation cascade, and factor Xa in the common coding for Kunitz type inhibitors is thus very old and pathway. 65 very widely distributed.
By "hookworm" is meant any nematode that sucks Also encompassed by this invention are synthetic
blood from the small intestine including, but not limited hookworm anticoagulants exhibiting activity and struc
to, the major hookworms that infect humans, Ancylos- ture similar to the isolated and purified protein. Since
DETAILED DESCRIPTION OF THE
the protein is small, it can be prepared from its constitu- tial, terminal or intermediate DNA sequences which ent amino acids by sequential formation of peptide facilitate construction of readily expressed vectors, bonds using any chemical means. Alternately, the amino Correspondingly, the present invention provides for acid sequence, can be used to prepare cloned comple- manufacture (and development by site specific mutamentary DNA sequences defining the hookworm anti- 5 genesis of cDNA and genomic DNA) of DNA sequencoagulant of this invention, which can then be used to ces coding for microbial expression of anticoagulant transform or transfect a host cell for protein expression analogues which differ from the forms specifically deusing standard means. Also encompassed by this inven- scribed herein in terms of identity or location of one or tion are DNA sequences homologous or closely related more amino acid residues (i.e., deletion analogues conto complementary DNA described herein, namely 10 taming less than all of the residues specified for anticoDNA sequences which hybridize, particularly under agulant, and/or substitution analogues wherein one or stringent conditions, to hookworm anticoagulant more residues are added to a terminal or medial portion cDNA, and RNA corresponding thereto. In addition to of the polypeptide), and which share the biological the anticoagulant-encoding sequences, DNA encom- properties of hookworm anticoagulant described passed by this invention may contain additional sequen- 15 herein.
ces, depending upon vector construction sequences, DNA (and RNA) sequences of this invention code
that facilitate expression of the gene. for all sequences useful in securing expression in pro
Because of the degeneracy of the genetic code, a caryotic or eucaryotic host cells of polypeptide prodvariety of codon change combinations can be selected ucts having at least a part of the primary structural to form DNA that encodes the anticoagulant protein of 20 conformation, and one or more of the biological properthis invention, so that any nucleotide deletion(s), addi- ties of hookworm anticoagulant which are compretion(s), or point mutation(s) that result in a DNA encod- hended by: (a) the DNA sequences encoding anticoaguing the protein are encompassed by this invention. Since lant protein as described herein, or complementary certain codons are more efficient for polypeptide ex- strands; (b) DNA sequences which hybridize (under pression in certain types of organisms, the selection of 25 hybridization conditions) to DNA sequences defined in gene alterations to yield DNA material that codes for (a) or fragments thereof; and (c) DNA sequences the protein of this invention are preferably those that which, but for the degeneracy of the genetic code, yield the most efficient expression in the type of organ- would hybridize to the DNA sequences defined in (a) ism which is to serve as the host of the recombinant and (b) above. Specifically comprehended are genomic vector. Altered codon selection may also depend upon 30 DNA sequences encoding allelic variant forms of antivector construction considerations. coagulants included therein, and sequences encoding
DNA starting material which is employed to form RNA, fragments thereof, and analogues wherein RNA
DNA coding for the hookworm anticoagulant of the or DNA sequences may incorporate codons facilitating
invention may be natural, recombinant or synthetic. transcription or RNA replication of messenger RNA in
Thus, DNA starting material isolated from tissue or 35 non-vertebrate hosts.
