Note: Descriptions are shown in the official language in which they were submitted.
~ 0~27
W0 95/18632 2 8 . ~ 190
-1 -
Nucr~FoTmE ANn AMlNO ACll) SF.OUFNCF..'~ OF A r. r
RARF. OUTER MF~MBR~NF~ pROTFIN
ThisapplicationisaC~ntiml ~ in-paltApplicatiionofu~s~serialNo~o8l2s5~322filed
June 7, 1994, which is a l~-~ntin~ ir~n-in-part Application of U.S. Serial No. 081178,084
5 filed January 6, 1994.
ThisworkwassupportedbyU.S.GovernmentNlHgrantNos.A1213'i2andA129733.
BACKGROUND OF THE INVENTION
I . Field of tfie Invenfion
This invention relates to methods for isolation of rare proteins from bacterial samples.
10 More pa~ ,ulauly, this invention relates to a method for isolating rare outer membrane
proteins from the family Spirochae~aceae, such as genus Treponema and to the use of
such proteins in diagnosis and l)lu,ull~laHs of related diseases.
2. Description of Related Ar~
The genus Treponema (order S~,., .~..hu~.u~, family $;, .~ . ,. f ~), a type of gram-
15 negative bæteria, contains four human pathogens as well as at least six ~ ."~The pathogens are ..l~ ;, J by an extreme sensitivity to ~ VilUlUll~lLll conditionsthat renders them impossible to culture in vitro. Due to DNA homology the agents that
cause syphilis, yaws and endemic syphilis have been combined into one species and three
subspecies: T. pallidum subsp. pallidum (syphilis); T. pallidum subsp. pertenue (yaws);
20 and T. pallidum subsp. endemtcum (endemic syphilis). T. carateum, which is the
causative agent of pinta, remains a separate species. Syphilis is found world~ide, yaws
~- is endemic in the tropics, pinta is prevalent in tropical areas of Central and South
America, and endemic syphilis is restricted to desert regions. These treponemal
infections are very complex, each exhibiting distinct stages of ~yllllJtUllla~ic25 uuauurc~alivllD fo~lowed by a~lulu~Ullla~i~, periods. Without antibiotic therapy, these
W095118632 2 1 ~ ~ 4 2 7 . ~I/V~ S C .,~ --
-2--
diseases are chronic and may last for 30 to 40 years.
To date, the four pathogens have been considered _ulir,~ ,lly identical. An irldividual
~ulJ~ ,c;~ pecific antigen has not been identified and serological reactions ~l~ .. ,... ,~l, A '.
; " "~ " A~ g Al relatedness. Both Wassermann and anti-T. pallidum subsp. pallidum
5 antibodies develop in response to each treponemal disease, and known protective
;"..,. ,. ~c ~ are also related, as shown by cross-resistance (T.B. Turner, e~ aL, Biology
of fhe Tr r- ~'~ W.H.O Monogr. Ser. 35:1-277, 1957). Therefore, the
r,~ ~J~ location together with the clinical ., ; ~ ;.... of the patient have been
considered the key to diagnosis (Manual of Clinical Microbiology, 5th Ed., A. Balows,
el al., Eds., p 567, 1991).
Freeæ-fracture electron microscopy of outer membranes from pathogenic spirochetes has
revealed that their integral ~ ' " outer membrane protein density is one to two
orders of magnitude less than that of typical gram negative bacterial pathogens. It has
been proposed that this low outer membrane ~,VllI~)U~;IiVl4 and thus low surface exposure
15 of antigenic target molecules, allows these organisms to effectively evade the host
immune response, r....;~;l...:i,,A to the chrvnic nature of infection exhibited by all
spirochetal pathogens.
As is well known, the outer membranes of spirochetes, including that of Treponema
pallidum subsp.pallidum, the agent of syphilis, are fragile structures as compared to those
20 of typical gram negative bacteria. C~ /, separaùon of the outer membrane fromthe inner membrane has prvven extremely difficult. Certain other medically relevant
spirvchetal bacteria with outer membrane structure and, hence, protein structure, similar
to those of T. pallidum include Borrelia burgdorferi (Lyme Disease), Borrelial species
(relapsing fever), and Leptospiral species (I~v~ilv~;s). ,
25 The outer membrane of T. pallidum has been found to be ~ ly inert amd resistant
to specific 1. ~ antibody (Radolf, et al., ~nfect. Immun., ~2:579, 198Z; Hovind-
2 1 80427
wo 95118632 r~ c
--3-
Hougen, e~ aL, ~c~a Pathol. Microbiol Scand ., ~263, 1979; Nelson, et al., J Exp.
Med, 89:369, 1949). Yet freeæ-fracture electron microscopy has shown th;lt certain rare
outer membrane protein (tromp) molecules of T pallidum have surfæe exposed antigenic
sites that bind antibody present in the serum of challenge immune animals (Blanco, et aL,
J. I~nmunol., 144:191~1921, 1990). Taken together the spirochetal bacterial pathogens
imperil the health of a ~ portion of the human population, yet .1~ lvlu~ of
effective and specific væcines and isolation of antigens capable of generating protective
immune response has been hampered by a . ~. ,-: 1. . nl ~Ir number of problems associated
with this organism: the ;~ y of culturing the T. pallidum in vitro, the limited
numbers of organisms that can be obtained from infected animals, the ~ l of
treponemes by host tissue . .., ,1., . - . ,1~ the fragility of the treponemal outer membrane,
and the difficulty of isolating and identifying the outer membrane proteins of pathogenic
spirochetes.
Previous studies attempting to identify Ll. ' outer membrane proteins of
15 pathogenic spirochetes have utilized various detergent g~ 7~finn approaches.
Spirochetal outer membranes bleb from the underlying IJ-U~U,~ UUC cylinder underrelatively mild conditions, including dilute detergents and hypotonic ~ lIU. .I~.lt~.
However, such apporaches have identified only abundant subsurface located proteins,
including various lipoproteins which by definition are not 1 - ,~.. ,.. ,.1..,.. ,~ molecules amd
20 do not form particles viewed by free_e-fræture analysis.
Therefore, the need exists for new and better vaccines based upon the irhntif /~nfion of
virulence related outer membrane molecules to be used in diagnosis amd for ~
of diseases related to the pathogenic spirochetal bæteria, especially the genus T pallidum.
W09Stl8632 2 1 80 427
4-
SUMMARY OF THE INVENTION
A novel method is provided for isolating outer membrane of pathogenic S~;, ùc~la~f~family without use of detergents. The pathogen is purified from ~... - - .,:., ~;.,~ host
usmg a density gradient ~, "-.r"L~ medium that is stable at pH from
5 about 3.2 to about 3.0, preferably a Ficoll step gradient. The purified pathogen is treated
with a lipid soluble ~L1UIIIUIJIIUIC that intercalates into outer membrane to provide a visual
marker of membrane matter. Outer membrane is released from ~UI uLu~ulaDlll;c cylinders
using a hypotonic, low pH buffer, preferably citrate, followed by density gradient
l; r, l~ which yields, for example, the ' u..lul~h~lL labeled bands at 7% and 3510 % sucrose (wt/vol) for T. pallidum and T vincentii, I~ IY Freeze-fracture electron
microscopy of membrane vesicles from the spirochete reveals an extremely low density
of protein particles. For instance, the particle density of T. pallidum is ~I,UUI. ~/ SiX
times less than that of T. vincentii. Comparative; " , .. .l ,l. .1 analysis of the T. vincentii
membrane material to that of whole organisms showed a li~uu~uùl~Da~",l~;~ (LPS) ladder
15 consistent with 20% recovery ofthe outer membrane. l,.,., ..~l .l.,l, of T. vincentii outer
membrane also showed two amtigenic proteins at 55- and 65-kDa.
penicillin, which binds only to inner membrane and not to outer membrane, was used
to detect the presence of any inner membrane in the outer membrane preparation isolated
according to the method of the invention. No penicillin binding proteins in the T
20 pallidum membrane material were detected, indicating the absence of inner membrane
T".".. bl.,l analysis of T. pallidum outer membrane using antibodies specific for
~I;,ulaD.l.;c associated proteins showed no detection of known 19-kDa "4D" protein or
the 47-kDa lipoprotein and only trace amounts of ~ n ~ protein. Rare outer
25 membrane proteins associated with the T. pallidum were detected by one and two
,l:"" ,~;".. l reducing SDS-PAGE separation and ;~ l--l analysis, using gold
stainirlg and serum from rnfected and challenge immune animals. As compared to whole
2 1 80427
WO95/1863:Z l~l/.J.. _ Jtl~
--5 -
organism lu~c,u~lAliulls, four of the isolated proteins were obtained in significantly
enriched amounts from the outer membrane preparation.
Methods are provided for the use of outer membrane proteins of pathogenic
Spirocizaetacae family for detection and A111. ~ I of associated disease states.
5 The isolation of the T. pallidum outer membrane and irl .;il~ of its protein
.. " .~l ;l . ,...~ . has been . ~ " ' by the fragility of this structure, the limited number of
IL ~AUu..~ that can be acquired by rabbit infection, and the significant amount of host
protein following extraction of organisms from infected animals.
Moreover, freeze-fracture electron lI. _lU~U,Uy has revealed that the outer membrame of
10 T. pallidum contains two orders of magnitude less integral membrAne protein than typical
gram negative bacteria (Radolf, ef al, Proc. Nafl Acad. Sci. Ui'~;A, 86:2051-2055, 1989;
WaLker,efaL,~ Bacferiol., 171:5005-5011,1989). BecauseofthepaucityofTpall~dum
rare outer membrane protem ( I~OMP), it is likely that previous studies using detergent
extraction of T. pallidum to identify ll~.. l.~.l.l.. A.. ~ outer membrane proteins have
5 mistakenly identified as outer membrane proteins abundant subsurface molecules,
including li,uuulut~,;ll~ anchored in the inner membrane that are released by such
treatments (1'l ' ' ef aL, Infecf. Immun., 57:2872-2877, 1989; Penn, ef aL,
Immunolo,~,~Lti:9-16, 1982;Penn,efaL,J. Gen Microbiol., 131:2349-2357,1985).
It b,as previously been shown that while 0.1% Triton X-l 14 can selectively solubilize the
20 T. pallidum outer membrane, some subsurface molecules, including the 47-kDa
lipoprotein, are also released (Radolf, ef aL, Infecf. Immun., 56:490498, 1988).C~.. IIA~ IIC of Triton X-l 14 of up to 2% have been shown to release additional T.
pallidum li~uuulut~,;lla ((~ _' ef al., J. BacterioL, 170:5789-5796,1988; Radolf,
ef aL, supra, 1988).
25 The present invention provides a method for isolating the outer ~ from
LLC~O~ and other spirochetes with rare outer membrane proteins in the absence of
2 1 80427
W0 9S/18632 F~ ~ ' 19u
-6 -
detergents. In the examples herein this procedure was applied to T. vincentii, which,
because of the LPS content of its outer membrane, was used as a marker for outermembrane recovery. Preliminary studies showed that while a hypotonic osmotic
CllVilUlUlo.~ caused significant blebbing of the treponemal outer membrane, only a small
5 amount of outer membrane was released. F.n~1rlflr~ filaments may physically interact
v.~ith the outer membrane in the process of motility (Berg, J. Theor. Biol., 56:269-273,
1976; Goldstein, ef al., Cell Mofility and fhe Cyfoskelefon, 9:101-llO, 1988). These
structures may limit the release of outer membrane under hypotonic conditions.
Therefore, in the present invention a low pH hypotonic buffer is used to dissociate
1 0 en~ fl~elhlr filaments (Blanco, ef al., Infect. Immun. , 56: 1 68-1 75,1 988). As a result,
the outer membrane is completely released as viewed by electron IlI;~lU~-~UI~y . The low
pH treatment, however, is ;~ with purification of T. pallidu~n by the
cullv~lL;ullal Percoll procedure due to the adverse effects of low pH on residual Percoll,
which solubili_es in low pH conditions. Therefore, in the practice of this invention, T.
15 pallidum is purified using a continuous or di c.-. " ,l; .l l~ density gradient separation in
a medium that is stable in the pH range from 3.2 to 3.0, removes ~ host
, and is also compatible with the subsequent low pH incubation.
A second key step in the practice of this invention is treatment of treponemes with a
CIUUIIIU~JIIUI~;~ preferably one that intercalates into biological and liposomal20 The preferred ~IUUIIIUIJIIUIC is octyl-decyl rhodamine, but one skilled in the art will
appreciate that any CIUUIIIUIJIIUI~ of a si_e suited to intercalate into liposomal m~mhrs nrc
can be used so long as it is naturally lipophilic or can be substituted with
lipid-c~ hili7in~ moieties containing between 8 and lO carbon atoms. In addition, the
lipid-soluble e~Ulllv~ullul~ should be selected so as not to " ~ l~, alter the
25 membrane particle density. Use of the CIUUIIIU~UIIUIC provides a visual marker to follow
the disposition of released outer membrane. To determine whether the chosen
lipid-soluble UIIIUIIIUI1IIUI~ alters membrane palticle density, a pathogenic spirochete
having an order of magnitude greater amount of outer membrane protein than the one
being isolated can be used. For instance, using Borrelia b... X.IU,J~, i, a pathogenic
2 i 80427
wo 95/18632 P~
--7-
spirochete which has an order of magnitude greater amount of outer membrane protein
than T. pallidum (Walker, e~ al, supra, 1991), it was found that octyl-decyl rhodamine
did not change its outer membrane particle density (data not shown), suggesting that the
outer membrane proteins of T. pallidum and T. vincentii were also not affected by this
5 reagent.
The f nding herem that T pallidum outer membrane banded in a sucrose gradient at a very
low density (7%) is consistent with membrane that contains a small amount of protein
(Tomlinson, et aL, Biochem., ~:8303-8311, 1989). T_is finding was further confirmed
by freeæ-fracture electron microscopy of purified T. pallidum membrane vesicles, which
10 showed fracture faces that contained extremely rare ;"1,~.,...,.i",...~",~ particles. This
result is similar to the low particle density observed by others for the native outer
membrane of T pallidum (Radolf, et al. supra; Walker, ef aL, supra). By ~
the T vincenfii outer membrane banded in a sucrose gradient at a higher density (35%)
as is consistent with the greater amourt of ".1,,.. ,.. I.. ~.. ,u~ particles observed in its
membrane and/or is consistent with a membrane that contains li~vlJol~ .u.,.,llu.;d~ (LPS).
The selective isolation of the T. pallidum outer membrane from the ~ vl ,~ - -- ;- cylinder
was determined by the use of penicillin binding proteins (PBPs) as a marker to visualize
inner membrane associated proteins. Previously studies have shown that T. pallidum
PBPs remain with the l -v~vl~lu~ uic cylinders following s~ hili7oti~n of the outer
membrane in the detergents Triton X-114 or Triton X-100 (C.--nnin~h~ n~ ef al., J.
Bacteriol., 169:5Z98-5300, 1987; Radolf, ef al., Infect. Immun., 57:1248-1254,1989).
No PBPs were detected with purified outer membrane prepared according to the method
of t_is invention, indicating that the procedure selectively removes only the outer
membrame, free from ~... '~ . .- ~;..,. by inner membrane.
25 Of particular ~i~nifi~ n~ is the complete absence of the T. pallidum outer membrime
preparation of the 4D protein and the 47-kDa major lipoprotein, and the flndmg of only
trace amounts of ~nril~fl~ protein, indicating little to no i..,.'---.;. ~;.... by these
WO 9Y18632 2 1 8 0 4 2 7 ~ .'C J30
-8-
periplasmic ~ The 47-kDa lipoprotein, one of the most abundant T pallidum
molecules, was not detected in the outer membrane preparation, thus confirming that
inner membrane anchored 1;~ were not released by this procedure.
Coomassie stained SDS-PAGE and; ~ n~ amalysis of 1 X 109 T. vincentii
5 equivalents of outer membrane revealed two major antigenic protein species of 65- and
55-kDa. In contrast, Coomassie stained SDS-PAGE of a 5-fold greater amount of T
pallidum outer membrane showed no detectable protein. These findings are consistent
with the observations of freeæ fracture electron Illh,l~cv~y indicating that the outer
membrane particle density of T pallidum is six times less than that of T. vincentii. From
10 the outer membrane particle density of T. pallidum, which has been determined to be 170
pallh,lc;,/uu..' and the surface area of T. pallidum, which is ~ / 4um2, it is
calculated that S X 109 T pallidum should contain only 250 nano grams of outer
membrane protein based upon a single species of 50K molecular weight. Therefore, the
amount of a single species of TROMP isolated using the method of this invention is
15 several hundred times less than was previously ~ u~ly identified by prior art methods (Norris, et aL, Microbiol., 57:750-?79, 1993).
Enhanced, ' ' (ECL); - - -, . ,. Il 11.1. .1 l; . ~L has the sensitivity of detecting pico
grams of antigen (ECL Western Blottmg protocols, Bl ' , ' ' c, England, 1993).
Therefore, this technique is preferably employed in the method of this invention for
20 detecting outer membrane associated protein. Most preferably, using ECL, ' ' '~
of outer membrane samples are prepared in urea and elc~ ci7~d in one dimension
or following two l, ..ci~ (2D) ele~ , ' and probed with sera from rabbits
with immunity to the pathogenic treponeme of interest. Using this technique upon T
pallidum showed two major antigenic protein bands at 17- and 45-kDa. The 17-kDa -
25 protein had a pl of greater than 7.0, showed higher oligomeric forms, and selectivelypartitioned mto the 11~ ' ' phase following Triton X-l 14 detergent extraction (data
not shown).
2 1 80427
Wo95/18632 r~l~v..,~ iJ~
J
These findings are consistent with the properties of the native and .~c. ." ,1,;, IA 1~1 1 7-kDa
- lipoprotein of T. pallldum (Atkins, et al., Infect. Immun., 61:1202-1210, 1993). It was
also shown using specific mAInA~ antibodies that the 45-kDa protein was the
previously I I~ I TmpA lipoprotein (Schouls, et al., Microb. Pathog., 1:175-188,1989; Hansen, et al., J. Bacteriol, 162:1227-1237, 1985). While the vast majority of
these two proteins remain associated with the l~luluul~ ic cylinder following outer
membrane removal (data not shown), some of the 17- and 45-kDa li,uu~u~ are
specifically associated with outer membrane.
In additiûn to the strongly antigenic 17- amd 45-kDa l;~u~ t~ ls of T pallidum isolated,
gold-stained 2D blots of 3 X 101 treponemal equivalents revealed four additional T.
pallidum proteins, includmg one each at 28- and 65kDa, and two at 31 -kDa. All of these
proteins have been found to contain antigenic sites reactive with sera from immune
rabbits. r--mr ~ nn of the pI's of these found proteins to those on 2D blots of 5 X 10~
whole organisms have shown that the 31-kDa (acidic pI) amd 28-kDa proteins correspond
to prominent T. pallidum protein spots on imrnlln~Alhlnts and may be additional outer
membrane associated li~u,ulut~,;..~ not heretofore identified. In contrast, the 31-kDa
protein (basic form) cu.l~,uul.l~ to a minor and faintly detectable protein spot on 2D
blots of whole organisms, while the 65-kDa protem does not correspond to amy previously
identified T. pallidum protein. In view of their sigluficant enrichment following outer
membr~me isolation, the 31-kDa (basic pl) and 65-kDa proteins are identified as rare outer
membrane proteins.
The outer membrane proteins of typical gram negative bacteria include an export sigmal
cleaved by leader peptidase I, and: . ', ' ' beta pleated sheet st~ucture throughout
the secondary sequence that generates membrane spanning regions (Vogel, et aL, J: Mol
. Biol ., 190:191-199, 1986; Weiss, et al., Science, 2~4:1627-1630, 1991; Von Heijne, J:
Mol. Blol., 184:99-105, 1985). Recently, the gene encoding a surface exposed 31-kDa
protein of ~ eptospira alstoni, designated Omp-LI (outer membr~me protein of ! rptA crirA)
has been cloned, sequenced, and expressed (Haake, et al., ~ BacterioL, 175:42254234,
2 1 gC427
wo 95/18632 1 ~ Iso ~
--10-
1993). The deduced amino acid sequence of this protein shows an e~port and
beta pleated sheet topology resulting in 10 membrane spalming domains
(Haake, et aL, supra). The structural similarity between this putative outer membrane
protein of Leptospira and those of typical gram negative bacteria suggests that other
5 spirochetal outer membrane proteins may be structurally similar to those of typical gram
negative bacteria. The Leptospira outer membrane has been isolated using the method
of tbis invention. The membrane material purified was found to selectively contain
lipopol~ ~;;le like substance (LLS), which is unique to the Leptospira outer
membrane (Zeigler, et al, Can. .1: Microbiol, 21:1102-1112, 1975), and several proteins
10 including OMP-LI. These findings provide additional evidence that the membrane
material and associated protem isolated by the method of this invention from T. pallidum
is outer membrane in origin.
The bmding of antibody in immune serum to virulent T pallidum results in aggregation
of TROMP particle as viewed by freeze fracture electron microscopy (Blanco, et al.,
15 supra, 1990). These fundings have recently been confirmed and extended using serum
obtained from animals with varying degrees of challenge immunity. Particle :~gr~ n
directly correlates with the ~ v~ t of challenge immunity, suggesting that TROMPare key targets for a protective host immune response.
20 In addition, due to isolation and purification of the T pallidum outer membrane, the
amino acid sequence of the protein has been obtained and of DNA encoding it and for
cloning of TROMP molecules. The ~ ollLil~lt expression of these rare outer membrane
proteins can be used for ~ biology studies to address directly the molecular
basis for T. pallidum ~fl~ - cic for diagnostic tests to detect syphilis and for25 d~ lv~ li of host immunity during syphilis.
2 1 ~0427
WO 95/18632 r~ I ,v.. ~ ~ l9O
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE I is an SDS-PAGE ~-- t~ showing CU~ ivt~ analysis of S X lû3
unpurified and Ficoll purified T. pallidum (Tp). The molecular weights (X 103) of marker
standards (MKs) are indicated.
5 FIGURE IA shows a Coomassie stained SDS-PAGE gel.
FIGURE I B shows illlll.~obl~l~ probed with anti-rabbit serum proteins.
FIGURE 2 shows electron Illi-,lu~ Jlla of outer membrane material isolated from T
pallidum (Tp) md T. vincenfii (Tv).
FIGURE 2A shows a whole mount electron ~ ,lu~ ,ulla of Tp purified on Ficûll and10 labeled with rhodamine and Tv washed with PBS and labeled with rhodamine.
FIGURE 2B shows a whole mount electron llfi.,lu~ ~ of Tp and Tv organisms treated
with acidic citrate buffer, showing release of outer membrane.
FIGVRE 2C shows a whole mount electron Ill;~,~uyl~h~ of Tp and Tv outer membranevesicles purified on sucrose gradient.
1~ FIGURE 2D shows a freeæ fracture electron III;~IU~I~I,UIID of purified Tp and Tv outer
membrane vesicles. Bar indicates 0.5~Lm.
FIGURE 3 shows an 1 l analysis of T. vincenfii, untreated and treated with
proteinase K (PK), showing 2 X 10~ equivalents of whole organisms (WO) and
lulutu~l~lll;c cylinders (PC) and I X 109 equivalents of outer membrane (OM). The
20 molecular weights (X 103) of marker standards are indicated.
` 21 80427
WO95/18632 I~
-12-
FlGURE 4 an SDS-PAGE ~ , showing penicillin binding proteins from l X
108 whole organisms (WO) and ~Iv~u~laa~ll;c cylinders (PC) of T. pallidum and from S
X 109 equivalents of outer membrane (OM). The molecular weights (X 103) of marker
standards are indicated.
5 FIGURE 5 shows an ' '~' analysis of outer membrane material of T. pallidum
probed with antisaa against three periplasmic associated proteins: whole organisms (Tp),
outer membrane (OM) are probed with anti-~nrlr~fl~Pll~r serum (~EF); anti-l9-kDA"4D" serum (a4D); and monoclonal antibody against 47-kDa lipoprotein (1~47 MAb). The
molecular weights (X 103) of marker standards are indicated.
10 FIGURE 6 is an i~n~n~hlot showing immume rabbit serum (IRS) detection of T.
pallidum outer membrane associated proteins. The molecular weights (X 103) of marker
standards are indicated (+ indicates treatment with 8M urea).
FIGURE 7 is an SDS-PAGE ;.. ,.. 1,1~.1 showing immune rabbit serum (IRS) detection
of T. pallidum outer membrane associated proteins separated by two .1; .. ~ gel15 Cl~,llulJI-vlca;a. The molecular weights (x 1 03) of marker standards are indicated. The
isoelectric focus (EIF) range from 7 to 5 is indicated by a bar at the top.