tissue culture, constructed from oligonucleotides using Isolation and purification of microbially expressed conventional methods, obtained commercially, or pre- proteins provided by the invention are by conventional pared by isolating RNA coding for anticoagulant pro- means including, for example, preparative chromatotein, and using this RNA to synthesize single-stranded graphic separations such as that illustrated in the ExamcDNA which is used as a template to synthesize the 40 pies, and immunological separations, including monocorresponding double stranded DNA can be employed clonal and/or polyclonal antibody preparations, to prepare DNA encoding the anticoagulant of this The hookworm anticoagulant protein of this inveninvention. tion exhibits a number of desirable characteristics. Un
DNA encoding the protein of this invention, or RNA like other anticoagulants derived from blood feeding corresponding thereto, are then inserted into a vector, 45 parasites such as the tick Ornithodoros moubata (Waxe.g., a pBR, pUC, pUB or pET series plasmid, and the man, L., et al., Science 248: 593-596 (1990)), the blackfly recombinant vector used to transform a microbial host Simulium vittatum (Jacobs, J. W., et al., Thromb. Haorganisms. Host organisms useful in the invention are emost. 61: 235-238 (1989)), and two species of leeches, bacterial (e.g., E. coli or B. subtilis), yeast (e.g., S. cer- Haementeria officinalis (Nutt, E., et al., /. Biol Chem. visiae) or mammalian (e.g., mouse fibroblast). This in- 50 263: 10162-10167 (1988)) and Haementeria ghilianii vention thus also provides novel, biologically functional (Condra, C, Thromb. Haemost. 61: 437-441 (1989)), viral and circular plasmid RNA and DNA vectors in- most of which are capable of inhibiting components of corporating RNA and DNA sequences describing the the common pathway (factor Xa and thrombin), hookhookworm anticoagulant generated by standard means. worm anticoagulant binds to and inhibits the activity of Culture of host organisms stably transformed or trans- 55 clotting factor Vila, the enzyme responsible for initiatfected with such vectors under conditions facilitative of ing the human coagulation cascade. Moreover, the prolarge scale expression of the exogenous, vector-borne tein of this invention is small, soluble and potent. Thus DNA or RNA sequences and isolation of the desired it has utility as a therapeutic agent for the treatment of polypeptides from the growth medium, cellular lysates, numerous vascular disorders including peripheral vasor cellular membrane fractions yields the desired prod- 60 cular disease, stroke, coronary heart disease, hypercoucts. agulable states, and other clotting disorders.
The present invention thus provides for the total As the major morbidity associated with hookworm
and/or partial manufacture of DNA sequences coding infection is a reflection of the gastrointestinal blood loss
for hookworm anticoagulants, and including such ad- caused by the adult worm, interventions aimed at inhib
vantageous characteristics as incorporation of codons 65 iting the anticlotting mechanisms of these intestinal
preferred for expression by selected non-mammalian helminths may significantly lessen the sequelae of
hosts, provision of sites of cleavage by restriction by chronic infection. The isolation and purification of
endonuclease enzymes, and provision of additional ini- hookworm anticoagulant thus also provides a means to
develop hookworm alternative therapies to prevent blood loss during infection. The isolation and purification of hookworm anticoagulant also provides a polypeptide marker, for diagnostic purposes.
In addition, enhancement of an immune response 5 aimed at the hookworm anticoagulant represents a viable strategy for vaccine development focused on reducing the burden of hookworm infection in populations at risk. Hookworm infection is one of the most clinically important soil-transmitted helminthiases, and third 10 world children suffer most from this usually insidious hemorrhage (Hotez, cited above). As an antigen, the protein of this invention offers potential for vaccination.
The following examples are presented to further illustrate and explain the present invention and should not 15 be taken as limiting in any regard.
Adult hookworms of the genus Ancylostoma caninum were obtained from the intestines of an infected dog as 20 described by Schad, G. A., Exp. Parasitol. 47: 246-253 (1979). Briefly stated, infected dogs were sacrificed when they exhibited peak parasite populations (determined by counting the number of hookworm eggs per 2J gram of feces, about 22 to 31 days post-infection). The isolated worms were stored frozen at —70° C. Crude hookworm extracts were prepared by suspending ~ 100 adult worms at a time in 1 mL of 0.05M Tris-HCl buffer, pH 7.5 (hereinafter referred to as "buffer"), and grind- jQ ing in a glass homogenizer for 10 minutes on ice. This suspension was then centrifuged at 8000 g for 2 minutes, and the supernatant was collected. The protein content of the extracts was then determined using Bradford's method, which involves the binding of Coomassie blue 35 to proteins, resulting in a shift in absorption maximum of the dye (Bradford, M. M., Anal Biochem. 72:248-254 (1976)). Extracts were frozen at —20° C.