FIGURE 8 shows gold staining detection of T. pallidum outa membrane associated
proteins separated by two 1;~ gel clc~ ulJllvl~;a. Arrows and bracket indicate
T. pallidum proteins identified, including L 1~ n ~ (EF) and TmpA. The molecular
20 weights (X 103) of marka standards are indicated. The isoelectric focus (EIF) range from
7 to 5 is indicated by a bar at the top.
FIGURE 9 shows the nucleotide sequence for an open reading frame identifJed in the 872
bp HindIII fragment in the DNA of the 31 kDa TROMP protein (pl 6.7). The nucleotide
sequence encodes a precursor TROMP protein of 288 amino acids known as TROMPI
25 (SEQ ID NOS:I and 2).
21 8~427
WO 9S/18632 1 ~ ..,5.'t
-13-
DETAILED DESCRIPTION OF THE I~VENTION
The present invention provides iso~ated and purified r-Are outer membrane proteins and
protein fragments of tbe pathogenic Spirochaefacae family, especial~y of the genus
Treponema, and a method for their isolation. These , - proteins are useful in
5 a ~ for mducing an immune response to pathogenic
Spirochaetacae from which they are derived. Hence, the rare outer membrane proteins
of this invention are useful for Am~linrAfin~ the effects of disease states associated with
the pathogen from which they are derived. In addition, the rare outer membrane proteins,
which contain antigenic epitopes for antibodies found m the blood of infected mammals,
10 such as humans, can be used to detect individuals infected with disease states associated
with the pathogens from which the proteins are derived Alternatively, antibodiesgenerated from; " .. , .. ., A~ 1~ .. 1 of an individual with the rare outer membrane proteins can
be used to detect exposure to or infection by the pathogen in another individual.
The preferred outer membrane proteins of this invention are isolated from the outer
15 membrane of the genus ~reponema pallidum subsp. pallidum, the pathogen responsible
for causing syphilis in humans, and are . l. --... l. ;,. .1 by the following isoelectric focus
points and molecular weights, as determined by reducing SDS-PAG~ as showrl in Table
1.
TABLE 1
r~
ROM I 31 kDa 6.6
ROM 2 65 kDa 5.9-6.0
ROM 3 28 kDA 6.9-7.0
ROM ~ 3 l kDa 6.5
WO95~18632 21 8~427 ~ 5lCI9~ --
-14-
A novel method for isolating outer membrane of pathogenic Spirochaetacae family is
presented herein. The pathogen is purified from c~ I host ~ such as
tissue, blood, bodily secretions, and the like, preferably using a .l;~ .. .l;, l. .., ~ Ficoll step
gradient of at least four steps. It is preferred that the densities of the .1: ;.. " .. ; . l, .. ~ step
gradient be as follows: 1.045, 1.055, 1.065 and 1.085 g/ml. The purified pathogen is
treated with a lipid soluble cLvlllv,ul.ul. that intercalates into outer membrame, to provide
a visual marker of membrane matter. The lipid-soluble ~,luu~lv~l~v~ is preferably a dye
marker, most preferably a fluorescent dye marker substituted with one or more branched
or unbramched alkyl chains, each containing from about 8 to 10 carbon atoms.
Outer membr~me is released from ,UlULU,Ul_~llliC cylinders without use of detergent using
a hypotonic, low pH buffer followed by density gradient ~ l i rl.G.. ;. .l . to obtain the
~.Ill.. l.l.. ~ Iabeled band. The buffer is kept at pH from about 3.2 to about 3.0, and the
buffer is preferably citrate or acetate. Preferably, the buffer has an ionic strength of 50
mM to 100 mM.
15 The density gradient c. . .l . ; r, ~GA; ;l ~ ~ medium must be selected to be stable within the low
pH range ofthe buffer. Generally, amy polymeric density gradient ~. ~ ;r.,GA:;. .,. medium
havmg stability at pH within the r~mge from about 3.2 to 3.0 can be used, but preferably
the medium is a polymeric saccharide medium such as FICOLL~v, a synthetic polymer
of sucrose, or FICOLL HYPAQUE~v density gradient medium. The . ..I.ir.~,A;;~..,
20 medium can be either continuous or 1: .... ~;.l....ll~ but preferably in the practice of this
invention the density range is from about 1.045 to about 1.085 g/ml. Although any
workable means can be used, the band containing the ~h1VI1IU~UI~ Iabeled bamd ispreferably separated from the sucrose gradient medium by needle aspiration.
Arltibodies provided in the present invention are ill~ lllUl~_liV~ with at least one
25 Spirochaetales ROM protein of the pathogen of interest. Antibody which consists
essentially of pooled monoclonal amtibodies with different epitopic ~l ~ ; r ~ as well
as distinct ~ antibody ~Ul~,uf,la~ivll~ are provided. ~ l - AI amtibodies are
2~ ~5~
WO gS/18632
-15-
made from antigen-containing fragments of the protein by methods well known in the art
- (Kohler,etal.,Nature,2~:495,1975;CurrentProtocolsinMolecularBiolo,~,Ausubel,
et al., ed., 1989). The term antibody, or ;~ . l lh~ ;" as used in this invention
includes intact molecules as well as fragments thereof, such as Fab and F(ab')2, that are
5 capable of bmding an epitopic ~ t~rrnin ~t on a S~, u~,'.uGt..leJ ROM, such GqS the ROMs
of T. pallidum shown in Table I .
Minor m~A,rlifiAAtinnc ûf primary amino acid sequence may result in proteins that have
substantiaily equivaient fimction compared to the ROM proteins described herein. Such
may be deliberate~ a5 by 5ite-directed ..~ -g~ , or may be ! . '
10 All proteins produced by these ",~ 1 are included herein as long as the antigenic
fimction of the modified ROM.
Modifications of the ROM protein primary amino acid sequence aiso include
uu~ vaiiv~ variations. The term "cu~ ive variation" as used herein denotes the
.ri.lA... ,.~ ... of an amino acid residue by another, biologically similar residue. Examples
15 of co~ A,ive variations include the c~hctihlti..n of one llydlul,llol,;c residue such as
isoleucine, vaiine, leucine or methionine for amother, or the ~ ., of one polar
residue for another, such as the CllhstitlltiA~n of arginine for Iysine, glutamic for aspartic
acids, or glutamine for asparagine, and the like. The term "cu.~ iV~ variation" aiso
includes the use of a substituted amino acid in place of an I ' ~ parent amino acid
20 provided that antibodies raised to the substihuted ~uoly~ id; also with the
..,.~..l.~:;l.. . ~ polypeptide.
The invention extends to any host modified æcording to the methods described, ormodified by any other methods, commoniy hnown to those of ordinary shill in the art,
such as, for example, by hansfer of genetic materiai using a Iysogenic phage, and which
25 result in a prokaryote expressing the Spirochaetales gene for protein. Pluh~yuL~s
~,,.,.~r..,.,.. ~1 with the S/~ hu.~.1~s gene encoding the ROM protein are particular~y
2 1 80427
WO 95/l8632 P~llI l~
-16-
useful for the production of ~f ly~Jlid~D which cam be used for the ;" "., .; ,~i"" of an
animal.
In one rll l o ~ the invention provides a ~.I.--..vl. . .,l;. I C"".l,f.~;l;..,l useful for
inducing an immune response in an animal to pathogenic Spirochaefales, preferably a
5 Treponema, most preferably T. pallidum. The i,..l...~;l;.... comprises an
;""",..,nlf~ lly effective amoumt of am amtigenic outer membrane protein in a
l,l,A.".A~.,l;. _lly acceptable carrier. The term ";~ c ~; ~lly effective amount," as
used in describing the mvention, is meamt to denote that amount of $., I.,l...G~L~D amtigen
that is necessary to induce ir an animal the production of an immune response to10 Spirochaefales. The}areoutermembraneproteinoftheinventionare~Jf~l. ' `yusefulrhl sensitizing the immume system of an animal such that, as one result, am immume
response is produced which ameliorates the effect of Spirochaefales infection. For
instmce, an immune response to T pallidum cam be produced by I ' g to an
animal the ROM proteins of Table I isolated by the method of this invention.
In another ~,,.I,f,.l;,.. l as shown in Figure 9, the invention provides the arnino acid
sequence of a 288 amino acid precursor protein fragment (TROMPI) of the 31 kDa
protein ofthis invention (SEQUENCE I.D. NO. 1). TROMPI is encoded by a DNA open
reading frarne of 867 bp (SEQUENCE I.D. NO. 2) isolated usmg tryptic digest amino
acid sequence analysis of the genomic DNA of T. Pallidum, which had been previously
20 digested with EcoRI restriction enzyme. A segment comprising the first 32 residues from
theN-terminUsOfthepreCUrsOrprOteillhas~ h.;~1;.`Ofallydl~.' '' signalpeptide
including a 13 residue N-region contGinmg four basic charged residues (Histidirle, Lysine,
Histidine, and Arginine), an H-region containing 11 consecutive II,~'dl~JIlfJb;C amino
æids, and a C-region contrDining a putative concensus leader peptidase I cleavage site of
25 Threonine-Histidine-Alanine. The mature processed protein consists of 256:amino acids
with a calculated mass of 28,182 Da.
W0 95118632 2 1 8 0 4 2 7 F~ n _ 19()
-17-
A T. Pallidum rare outer membrane protein can be d~Lll;u;atl"c~ c~ y by injection,
rapid infusion7 l~da~lllyll~dl absorption~ dermal absorption~ or orally. PI ~ IY
accept~ble carrier ~ J~aL;~ for parenteral Al~ include sterile or aqueous or
non-aqueous solutions, CIICr.~nciA,nc and emulsions. Examples of non-aqueous solvents
5 are propylene glycol, pol.~ lyl~ ., glycol, vegetable oils such as olive oil, and injectable
organic esters such as ethyl oleate. Carriers for occlusive dressings can be used to
increPAse skin ~.,llll.,~;l;~y and enh_nce antigen absorption. Liquid dosage forms for oral
All l n~ may generally comprise a liposome solution containing the liquid dosageform. Suitable forms for suspending the liposomes include emulsions, ~
10 solutions, syrups, and eliAYirs containing inert diluents commonly used in the art, such as
purified water. Besides the inert diluents, such .-. ~ can also include adjuvants,
wetting agents, ~ la;ryi..g and suspending agents, and a~ , flavoring, and
perfuming agents.
It is also possible for the antigenic preparations containing the Spirochaetales ROM
15 proteins of the invention to include an adjuvant. Adjuvants are substances that can be
used to ~ lly augment a specific immune response. Normally, the adjuvant and
the antigen are mixed prior to l " . ~ .. . to the immune system, or presented separately,
but into the same site of the animal being 1. Adjuvants can be loosely divided
into several groups based on their , These groups include oil adjuvants (for
20 example, Freumd's Complete and Incomplete), mineral salts (for example, AIK(SO4)2,
AlNa(SO4)2, AINH4(SO4), silica, alum, Al(0E~)3, Ca3(PO4)2, kaolin, and carbon), poly-
nucleotides (for example, poly IC and poly AU acids), and certaih natural substances (for
example, wax D from A~j~ ob.JctL, tuberculosis, as well as substances found in
C." ~ bL~ L, parvum, Bordete& pertussis, and members of the genus Brucella).
25 In amother ~ ' t, a method of inducing an immune response to pathogenic
Spirochaetales, especially Treponema such as T. pallidum subsp. pallidum; T. pallidum
subsp. pertenne; T. pallidum subsp. r-- ~~--- , and T. cerateum in animal is provided.
Many different techniques exist for the timing of the ;, ~ when a multiple
WOg~/18632 21~b427~ v
-18-
regimen is utilized. It is possible to use the antigenic preparation of the
invention more than once to increase the levels and diversity of expression ofthe immune
response ofthe immunized arlimal. Typically, if multiple ;".."...,;, ~ are given, they
will be spaced two to four weeks apart. Subjects in which an immune response to
5 S~ o~u~;~ùles is desirable include domestic animals and humans.
Generally, the dosage of ROM protein ~ln . .~,..,l to an animal will vary depending on
such factors as age, condition, sex and extent of disease, if any, and other variables which
can be adjusted by one of ordinary skill in the art.
The antigenic ~ s of the invention can be Al~ as either single or
multiple dosages and can vary from about 10 ug to about 1,000 ug of the Spirochae~ales
ROM antigenic protein per dose, more preferably from about 50 ug to about 700 ug of
ROM antigenic protein per dose, most preferably from about 50 ug to about 300 ug of
ROM antigenic protein per dose.
When used for; .. - - . .Ih ~ , the ~ antibodies specific for Spirochaetales
15 ROM proteins of the invention or fragments thereof may be unlabeled or labeled with a
therapeutic agent. These markers can be coupled either directly or indirectly to the
~"...,.~rl.~ 1 antibodies of the invention. One example of indirect coupling is by use of
a spæer moiety. These spæer moieties, in turn, can be either insoluble or soluble
(Diener, e~ aL, Science, ~l: l 48~ 1986) and can be selected to enable drug release from
20 the mnnn^lrm ~ antibody molecule at the target site. Examples of diagnostic markers that
can be coupled to the n^r~nn^ln^ I antibodies of the invention for ;... ,..~ of
disease states associated with Sl,i",.h,,~:al~ such as T. palZidum are drugs, . Arli. .~
lectins, and toxins. The labeled or unlabeled monoclonal antibodies of the invention can
also be used in ~. ,...1~ - A ;~1~ with therapeutic agents such as those described above.
25 When the ".1,..~ IA1 antibody of the invention is used in ~ .., with various
therapeutic agents, such as those described herein, the A.~ ,AI ;, ... of the ".. ."A,,.I. .. ,AI
W095118632 21 8Q~7 r~
-19-
antibody and the therapeutic agent usually occurs substantially ~ ly. The
terrn "substantially . .." . .,.I.n.a". v,.~ly" means that the monoclonal antibody and the
therapeutic agent are a~ r-d reasorlably close together with respect to time.
Usually, it is preferred to administer the therapeutic agent before the mnnnrlnnrl
5 antibody. For ex3mple, the therapeutic agent can be a~ 1 to 6 days before the
mnnn~Jrm~ll antibody. The ~.I . .;~.,,I;. .,. ofthe therapeutic agent can be daily, or at any
other interval, dependirlg upon such fætors, for exa}nple, as the nature of the disorder, the
condition of the patient and half-life of the agent.
The dosage ranges for the .- ' nn of ,. ..~ antibodies of the invention are
10 those large enough to produce the desired effect in which the onset symptoms of the
Spi~ochaetales disease are A".~l;..."lr~l The dosage should not be so large as to cause
adverse side effects, such as unwanted cross-reactions, ~la~ lal,Li~; reactions, and the
like. Generally, the dosage will vary with the age, condition, sex and extent of the disease
in the subject and can be determined by one of skill in the art. The dosage can be adjusted
15 by the individual physician in the event of any ~ . . Dosage can vary from about
0.1 mgllcg to about 2000 mg/lcg, preferably about 0.1 mg/lcg to about 500 mg/lcg, in one
or more dose ~ daily, for one or several days. Generally, when the
..... ---cl.. ~l antibodies of the invention are ~' ' conjugated with therapeutic
agents, lower dosages, ~ , ' ' to those used for in vivo diagnostic imaging, can be
20 used.
The mnnn~ lnn I antibodies of the invention can be ~ l parenterally by injectionor by gradual perfusion over trme. The ".. n~ antibodies of the inventior~ can be
r ciJ ;IILIav~ ly~ , ~y, ;~ --ly ~ ly
intracavity, or I . Al ~`'i' ' I I~illly~ alone or in ~ . " "~.; . . ~ ;. .., with effector cells.
25 rl~al~lliv..~ for parenteral ~ .,.l;nn include sterile aqueous or non-aqueoussolutions"~ and emulsions. E~xamples of non-ælueous solvents are propylene
glycol, ~vl~.,LIIyl~ glycol, vegetable oils such as olive oil, and injectable organic esters
WO 9!i/18632 ~ IJ.I --
2~ ~0427
-20-
such as ethyl oleate. Aqueous calriers include water, alcoholic/aqueous solutions,
emulsions or ~lly. .~ , including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lætated Ringer's intravenous vehic~es include fluid and nutrient ~ ., electrolyte
5 l~ f.~ l, (such as those based on Ringer's dextrose), and the like. rlc ,~l ~aLi~ and
other additives may âlso be present such as, for example, a.lLil.~i.,.ul,;als, anti-oxidants,
chelating agents and inert gases and the like.
In a further ~,.,l.nll;..,...l the invention provides a method of detecting a pathogenic
Sp~, ù~7.u~...7~-associated disorder in a subject comprising contacting a ROM protein of
10 the Splrochaetales with an antibody specific therefor. The antibodies are detectably
labeled, for example, with a diagnostic .~ u,u~, a fluorescent compound, a
hinl compound, a ~1. .";1"",;".~...,1 compound, a metal chelator or an
enzyme. Those of ordinary skill in the art will know of other suitable labels for binding
to the antibody, or will be able to ascertain such, using routine ~AIJI ' ' "
15 For purposes of the invention, an antibody or nucleic æid probe specific for a
Spirochaelales ROM protein may be used to detect the presence of ROM protein or
fragment thereof in biological fluids or tissues. Any specimen containing a detectable
amount of ROM protein antigen or pol~llu~,luuLid~, can be used A preferred specimen of
this invention is blood, urine, ~.~.u~l,..ld fluid, or tissue of skin (epidermis, dermis, and
20 , l .~ ), spleen, liver, heart, brain, and bone origin.
Another technique that may also result in greater sensitivity consists of coupling
antibodies to low molecular weight haptens. These haptens can then be specifically
detected by means of a second reætion. For example, it is common to use such haptens
as biotin, which reacts with avidin, or d~LIu~ pyridoxal, and fluorescein, which25 can reæt with specific antihapten antibodies.
WO95/18632 2 ~ 8~27 .~ 3~
-21 -
Altematively, the ROM proteins of tbis invention, or antibody-binding fragments thereof,
can be used to detect antibodies to Spiroc~aetales ROM proteins in a specimen. The
ROM protein of the invention is particularly suited for use in;"", ,. ~ in which it
can be utilized in liquid phase or bound to a solid phase carrier. In addition, ROM
5 proteins used in these assays can be detectably labeled in various ways.
Examples of ;~ A.rD that can utilize the antibodies or ROM proteins of the
invention are competitive and ~...,. ... ,l,~:;l;v~;" ..,.... ,.~-- - ..~, in either a direct or indirect
format. Examplesofsuchi,,, ~ ^JDarethe ' y(RIA),thesandwich
(;,.". ."."". l, ;c assay) and the Western blot assay. Detection of antibodies which bind
10 to the ROM proteins of the invention can be done utilizing ;", ,. I~ Ay ~i that run in
either the forward, reverse, or ! ' '' , modes, including l . , . l
assays on physiological samples. The: of ROM protein used will vary
depending on the type of .~ and nature of the detectable label used. However,
regardless ofthe type of ;"",- lI~ A,y used, the ~... ...,I.AI;. ,., of ROM protein utilized
15 can be readily detemlined by one of ordinary skill in the art using routine
~.All. . ;,. .,:-:;nn
The S~" v~ O ROM protein or antibody-binding fragments thereof of the invention
can be boumd to many different carriers and used to detect the presence of antibody
specifically reactive with the protein. Examples of well-known carriers include glass,
20 polystyrene, polyvinyl chloride, pvl~ u~yl~"le, pOl~.,;hJI~ " pOIy~.all ', dextran,
nylon, amyloses, natural and modified celluloses, pOl.~a.,l~lallfid~,~, agaroses, and
magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the
mvention. Those skilled in the art will know of other suitable carriers for binding the
ROM proteins of the invention or will be able to ascertain such, using routine
- 25 ~.l,.. ;", ,: l;",~
There are many different labels and methods of labeling known to those of ordinary skill
in the art. Examples of the types of labels which can be used in the present invention
WO 95/18632 2 1 8 ~ 4 2 7 PCTNS95/00190
-22-
include enzymes, ~ vlv~ colloidal metals, fluorescent C~mrVlln~c
rhrmil.,.";". ~.,1 rnmrvl-n-lc andl,;~l,.",;~ rnmrol~n~lc
For purposes of the invention, the antibody which binds to ROM protein of the invention
may be present in various biological fluids amd tissues. Any sample containing a5 detectable amount of antibodies to ROM protein can be used. Normally, a sample is a
liquid such as urine, saliva, v~,le'v.ua,u;ll~.l fluid, blood, serum and the like, or a solid or
semi-solid such as tissue, feces and the like.
The ~ "~l antibodies of the invention, directed toward Spirochaetales ROM
proteins, are also useful for the in vivo detection of antigen. The detectably labeled0 ""....~ l antibody is given in a dose that is ~ y effective. The term
lly effective" means that the amount of detectably labeled mnnrlr~lfm~l
antibody is I ' ~l in sufficient quantity to enable detection of S,v;, v~I.a~
protein antigen for which the ' ' antibodies are specific.
The ..~ ;l- of detectribly labeled ." - ~ antibody ~ ' ~v should be
15 sufficient such that the binding to those cells, body fluid, or tissue having ROM protein
is detectable compared to the; 'Cv ' Further, it is desirable that the detectably
labeled, .... -, I..., ~ antibody be rapidly cleared from the circulatory system in order to
give the best target-to-background signal ratio.
As a rule, the dosage of detectably labeled ml~n~lrlf~n^l antibody for in vivo diagnosis will
20 vary depending on such factors as age, sex, and extent of disease of the subject. The
dosage of,, ..., ,,., I....,.l antibody can vary from about 0.001 mglm2 to about 500 mg/m2,
preferably 0.1 mglm2 to about 200 mglm2, most preferably about 0.1 mglm2 to about 10
mglm2. Such dosages may vary, for example, depending on whether multiple injections
are given, and other factors known to those of skill in the art.
WO 95/18632 2 ~ ~ $ 4 ~ lgo
-23-
For in vivo diagnostic imagmg, the type of detection instrument available is a major factor
in selecting a given ~J;u;.uLu~. The " 1if i~uLu~f chosen must have a type of decay
which is detectable for a given type of instrument. Still another import~2nt factor in
selecting a, A~ U~U~lf- for in vlvo diagnosis is that the half-life of the, ,..~ u~ul~ be
cj long enough so that it is still detectable at the time of maximum uptake by the target, but
short enough so that deleterious radiation with respect to the host is minimized. Ideally,
a, Afli. .;~. .lul.~ used for in vivo imaging will lack a particle emission, but produce a large
number of photons in the 140-250 key range, which may be readily detected by
~u~ ..iulldl gamma cameras.
10 For in vivo diagnosis, ~ ulul~ ~ may be boumd to i .. " ., ., ..~gl.:.l,. ,1; " either directly or
indirectly by using an ' functional group. ~ ' functional groups
which oflen are used to bind radioactive metallic ions to ;,."..."..~gl~ "c are the
.;r,... ~ chelating agents such as di~ acid (DTPA) and
ethyl.. -~ acid (EDTA) and similar molecules. Typical examples of
15 metallic ions which can be bound to the monoclonal antibodies of the invention are " 'In,
9'Ru 6'Ga 68Ga '2As, 89zr, 20'TI, and 99mTc.
The ",..,~f.~ antibodies of the invention can also be labeled with a 1~ ll. ;f
isotope for purposes of in vivo diagnosis, as in magnetic resonance imaging (MRl) or
clectron spin resonance (ESR). In general, amy l,u..~. ' method for visualizing
20 diagnostic imaging can be utilized. Usually gamma and positron emitting ,A.1;..: ~vlul . ~
are used for camera imaging and ,u - ~ ~L ; f isotopes for MRI. ~lements which are
~uolLi~,ul~ul~ useful im such techniques include ~5~Gd, 55Mn, 162Dy, 52Cr, and 56Fe.
The ,.. ~ l antibodies of the invention can be used to monitor the course of
Al 11. i', ~ 11 of S,[,;, u~huritul~,~ associated disorder. Thus, by measuring the increase or
25 decrease of antibodies to ROM protein antigen present in various body fluids or tissues,
it would be possible to determine whether a paTticular therapeutic regiment aimed at
the disorder is effective.
WO 95/18632 2 1 8 ~ 4 2 7 ~ 190
-24-
The materials for use in the method of the invention are ideally suited for the preparation
of a kit. Such a kit may comprise a carrier means being ~,UIII,Ual Llll~,ltd~ to receive in
close ~ . " ,1;,.. . ". . ,1 one or more container means such as vials, tubes, and the like, each of
the container means comprising one of the separate elements to be used in the method.