A chromogenic assay employing commercially purified human coagulation factor Xa and chromogenic 40 substrate (Chromozym X, N-methoxycarbonyl-NleGly-Arg-4-nitranilideacetate) purchased from Boehringer-Mannheim (Indianapolis) was employed to characterize the hookworm protein extracts and identify proteins subsequently purified from it. Ten ul of Factor 45 Xa (0.01 units) were incubated with 20 ul of hookworm extracts, protein or buffer A (0.05M Tris-HCl, pH 8.2, 0.1% bovine serum albumin) for 10 minutes at 20° C. Nine hundred ul of buffer A was added, followed by 20 ul (40 ug) of Chromozym X. The mixture was allowed 50 to react for 6 hours and optical density was then measured at 405 nm. Negative controls lacking purified factor Xa showed minimal hydrolysis under the same conditions
Identical conditions were used for measuring the 55 effect of hookworm protein on the chromogenic hydrolysis of 40 ug of substrate (Chromozym TH: Tosyl-GlyPro-Arg-4-nitranuide-acetate) by commercially purified human thrombin (0.02 units), both purchased from Boehringer-Mannheim. 60
Factor X clotting time was determined by adding 50 uL pooled human plasma to 50 uL of hookworm extracts or protein in 150 uL buffer. To 100 uL of this was added 150 uL bovine factor X-deficient plasma (Sigma, St. Louis), 100 uL of Stypven (Sigma, 1:10 dilution), 65 cephalin (Sigma), and CaCh (0.035M) as described by Bachmann (Bachmann, F., et al., Thromb. Diathesis Haem. 2: 24-38 (1958)). Time to clot was measured and
compared to both standard curve and controls using buffer in the absence of hookworm protein.
As depicted in FIG. 1, factor Xa activity was reduced by 50% in the presence of 40 ug of crude soluble hookworm protein. Using the factor X clotting time bioassay, factor X clotting time was increased by 71% relative to control plasma alone in the presence of 500 ug of crude hookworm extracts.
Prothrombin time (PT) and partial thromboplastin time (PTT) were determined by adding hookworm extracts or protein to 400 uL of pooled human plasma at 20° C. and then measuring PT and PTT using Dade Thromboplastin C Plus ® Dade Actin FSL (ft) (Baxter Healthcare, Miami), respectively, in a MLA 1000 automatic clotting time recorder (Medical Laboratory Automation, Mount Vernon, N.Y.). Results were expressed as percentage increase in clotting times compared to controls in which buffer was substituted for hookworm protein. Using these procedures, adding 0.6 mg of crude soluble hookworm protein to 0.4 ml of human plasma, PT was prolonged by 125% (from 9.9 seconds to 22.3 seconds), and the PTT was prolonged by 57% (from 29.2 seconds to 45.9 seconds).
The supernatant containing soluble hookworm protein was applied to a 2.7X9 cm Q-Sepharose ion exchange column (Sigma, fast flow, wet bead size 45 to 165 u) equilibrated in buffer containing 1.0 mM orthophenanthroline, 1.0 mM dithiothreitol, 0.1 mg/ml 13,000 to 23,000 molecular weight polyvinyl alcohol, and 0.1% polyoxyethylene 23 lauryl ether (30% wt/vol). Bound protein was eluted with a 0 to 2.0M NaCl gradient, and those fractions in the elution (FIG. 2) which were capable of inhibiting factor Xa in a chromogenic assay described above were pooled and frozen.
The column fractions that exhibited the most significant factor Xa inhibitory activity in vitro also prolonged the factor X clotting time by 33%. Fractions were also assayed for their ability to prolong the PT and PTT. As shown in FIG. 3, the column fractions that contained the factor Xa inhibitory activity were identical to the column fractions that prolonged both the PT and PTT. No other column fractions inhibited the activity of purified factor Xa or caused prolonged of the PT/PTT.
Specific inhibition of factor Xa hydrolytic activity was enriched fivefold after Q-Sepharose, from 7.75 inhibitory units (IU)/mg of protein to 42 IU/mg of protein. One IU was defined as the amount of hookworm protein that would cause a 1% reduction in the rate of chromogenic substrate hydrolysis by purified factor Xa, compared to controls. Pooled fractions obtained from the Q-Sepharose column that inhibit factor Xa were dialized against buffer containing 0.1M NaCl and applied to an affinity column consisting of purified human factor Xa bound to agarose resin. The protein that bound to this Factor Xa was eluted with 0.17M acetic acid. Those fractions which were eluted from the column and contained factor Xa inhibitory activity were pooled, and the buffer changed to buffer containing 0.1M NaCl using a Centricon 3 ® microconcentrator (Amicon).
The pooled fractions were applied to a Superose 12 (Pharmacia) gel filtration column (24 ml) using fast pressure liquid chromatography. The fractions containing factor Xa inhibitory activity, which exhibited over a 100-fold increase over the original extract, were pooled and lyophilized. At this point, the protein was visualized as a single band on SDS-polyacrylamide gel elec