5 For example, one of the container means may comprise a S~;, v~ ~ / ROM proteinbinding reagent, such as an antibod~. A second container may further comprise ROM
proteins or fragments. The ~ may be present in liquid or Iyophilized form, as
desired.
Mr)n~ nAI antibodies are made from antigen containing fragments of the protein by
10 methods well known in the art (Kohler, e~ aL, supra, 1975). The term "antibody" as used
in this invention is meant to include intact molecules as well as fragments thereof, such
as Fab and F(ab')2, which are capable of binding the epitopic d ~ .",;..~.,1 as well as
genetically engineered antibody molecules such as single chain, chimeric, CDR grafted
antibo~ies, and variants thereof known to those skilled in the art.
15 The arltibodies of the invention can be used in ~ ~ , . ' y for the
isolation of protein fragments and amino acid sequences in ROM proteins containing
amtigenic activity of the present invention. One way to utilize ~ aJ
..1".." '. ~5~..1~1~Y can be utilized is by the binding of the antibodies of the invention to
CNBr-S~l.a.u ,~-1B or Tresyl activated Sepharose (Pharmacia). These solid phase-
20 bound antibodies can then be used to specifically bind sequences containing the antigenicactivity of ROM proteins from mixtures of other proteins to enable isolation and
purification thereof. Bound sequences can be eluted from the affmity ~,L.,
material using techniques known to those of ordinary skill in the art such as, for example,
chaotropic agents, low pH, or urea.
25 The invention provides polyl-u~ vLid~ encoding the isolated ROM proteins, preferably
those of the genus Treponema, most preferably the ROM proteins of T. pallidum shown
in Table I above. These ~uvlyllu~ uLi~ include DNA, cDNA and RNA sequences which
WO9S/18632 2 1 ~ Q 4 2 ~ P~l/u~ o
-25-
encode ROM proteins or antigenic fragments thereof. It is understood that all
polynucleotides encoding all or a portion of ROM proteins are also included herein, so
long as they encode a polypeptide with antigenic activity. Such polyllu.,l~,vLi.lcs include
both naturally occurring and intentionally ~ l polym~ otiA-~ For example, a
5 ROM protein may be subjected to site-directed v - The polyllu~,L,-vLi~ of the
mvention include sequences that are degenerate as a result of the genetic code. There are
only 20 natural amino acids, most of which are specified by more than one codon.Therefore, as long as the amino acid sequence of the ROM protein is unchanged, or
although changed retains the antigenic activity of the Cvllc, " ~ ROM protein, all
10 degenerate nucleotide sequences are included in the mvention.
As used herein the term "antigenic activity" shall mean the protein or povly,u~lv~idc binds
with suitable affinity under physiologic conditions to an antibody known in the art to be
associated viith the disease state caused by the pathogen from which the ROM protein or
polypeptide is derived. Alternatively, "antigenic activity" means that the protein or
15 poly,u~"ulhle binds with such affinity to an antibody specific to a S,v;lu~ ROM
protein isolated by the method of the present invention. DNA sequerlces of the invention
can be isolated by several techniques known in the art. These mclude, but are not limited
to: l) L~lvl;vi~iivll of probes to genomic or cDNA libraries to detect shared nucleotide
sequences, and 2) antibody screening of expression libraries to detect shared structural
20 features.
Screening procedures which rely on nucleic acid ll~VI;vi~dtiUII make it possible to isolate
any gene sequence from any organism, provided the appropriate probe is available. For
example, ~ l v~i-lr probes, which correspond to a part ofthe sequence encoding the
protein in question, can be synthesiæd chemically. This requires that short, ..liV. ~
25 stretches of amino acid sequence must be known, preferably of at least l 7 nucleotides in
length. The DNA sequence encoding the protein can be deduced from the genetic code,
however, the degeneracy ofthe code must be taken into account. It is possible to perform
a mixed addition reaction when the sequence is degenerate. This includes a ll~ uv.,.luu~
WO95/18632 2 1 804 27 /~ 5~. --
-Z6-
mixture of denatured double-stranded DNA. ~or such screening, hybridization is
preferably performed on either single-stranded DNA or denatured double-stranded DNA.
This is especially useful in the detection of cDNA clones derived from sources where an
extremely low amount of mRNA sequences relating to the polypeptide of interest are
5 present. In other words, by using stringent ll~bl;di~d~iull conditions directed to avoid
non-specific binding, it is possible, for example, to allow the A~
vi~uali~livll of a specific cDNA clone by hyhlhli~iull of the target DNA to the single
probe in the mixture which is its i...,..l,l~ ,. ~ (Wallace, e~ al, Nucleic Acid Research,
9:~79, 1981).
10 A cDNA expression library, such as lgtll, can be screened indirectly for ROM
,uuly,u~,~Jtid~,~ having at least one amtigenic epitope, using antibodies specific for a ROM
protein isolated from live pathogen according to the method of this invention orantibodies from the blood of individuals infected with the pathogen of interest previously
identifled as specific to the pathogen of interest. Such antibodies can be either
15 mr~ntlrl--n~l or polyclonal and used to detect an expression product indicative of the
presence of a ROM cDNA.
A ROM protein cDNA library can also be screened by injecting different cDNAs into
oocytes. After expression of the cDNA gene products occurs, the presence of the specific
cDNA gene product can be identified by antibody screening with antibody specifically
20 illllllullul~ liv~ with ROM ~ul,y,u.,~vlid~,~, for example. Alternatively, functional assays
for ROM proteins of toxicogenic activity could be performed to identify ROM proteins
producing oocytes.
Specific DNA sequences encoding Spirochaetales ROM protems can also be obtained by:
(I) isolation of double-stranded DNA sequences from genomic DNA; (~) chemical
25 Illall~Lul~ of a DNA sequence to provide the necessary codons for the polypeptide of
interes~ and ~3) in vitro synthesis of a double-stranded DNA sequence by reverse
2~ 27
wo sstl8632 P~ ,~ 190
-27-
tr~ln~ rirti.7n of mRNA isolated from a eu iaryotic donor cell, resulting in a cDNA, or
Y DNA.
- Synthesis of DNA sequences is frequently the method chosen when the entire sequence
of amino acid residues of the desired ~ùlylu~ Lidc product is known. When the entire
5 sequence of amino acid residues of the desired pul~ /tilc is not known, the direct
synthesis of DNA sequences is not possible, and the method of choice is the formation
of cDNA sequences. Among the standard procedures for isolating cDNA sequences ofinterest is the formation of plasmid or ba~ ;u~ ~ based cDNA libraries in which
mRNA is reverse tr.mscribed from donor cells with a bigh level of genetic expression.
10 When used in ~"".1,;.,..;,~" with polymerase chain reaction (PCR) techmology, less
common mRNA species (cDNA) can be cloned as well. ~then sigluficant portions ofthe
amino acid sequence of a polypeptide are known, labeled single or double-stranded DNA
or RNA probes which represent a sequence present in the target cDNA, may be used in
DNA/DNAl..yl,.;d~Liu..procedureswhichareperformedonclonedcopiesofthecDNA,
15 which have been denatured into a single-stranded form (Jay, et aL, Nucleic Acid
Research, 11:2325, 1983).
Since the novel DNA sequences of the invention encode a unique sequence of ROM
protein, it is now a routine matter to prepare, subclone, and express smaller polypeptide
fragments of DNA from this or ~ullc~.vl.d.llg DNA sequences. Alternatively, by
20 utilizing a DNA fragment, it is possible, in uu; with known techniques, to
determine the DNA sequences encoding an entire ROM antigenic protein. Such
techniques are described in U.S. Patent Nos. 4,394,443 and 4,446,235, which are
ill~.UI UUl...~,d herein by reference.
The poly~L;~e resulting from expression of a DNA sequence of the invention can be
25 further ..1, ~ 1 as being free from asssciation with other eukaryotic polypeptides
or other ~ '. that might otherwise be associated with the ROM protein in its
natural cellular ~,IIV;IU~ t. Isolation and purification of microbially expressed
wo95/18632 2 t 8 ~ ~ 2 ~ P~l/u ~ cc~
-28-
polypeptides provided by the invention may be by ~U~ iU~ means including,
preparative ~LI~ separations and illllllullOlogi~,al separations involving
,,,,. ncl. ,,~l and/or polyclonal antibody preparation.
For purposes of the present invention, ROM pùl~ that are hnn1nln~ollc to those
5 of the invention can be identified by st~uctural as well as functional similarity. Structural
similarity can be L:h" I, for example, by assessing pOI~ 1 ~- strand
hybridization or by screening with antibody, especially a ~ ..nrl-~ antibody, which
recog~uæs a unique epitope present on a ROM protein disclosed in this invention. When
llyl~lhl;LdL;ull is used as criteria to establish structural similarity, those pol~llu~ ,uL;d~
10 sequences that hybridiæ under stringent conditions to the polynucleotides of the
imvention are considered to be essentially the same as the l~ul y ' ' sequences of the
irlvention.
A wide variety of ways are available for introducirlg a polyl...~,~eul;de expressing a
Spirocllaetales ROM protein into the uulOalfi....l host under conditions which allow
15 for stable and expression of the gene. DNA constructs are available which
irlclude tbe ~ and h ~n~l ~inn I regulatory signals for expression of the ROM
pol~.lu,,l~uLill~, the ROM gene under their regulatory control, and a DNA sequence
Il..., ..~1-."....~ with a sequence in the host organism, whereby integration will occur; and/or
a replication system which is fimctional in the host, whereby integration or stable
will occur.
The ~ initiation signals will include a promoter and a 1.~ ,~...;1~1;,.,.~1
irlitiation start site. In some instances, it may be desirable to provide for regulative
expression of the ROM ~JUI,yllU ,Iw~;de. This can be achieved with operators or a region
binding to an activator or enhancers that are capable of induction upon a change in the
25 physical or chemical ~ ;IUIIII~ of the host. For example, a t~ sensitive
regulatory region may be employed where the organisms may be grown up in the
laboratory without expression of the ROM protein, but upon change m the growth
2 t 8~
WO 9S/18632 . ~, I "~ .
-29-
conditions or ~lv;lu~ lL, expression would begin. Other techniques may employ a
specific nutrient medium in the laboratory, which inhibits the expression of the ROM
protein, where the nutrient medium in the later .,..vi., would allow for expression
oftheROMprotein. Forl,r,,~l-l;,.,,-'initiation,aribosomalbindingsiteandaninitiation
5 codon will be present.
Various ~ c may be employed for enhancing the expression of the mRNA,
~ uLi~,ukuly by using an active promoter, as well as by emplûying sequences, which
enhance the stability of the mRNA. The initiation and ~ rl termination region
will involve stop codon(s), a terminator region, amd optionally, a polyadenylation signal.
10 In the direction of l~ , namely in the 5' to 3' direction of the coding or sense
sequence, the construct will involve the l, ,.. ~ . ;I-l ;. ." -l regulatory region, if any, and the
promoter, where the regulatory region may be either 5' or 3' of the promoter, the
ribosomal binding site, the initiation codon, the structural gene having an open reading
frame in phase with the mitiation codon, the stop codon(s), the pOI~ad~ Liul~ signal
15 sequence, if any, and the terminator region. This sequence as a double strand may be
used by itself for l l ,., .~ r .. "- ~;. ,., of a IlI;l~lUUl~ .. host, but will usually be included
with a DNA sequence involving a marker, where the second DNA sequence may be
joined to the expression construct during ud,,_Liu~ of the DNA into the host.
A marker structural gene may be present that provides for selection of those hosts tbat
have been modified or r ~ The marker will normally provide for selective
advantage, for example, providing for biocide resistance, for example, resistance to
antibiotics or heavy metals; ~ , ' so as to provide prototropy to an
ULIUIJllic host, or the like.
Where no functional replication system is present, the construct will also include a
sequence of at least 50 basepairs (bp), preferably at least about 100 bp, and usually not
more than abou~ 1000 bp of a sequence l " r~ g~ with a sequence in the host. In this
WO 95/18632 2 1 8 ~ 4 2 7 P~ t t l,t,
-30-
way, the probability of legitimate ~ AIinn iS enhanced, so that the gene will beintegrated into the host and stably maintained by the host. Desirably, the ROM protein
gene will be in close proximity to the gene providing for ~ i..., as well as thegene providing for the competitive advantage. Therefore, in the event that a ROM protein
gene is lost, the resulting organism will be likely to also lose the .:.. I.l.. ,.. ,I;.~ gene
amd/or the gene providing for the competitive advantage, so that it will be unable to
compete in the ~,.vi.u. Il~l.. with the gene retaining the intact construct.
A large number of ~ regulatory regions are available from a wide variety of
~ ,lUUl~;~lllialll hosts, such as bacteria, 1~ , algae, fungi, and the
10 like. Various ~ Al regulatory regions include the regions associated with thetrp gene, lac gene, gal gene, the lambda left and right promoters, the Tac promoter, the
naturally-occurring promoters associated with the ROM protein gene, where functional
m the host. See, for example, U.S. Patent Nos. 4,332,898, 4,352,832 and 4,356,270. The
t. rrnirotinn region may be the termination region normally associated with the
1 ~i 1~,,, ,~., ;,,1;,~,~ ~1 initiation region or a different trAnc~ rirfinn~l initiation region, so long as
the two regions are compatible and functional in the host.
Where stable episomal or mtegration is desired, a plasmid will be employed
tbat has a replication system that is functional in the host. The replication system may be
derived from the ~,LUIIIO~UII.~ an episomal element normally present in the host or a
20 different host, or a replication system from a virus that is stable in the host. A large
number of plasmids are available, such as pBR322, pACYC184, RSFIOIO, pR01614,
and the like (see, for example Olson, et al., J: Bacteriol. 150:6069, 1982, amd
p ~g,1 ~orion et aL, Gene, L:237, 1981, and U.S. Patent Nos. 4,356,270, 4,362,817, and
4,371 ,625.)
25 The ROM pol~ ' ' can be introduced between the ~ l and l,~
initiation region and the tr~nC~rirtinnol and 1., . . 1-. ;. ", ~ i region, so as to be
under the regulatory control of tbe initiation region. This construct will be included in a
WO95/18632 21~D4~7 r.l~u~ liJ~,
-31 -
plasmid, which wil~ include at least one replication system, but may include more than
one, where one replication system is employed for cloning during the u~ uy~ lL of the
p~asmid and the second rep~ication system is necessary for functioning in the u~timate
host. In addition, one or more markers may be present, as described above. Where5 integration is desired, the p~asmid wi~ desirab~y inc~ude a sequence homo~ogous with the
host genome.
The ~ can be iso~ated in accordance with ~v~ iullal techniques usua~y
employing selection of the desired organism as against unmodified organisms or
l,,...~r ,.;"ba organisms, when present. The j r ' then can be screened for
10 pesticida~ activity.
As hosts, of particular interest wi~l be the ,uluh u~ut~ and the lower eukaryotes, such as
fungi. I~ustrative ~luhdl~u~s, both G~am-negative and -positive, include
Ellltl.,~ , such as l~scherichia, ~rvinia, Shigella, .C ~ ~In and Proteus;
J~lri~ rono; Rhizobiceae, such as Rhizobium; Spirillaceae, such as r~
Z, . . Serratia, ~eromonas, Vibrio, Db3UIIb' V~b~l iO, Spirillum; 1.~7~ t~b~ itl~7r~10;
- p, . ~ -~- ?, such as P and ,4cetobacter; A",/"h~ .b~.~UL and
Ni~Jbu~ a~eu~. Among euharyotes are fungi, such as ~i,, , and A
which includes yeast, such as Sac"h..,, and S..~ ..hu", , and
Rn~ , _ yeast, such as Rhodotorula, ~ ' ' . S~,u, . ~ , , and the
20 ~ike.
Host organisms of particu~ar interest include yeast, such as Rhodotorula sp.,
Au, , L ~' Sp., Sac.hu, ., sp., and Sporobolomyces sp.; phy~lop~ane organisms
such as r~ ~. Sp., l~rwinia sp. and Flu v~bu~kl ' Sp.; or such other organisms
as Escherichia, ~.~7rt(~bori~ sp., Bacillus sp., and the lihe. Specific organisms inc~ude
25 P:~ZUU!b....J~ aeruginosa, r~ 1 fluorescens, Su..hu" , cerevisiae, Bacillus
6 ~ EDcherichia coli, Bacillus subtilis, and the ~ike.
WO95/18632 218042~ P~ 5[[~
-32-
In general, expression vectors containing promotor sequences which facilitate the efficient
;.". of the inserted genetic sequence are used in connection with the host. As
desc}ibed above, biologically fimctional viral or plasmid DNA vectors capable ofexpression and replication in a host are known in the art. Such vectors are used to
5 incorporate ROM protem encoding DNA sequences of the invention. Expression vectors
typically contain an origin of replication, a promoter, and a terminator, as well as specific
genes that are capable of providing phenotypic selection of the l ".. . r " . . ,~ ~l cells.
T,r ~ .n of the host cell with the ~ DNA may be carried out by
conventional techniques well known to those skilled in the art. Where the host is
10 prokaryotic, such as E. coli, competent cells wbich are capable of DNA uptake can be
prepared from cells harvested after exponential growth amd ~, ,l .~,.l.... ,l ly treated by the
CaC12 method using procedures well known in the art. Alternatively, MgCI2 or RbCI
could be used.
Where the host used is a eukaryote, various methods of DNA transfer c~m be used. These
15 include~ r l;....ofDNAbycalciumphosphate~ JIIV~ '' I mechanical
procedures such as III;~ ,Liull or el.,~ u~ liull, insertion of a plasmid encased in
liposomes, or the use of viral vectors.
Eukar,votic host cells may also include yeast. For example, DNA can be expressed in
yeast by inserting the DNA into appropriate expression vectors amd introducing the
20 product into the host cells. Various shuttle vectors for the expression of foreign genes in
yeast have been reported (" , J. et aL, Nature, ~11:205, 1989; Rose, et aL, Gene,
oO:237, 1987).
Isolation and purification of microbially expressed protein, or fragments thereof provided
by the invention, may be carried out by ~ull~ l means including preparative
el.. ~ .I.y and ;~,.. ,.. ~.. rlr~ l separations involving ~.. "~rl.,.. ,.l or polyclonal
antibodies. An~ibodies provided in the present invention are ;IlL..~.~u~ .,Live with the
W09~i/18632 2 1 8 0 ~ 2 7 . ~ . 5.~ ~i3~
-33-
Spiroc~aetales ROM proteins of the invention. Antibody which consists essentially of
pooled m(~nnrl~nql antibodies with different epitopic ~ ;r~ as well as distinct
mr,nrrlrnql antibody l~lC~ r~iUIIa are provided. Mrnrrlrnql antibodies are made from
antigen containing fragments of the ROM protein by methods well known in the art5 (Kohler, et aL, supra, 1975; Current Protocols in Molecular Biolo~, Ausubel, et aL, ed.,
1989).
Minor mrrlifirqtirn~ of the ROM protein primary amino æid sequence may result in~ol~ Jtid.,. that have substantially equivalent antigenic ætivity compared to the ROM
proteins and polypeptides described herein. Such ~ "r.~l; r~ may be deliberate, as by
10 site-directed ~ , or may be ~ All proteins produced by these
" ,r..l; r;. ~ . are included herein as long as the antigenic activity for antibodies specific
to ROM proteins is present.
The r...".l~r.~ ,. of the amino æids of the ROM proteins of this invention can be
determined by methods well ~qnown in the . rt, for instance, the rll~ ;llOll~da~ltuillc
(PTH) method of Edman, et aL (Europ. J. Bioc~zem., 1:30, 1967). Briefly, in this method
tbe purified protein is dried under vacuum and redissolved in a small volume of
æetonitrile 95% plus TFA (0.08/~). The, ' sample is then introduced into a
gaseous phuse sequencer connected to a I ' ~ ' ' r ' (PTH) analyzer. From the
amino æid sequence so obtained, a DNA sequence encoding the protein can be deduced
20 usmg routine methods well Iq~nown in the art.
Alternatively, to discover the nucleotide sequence of DNA material obtained using the
methods of this invention, double str..nded dideoxy sequencmg can be performed, for
example on a DuPont Genesis 2000, using the DuPont Genesis 2000 sequencing kit
according to the Illrllu~Lul~ hl~tlu~ . Post gel processing can be dorle with the
25 Base Caller 5.0 program (DuPont, Boston, MA). Alternatively, a DNA sequence of the
clone can be obtained usmg a S l~ II kit (United States Rir,rh~nnirql Cleveland,OH) on the automated DNA sequencer Genesis 2000 (Dupont, Wilmington, DE)
WO95/18632 2 1 8~427 P~ u...~
-34-
according to the ~ lra~,~UU~I'a instructions. The DNA encoding the gene may also be
chemically synthesized (Merrifield, J. Am. Chem. Soc, 85 pp. 2149 (1963)), or generated
by PCR.
The following examples iliustrate the manner in which the invention can be practiced.
5 rt is understood, however, that the examples are for the purpose of illustration and the
invention is not to be regarded as limited to any of the specific materials or conditions
therein.
~.XAMPLli'. I
Source of T. CA T. pallidum, subsp. pallidum, Nichols strain, was maintained
10 by testicular passage in New Zealand White rabbits as described previously (Miller, et al..
Br. .l Vener, Dis., 39:195, 1963). Animals used to prepare T. pallidum outer membrane
and antigen for sodium dodecyl sulfate (SDS)-,uul~ Aud~ gel ~ u~ul~u~cia;a (PAGE)
were injected ;.,~ ly with 10 mg of cortisone acetate (Merck Sharp & Dohme,
Rahway, NJ) per kg of body weight from days 3 through 12 after infection.
1~ T. vincentii was grown in spirolate broth (Gibco) l l ' ' with 10% heat-inactivated
rabbit serum. A~,u., ~, 300ml of culture containing 2 X 108, ~, /lul was
centrifuged at 10,000 X g for 15 min. The resulting treponemal pellet was ~ aua,u~lld~,;l
in 140ml of phosphate buffered saline (PBS), pH 7.2, and used for outer membraneisolations or l~c . . .: . i r, ,. ~I as described for use as antigen in SDS-PAGE.
Pul ' of T. pallidum. A total of 300 ml of PBS, pH 7.2, In 50 ml volumes was
used to extract treponemes from 20 infectea rabbit testicles. The treponemal cll~rPncion
containing alu,ull ~y 6 X 10~ organisms was subjected to two low speed
c.. ,,,; r, .~"~ i.. at 4000 X g in order to remove gross tissue debris. Treponemes were then
pelleted by ~ 1 .; r"~ ; .,. at 20K X g for 15 mm followed by 1~ ;. ,., m 40 ml of
~t8Q~
w0 95/18632 ~ .,5. ~ [ i90
-35-
PBS contaming 0.5% Bovine serum albumin (BSA; Intergen Co., Purchase, NY) and 7% FICOLL~M density gradient separation medium (Pharmacia, Piscataway, NJ). Ten
milliliters (l O ml) of suspension was layered onto 25 ml of a .1~ Ficoll/PBS
gradient with increasing buoyant densities of 1.045, 1.055, 1.065, and 1.075, and 1.085
5 g/ml. After c~ ,.;,;r"~ ., at 7K X g for 15 min, several bands were observed in the
gradient. Previous studies using darkfield and electron Ill;.~lU~Uyy of the fourI~l.,,;.y zones have shown "clean" single treponemes within tne two upper zones
(1.065-1.055 and 1.055-1.045) and some clumped and single ~I~,yoll.,~ plus host cell
debris within the two lower zones (1.085-1.075 and 1.075-1065). Only treponemes
10 recovered from the uppermost zone gradient by needle aspiration, followed by a 4-fold
dilution in PBS, were used for subsequent ~ The resulting treponemal
suspension was used ~-~~ ' '~ for extraction of the outer membrane.
F.XAMPLF. 2
Isolation of T. pallidum and T. vincentii Outer 1` 1 ' - To 140 ml of treponemal15 suspension obtained in Example I containing ayyl- ~/ S X 10~ l~cyull~ cj wasadded 200 ul of Rl 8 octyl-decyl Rhodamine chloride (Molecular Probes, Inc., Eugene,
OR). The suspension was incubated at rûom A,llly~ t~ for 10 min and then centrifuged
at 8K X g for 20 min. For removal of the outer membrane, the treponemal pellet was
Il ..~1.. "~r~l into 60 ml of ice cold 0.05M sodium citrate buffer, pH 3.2, and incubated
on a rocker with occasional vortexing for 2 hrs at room i . c to release the outer
membrane from the inner membrane. The suspension was then centrifuged three times
at 8K X g for 15 min in order to remove treponemal ylu~uyla~ ic cylinders. The
supernatant containing released outer membrane was then neutralized using IM Tris-HCI,
pH 9.0, and centrifuged at 150K X g for 16 hrs at 15C. The resulting membrane pellet
was l~ A into 2 ml of PBS, layered onto 36 ml of a continuous 5-40%
sucrose/PBS gradient for 7'. pallidu~n or 10-40% gradient for 1' vincentii, and centrifuged
at 100 K X g for 16 hrs at 15 C. Following ~ ,"; r~ ., the outer membrane band,identified visually by Rhodamine labeling, waS needle aspirated, diluted 7-fold with PBS,
WO g5/18632 2 ~ 8 0 4 2 7 r~
-36-
and 1~ ,, .; r~ at 1 50K g for 5 hr. The final purified membrane pellet was, r ~im 100 ul of PBS containing ImM EDTA, ImM PMSF, and stored at 4C. As shown in
Figure IB, the citrate buffer treated organisms showed re~ease of outer membrane.
~.X~MPLF 3
5 Electron ~fi..~ . ~ (EM) ~nd Freeze-Fr~cture EM. For electron IllL,lua~ul~y, aPARLODION(g) grid film cover (Mallinckrodt, Inc., St. Louis, MO) and carbon-coated
300-mesh copper grids (Ted Pella, Inc., Redding, CA) were floated for 5 min on 40 ul
specimen drops. After 3 washes in PBS and 2 washes in double-distilled water, the grids
were negatively stained with 1% uranyl acetate and examined in an electron III;~IU~ U~
10 (JEOL IOO CX) at 80 kV d'~ . 1. . ,.1;..~ voltage. Freeæ-fracture EM of outer membrane
vesicles was performed as follows. Fifty microliters (50 ul) of membrane suspension was
pelleted by - - I ~; r ~ . at 200K X g for 3 hrs and ~ 1 1 in I ul of 20% glycerol
in double-distilled water. A 0.5 ul sample was placed on a standard Balzars specimen
holder (Balzars Co., Redding, CA) and froæn by immersion in liquid propane (-190'C)
15 using a guillotine-type device. Frozen samples were transferred under liquid nitrogen to
the specimen stage of a Balzars 400K freeæ-fracture apparatus precooled to -150C.
Frozen samples were fractured at -120C by using a knife cooled at the t~ l,U.,.~ of
liquid nitrogen. The fracture surface was ' '~ replicated with platinum-carbon
at 45C and carbon at 90C. The replicas were floated in 3-4% sodium Ly~u~,lllul;.~. to
20 bleach the organic material and washed three times in double-distilled water. The replicas
were then placed on Formvar-coated freeæ-fracture grids (Ted Pella, Inc.) and observed
by electron IlllWU~U~,y as described above. The electron micrograph shown in Figure 2D
shows few ;~ protein particles.
2 ~ 80427
wo 95/18632 ~ , 5
-37-
T'XAI~rPT,T~'. 4
Isolation of ROMs from Outer r~ ` - Using One ~nd Two D- '
SDS-PAGE. SDS-poly~ ;d~ slab gels were run by using the ~ v~ buffer
system of Laernmli (Laemmli, Nature, London, U.K., 22:Z:680-685, 1970). Samples
containing 5 X 1 o8 whole organisms or I to 5 X 109 treponemal equivalents of membrane
material were boiled for 10 min in final sample buffer containing 4% SDS, 10%
2-,. .~ v~ l, and 0.01% blu~ lol blue in 62.5 mM Tris buffer, pH 6.8 (FSB);
for some samples, urea at a final ".,.. 1,"1;.,., of 8M (FSB-U) was included. In some
l .l.~ . ;. ,.. '~, samples were solubilized in FSB containing proteinase K (Sigma Chemical
Co., St. Louis, MO) at a .. .l ~ l of l00 uglml and incubated for 1 hr at 37~C before
boiling. Two-d;...- ,~ l gel el~ ul~l.v~ was performed as described by O'Farrell(J. BioL Chem., ~Q:4007-4021, 1975) with minor ,f~ Outer membrane
material containing from 5 X 109 to 3 X 10' treponemal equivalents was fr~st solubilized
for 1 hr at room t~ in Iysis buffer containing 9M urea, 2% Nonidet P-40 (NP40)
15 (Sigma Chemical Co., St. Louis, MO) and 2% carrier ampholytes at pH 95. Isoelectric
focusing was carried out for 18 hrs at a constant voltage of 600v in 0.2 cm x 12 cm tube
gels containing 2% pH 5-7 and 0.8% pH 3-10 Ampholines (BioRad, Richmond CA), 2%
NP40, and 9M urea. The second dimension consisted of standard SDS-PAGE as
described above. After el~ vl,hJl.,~;" gels were stained with Coomassie brilliant blue
20 v. L~ f~ topolyvi~.ylid~ defluoride(PVDF)membranes(Millipore,Bedford,MA)
as previously described (Towbm, e~ al., Porc. Narl. Acad Sci. USA, ~:4350-4354, 1979).
Following transfer, PVDF membranes were stained with 1% Amido Black or Aurogold
Forte (Amersham, UK). For ;,...,..,...~l.l..ll;,.p PVDF mPmh~nPc were incubated for I
hr with serum diluted 1:1000 m PBS containing 5% nonfat dry milk (Carnation Co., Los
25 Angeles, CA) and 0.1% Tween-20 (Sigma Chemical Co., St. Louis, MO) (MT-PBS).
Antibody-antigen binding was detected usmg the enhanced . '- ' (ECL)
system of Amersham (Amersham, UK). Blots were incubated for I hr m anti-rabbit Ig
or anti-mouse Ig conjugated to horseradish peroxidase diluted 1:2500 m MT-PBS. Blots
were next washed in PBS containing 0.1% Tween-20, mcubated for 1 min in the ECL
wo95/18632 2 ~ g 0 4 27 P~
-38-
developing reagents (Amersham, TJK), and then ~..~...,.rl,n~",.l,l,. ~1 with Kodak X-AR5
film. The Coomassie stained gel is shown in Figure IA, and the PVDF i~ ubluL is
shown in Figure 2A.
~,X~MPT.T~. 5
5 Detection of T. pallidum Penicillin Binding Proteins. Penicillin binding proteins
(PBPs) of T. pallidum were identified using '251Odine-labeled penicillin-V as follows.
Sodium ~L~ 51~L~ llu~ ,u-... liin (LillyT.-' EliLillyand
Company, Illd;alla,uOlis~ IN), was labeled with Nal2510dine using chloramine-T as
previouslydescribed(Preston,et al.,Antmicrob.AgentsChemot1~er.,34:718-721,1990).Equal volumes of ~251-penicillin-V were combined with I X 108 Ficoll purified T.pallidum, 1 X 108 T. pallidum ~uluLu~ulr~llf~ cylinders, and 5 X 109 treponemal equivalents
of outer membrane. Suspensions were incubated at room t~ al~c for 30 minutes
prior to .. '.; r~ at lOK X g for l 5 minutes for whole organisms and ~.uLu,ul~l.lic
cylinders, and at IOOK X g for I hr for outer membrane material. Pellets were
15 ~ ~u~.lld~,~ in FSB and c~ LIulJIIul~cd by the SDS-PAGE, using conditions described
above. Following el~,~,L u~l.u.. ,;" the gel was vacuum dried and then Al l~ f .1
v~ith Kodak X-AR5 film at room t.~ t~C for 24 hrs. The results are shown in Figure
4.
T~.X~MPl.F. 6
20 Pr~A " of Antisera from Syphilitic Rabbits. Serum from syphilitic rabbits
immune to challenge (immune rabbit serum; IRS) was acquired 6 months post-infection
;..1. ', ~1:, ,.1-.Iy with4X 10 T. pallidum. Artiserumagainstthe T. pallidum .~.,...1,;" .:
4D protein was prepared as described previously (Radolf, et aL, supra., 1986).
Mnnnrlnn~l antibodies against the T. pallidum 47-kDa lipoprotein (MAb IIE3) and
25 against the 42-kDa TmpA lipoprotein were kindly provided by Dr. Michael V. Norgard,
Univasity of Texas (('l ' ' et al., supra., 1989) and Drs. Jan vanEmben and Leo
2 ~ 80427
WO 95/18632 1r ~ . IJ V
-39-
Schouls, University of Bilthoven, N~ 8~ 1dS (Schouls, et al., Microb. Pathog., 1:175-
188, 1989), Il,a,u.,.,ii~ly. Anti-rabbit serum proteins were purchased from Sigma
Chemical Co., St. Louis, MO.
F.XAMP].F. 7
5 P, .. - of Control Antisera. Antiserum against T. vinçentii was generated in
rabbits as follows. A~u7.i~ y I X 109 PBS washed T. vincenfii organisms were
disrupted by sonication, combined with Freund's complete adjuvant, and injected both
IY (IM) and '~/ (SC). After 3 weeks, animals were boosted IM
and SC using a similarly prepared suspension in Freund's incomplete adjuvant. Animals
10 were bled one week following the boost
R~AMPL~. 8
Isolation of the r. pallidum and r. vincentii outer rl b - The present ûuter
membrane isolation procedure comprises the use ûf several novel steps including (I ) a
Ficoll gradient to purify T. pallidum, octyl-decyl rhodamine to label ,.. 1.. A~ (2) use
15 of a lipid-soluble dye marker, preferably a fluorescent dye marker that intercalates into
the outer membrane amd (3) a lûw ionic strength amd lûw pH buffer for the selective
remûval of the outer membrane.
Ficoll I ..... ri. ~ '.... of T pallidum resulted in significant removal ûf hûst ~...- - -:; ..~
proteins as detemlined by SDS-PAGL as shown in Figure IA, and ' I ~ using
20 anti-rabbit whole serum, as shown in Figure IB. Ficoll purified T. pallidum and PBS
washed T. vincentii were treated with 0.05M Citrate buffer which resulted in the release
of membrane as monitored by fluorescent microscopy of rhodamine labeled material (data
not shown) and by electron Illh~lU~U~ ' as shown in Figure 2B. After 45 mins, the
majority of treponemes had s;~lLlrl.,~l~ly na~rower diameters consistent with the removal
of their outer m.o~llh~m c The absence of .. 1.. n ~;, 11 - filaments, which are dissûciated
WO95/1863Z 2 1 8 0 4 2 7 r~"u~ ù
~o-
to flagellin at low pH (Blanco, et aL, supra., 1988), may have also contributed to the
release of outer membrane material. ('~mrsnc~.n by SDS-PAGE of the ~lu~ullla~llliC
cylinders from citrate treated treponemes with those of whole treponemes showed a
similar profile and intensity of stained proteins (data not shown) indicating that
5 II~JUII~ were not disrupted by this procedure. Sucrose gradient pllrifi~s~inn of
membrane material yielded a single rhodamine labeled band at the 7% sucrose gradient
for T pallidum amd at the 35% sucrose gradient for T. vincentii as determined byrefractive index analysis (data not shown). The membrane nature of this material was
,:' ' by electron ~ v~,u~Jy, which showed membrane vesicles that ranged in
diameter from au~ 8,1y 300 to 700 nm as shown in Figure 2C.
F.XAMPT.F. 9
Freeze-Fracture Eleetron r~li.,.~ , ~ of r'r ' - Vesicles. Purified membrane
vesicles were analyzed by freeze-fracture electron U:~,U,Uy in order to determine
particle ~ -' as shown in Figure 2D. Membrane vesicles from
15 both T. pallidum and T. vincentli contained extremely few protein particles. Of 200 T.
pallidum vesicles observed, only 8 were found to have fracture faces contaming particles;
the number of particles m these fracture faces ranged from I to 3. By ~nmr ~ic~,n of 50
T. vincentii vesicles observed, 22 had fracture faces containing at least I patticle. Total
particle ~ showed that the membrane particle density of T. pallidum was
20 a~ ' ' `y six times less than that of T. vincentii. In contrast, the fraction faces of T.
pallidum and T. vincentii ~IlU~U~Jla~ l;c cylinder inner membranes and of host tissue
n1 -nnhr m-nlc material acquired from -- r ' ~ rabbits contained a relatively high
density of particles (data not shown).
of r. vinccntii Outer r- Vesicles. The detection of the T.
25 vincentii LPS stepladder by; , . " ,l .l. .( analysis of proteinase K (PK) treated membrane,
,ulu~u,ula9111;~, cylinder, and whole organism fractions was used to assess the efficiency of
outer membrane recovery. As shown in Figure 3, the number and intensity of LPS bands
WO95/18632 11 r~l~u,. ~/C[I90
detected from I X 109 equivalents of outer membrane material was similar to that of 2 X
equivalents of whole organisms. By ç~mp i ~-n, 2 X 105 equivalents of l~l u Lu~ l;c
cylinders showed a marked decrease in tbe number and intensity of its LPS stepladder
bamds. These results indicate that a~J~Jl u~dlll_ ~l y 20% of the T. vincen f ii outer membrane
5 was recovered.
T 1 ~ ofthe T. vincentii outer membrane untreated with PK probed with amtisera
generated against whole orgamisms as described in Example 6 above and Coomassie
stained SDS-PAGE (data not shown) also detected two amtigenic proteins with molecular
masses of a~,ul~ 'S 65- and 55-kDa.
10 (~ A " of ~. pallidum Outer r~ ~ Vesicles. Detectionofinnermembrane
associated penicillin binding proteins (PBPs) was used to assess the purity of isolated
outer membrane (I' ~' et al., supra., 1987; Radolf, ef al., supra, 1989). I X I U8
whole T pallidum organism (Tp) and lJlululJlaDlll;~ cylinders amd S X 109 equivalents of
outer membrane (OM) were incubated with '251-labeled penicillin V prior to SDS-PAGE
and ~ ,I,y As shown in Figure 4, major PBPXs of 94-, 80-, 58-, 43-, and
38-kDa were detected in the whole organism and ~lutu~ , cylinder ~ alaL;UllD butnot in the treponemal equivalents of outer membrane material, imdicating the absence of
inner membrane,
In order to determine the extent of periplasmic protein l S X 109 treponemal
20 equivalents of outer membrane material and I X 108 whole organisms were probed on
;"" "... ,, .l .l..l, with specific antiserum against the 19-kDa proplasmic cylinder associated
protein 4D (Radolf, ef al., supra, 1989), specific antiserum against the ~nfl~fl~
(Champion, et al., Infect. Immun., 58:3158-3161, 1990), and a ~ ...f.rl~ antibody
against the 47-kDa major lipoprotein (1'1 ~ laill~ et al., supra, 1989). The results
25 shown in Figure S detect no 47-kDa lipoprotein or 4D protein. Further, only trace
amounts of ~on/l~flag~ were detected, cull~a~u..l;l.g to lrP- ~s~ 0.2%
~n~ fls~ r ~ I ;.. based upon a 1 0-fold decrease in the relative intensities of
WO95/18632 2tgQ427 1~".,~. 1 190
-42-
enfif\flA~ iDr bands detected as compared to those of I X 108 whole organisms. These
findings indicate that the outer membrane preparation was essentially free from
r,.".l,.,.,;.,~ by three ~ present in the periplasm of T pallidum.
Consistent with the known paucity of outer membrane protein in ~. pallidum (Radolf, ef
aL, Proc. Natl. Acad ScL USA, 86:2051-2055, 1989; Walker, et al., supra, 1989) and the
above freeze-fracture EM findmgs, Coomassie stained SDS-PAGE of a~ 1 5
X 109 treponemal equivalents of membrane material failed to detect any major protein
bands (data not shown). The initial itif ntifif otifm of membrane associated protein was
deterrnined ~ntivrr ~ y using immune rabbit antisera (IRS) and enhanced
chrmill.. ;.. f (ECL). The 5 X 109 equivalents of 1'. pallidum outer membrane
materiai were prepared in sample buffer, with and without 8M urea, before being probed
with IRS. As shown in Figure 6, three u~ul~ lLly reacting bands at 17-, 32-, and 45-kD
were detected in the absence of 8M urea. .~.~f~l..l .;1;,-: ;. ., . of membrane material in the
presence of 8M urea resulted in the loss of the 32-kDa band, but not the 1 7and 45-kDa
15 proteins, suggesting only two major antigenic species. This flnding was confLfmed by two
.~ . ".. ,~.... ,~1 (2D), " ", .. ,...~1)1. ~1 analysis as shown in Figure 7. The 1 7-kDa protein had a
pl of greater than 7 and showed additional oligomeric forms at 32- and 45-kDa, while the
45-kDa protein showed a single spot at a pI of f~ JlU~ ly 5.5. The 45-kDa protein
was ,..~ 1y identified as the TmpA lipoprotein (Hansen, et aL, supra, 1985) using
20 specificm~nncl~ antibodies(datanotshown).Longerexposed;...l.. li.~v,,....~ofthese
2D ;1,...l- I~'b~ also identified spots Cullc r ~' V to the pl's of the r~
proteins (data not shown).
In addition to the 17-kda, TmpA, and fnfif~flDvfll~r proteins, gold stained 2D
;",.. ,~.l.l..... l~ of SDS-PAGE gels (shown in Figure 8) containing 3 X 101 treponemal
25 equivalents of outer membrane which were subjected to isoelectric focusing (IEF) pH 5
to 7, showed five additional proteins, including two separate spots with different pl's at
31-kDa and single spots at 28-, 65-, amd 68-kDa. While the 68-kDa protein was shown
WO95/18632 2 1 8 ~427 ~ [l90
43-
by immlmohlot analysis to be rabbit albumin, all of the other proteins reacted specifically
with low dilutions of IRS, indicating their origin in T. pallidum (data not shown).
EY~MPI.F, 10
Cloning of the Trompl gene. To isolate the Trompl gene, two different degenerate5 mixed ol;r,..- ~ re designated 31-A and 31-C, were made to the amino acid
sequence of a tryptic digest amino acid sequence analysis peptide (AHDMQE) and
(EEAEFD), ~c~ ivcly~ generated from the 31-kda native protein (pl 6.7) obtained from
the outer membrane of Treponema pallidum. The primers were used in a PCR reaction
with the 7 to 9 kb fragment of genomic T. Pallidum DNA prepared by digestion with
10 EcoRI. A T. pallidum genomic library was made by partially digesting the DNA with
EcoRI, ~lul, Psal, and HaeIlI. Following digestion, the DNA was purified and EcoRI-
adapted. The restriction fragments were ~ ly cloned into the ~ ZAP Il phage
cloning system (Stratagene, San Diego, CA) and probed with the PCR product generated
using the above described 31-A and 31-C mixed ~ 1Pe From this library, 4
15 clones, designated 2A, 2B, 3, and 6, were identified with the probe. Following plaque
pllrifir~tion PCR was performed on each pbage clone to determine the actual insert size
of DNA. The rnsert size for clones 2A and 3 were both ~Iy~ , 1600 bp. Clone 2B
had an insert size of ~ , 1300 bp. The insert size for clone 6 was too large to
determine by PCR, but was estimated to be greater than 8 kbp.
20 Attempts were made to convert all 4 clones into the ~ errirt SK(-) plasmid form
(Stratagene, San Diego, CA) by in vivo excision. However, results of this attempt
indicated that clones 2A, 3, and 6 were expressing a product toxic to the E. coli cells
harboring the plasmids. The indication of toxicity was the very slow rate at which the
cells were growing. Upon continued cultivation on solid media, the cells began to lose
25 parts of the msert DNA, upon wbich subsequent growth became normal.
wo 95/18632 2 t 8 ~ 4 ~ 1 P~l/u~
-44-
lt was observed that clone 2A appeared to be slightly less toxic than the other two clones.
Therefore, this clone was chosen for further analysis. In order to obtain intactlC l DNA, clone 2A was grown for a very short time on LB agar plate solid
media (Gibco BRL, ~ h~ chl ~ MD.) and then cells were scraped offfor plasmid DNA
5 ulcl on The DNA appeared to be intact based on the insert size of h~u~ 'y
1600 bp when digested with l~coRI. In order to reduce the toxicity of the protein to the
host E coli, the 1600 bp fragment was ' . 'y restricted into three fragments of
872, 700, and 80 bp by digestion with HindlII. All three fragments were
~u~ rully subcloned back into pBluescript plasmid and were no longer toxic to E~ coli.
Tr~TPT,Tr. 11
DNA scquence of Trompl. An open reading frame was identified in the 872 bp HmalII
fragment 313 bp dv....,L.~ from tbe HindllI site. This fragment encoded 161 ammoacids. The remainder of the gene was shown to reside on both the 80 bp amd 700 bp
fragments. All together, the gene was found to consist of an open reading frame 867 bp.
15 The sequence ofthe 867 bp fragment (SEQUENCE I.D. NO. I) was obtained using well
known dideoxy sequencing techrliques of Sanger et al. (See Sambrook, et aL, Molecular
Cloning A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press,
1989). ThegeneencodesaprecursorproteinTROMPlof288aminoacids(31,742Da)
having the deduced amino acid sequence of SEQUENCE I.D. NO. 2.
20 The first 32 residues from the N-ter~ninus of TROMPI has ~l, ,l;~l;.~ of a
LydlulJl.ub;~signalpeptideincludingal3residueN-regioncontamingfourbasiccharged
residues (Histidine, Lysine, Histidine, and Arginine), an H-region cont3ining 11uuu~ ~,u~ivc l~ydlvlJllub;~ ammo acids, and a C-region containing a putative concensus
leader peptidase I cleavage site of Threonine-Histidine-Alanine. The mature processed
protein consists of 2~6 amino acids with a calculated mass of 28,182 Da. As determined
by inspection and Kyte-Doolittle hydropathy analysis, which identified areas of
Lydlvlullub;~ and hydrophilicity, the amino acid sequence analysis of the mature protein
2 T 804~7
Wo95/18632 r~ C I~ù
~5-
shows ~ l and regular ~ ;r beta-pleated sheet secondary structure
d;llg to outer membrane spanning regions. Moreover, the terminal beta-sheet
membrane spanning region shows features consistent with other well known grarn-
negative bacterial outer membrane proteins, including a glycine residue in the terminal
5 6th position and ~ lllIlldtill~ in phenylalanirle.
The foregoing description of the invention is exemplary for purposes of illustration and
~rlgn~tinn It should be understood that various ,~,n.l;~ can be made without
departing from the spirit and scope of the invention. Accordingly, the following claims
are intended to be interpr~ted to embrace all such mn~lifif~S~tinnc
Wo 95/18632 2 ~ 8 ~ 4 2 7
~1 6-
SUMMARY OF SEQUENCES
SEQ ID NO: I is the nucleotide sequence encodirlg a precursor TROMP protein.
SEQ ID NO:2 is the deduced a~nino acid sequence of TROMP1, a precursor TROMP
protein encoded by SEQ ID NO:1.
WO 95/18632 2 ~ 8 ~ ' l)b
'17-
5EQ~NCF LISTING
(1) GENERAL INFORMATION:
~i) APPLICAIIT: THE REGENTS OF T~E UNIVERSITY OF CALIFORNIA
(ii) TITLE OF I~VENTION: N~TCLEOTIDE AI~D AMINO ACID SEQUENCES OF A
T. Pallidum RARE O~TER MEMBR~NE PROTEIN
(iii) NUMBER OF SEQUENCES: 2
(iv) ~9J~;~I ADDRESS:
(A) ADDRESSEE: Spensley Horn Jubas & I.ubit$
0 (B) STREET: 1~80 Century Park East, Suite 500
(C) CITY: LOG Angeles
(D) STATE: ~'~lirn~n;~
~E) COUNTRY: USA
(F) ZIP: 90067
(v) COMPUTER REAI~BLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC ihl.~
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION N[~MBER: PCT/US95/
(B) FILING DATE: 6 January 1995
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Bostich, ~une M.
(B) REGISTRATION NUMBER: 31,23~
(C) REFERENCE/DOCKET NUMBER: FD-3744
(iX) TT~T. _, I'ATION I~FORMATION:
(A) TELEPHONE: (619) 455-5100
30 (B) TELEFAX: (619) 455-5110
WO 95/1863~ 2 1 8 0 4 2 7
48-
(2) INFORMATION FOR SEQ ID NO:l:
(i~ SEQUENOE t~ TR~T9TIcs:
(A) LENGTH: 957 base ~airs
(B) TYPE: nucleic acld
5 (c) STR7~ .: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vii) IMMEDIATE SOURCE:
(B) CLONE: TROMPl
10(ix) FEATURE:
(A) NAME/~EY: CDS
(B) LOCATION: 1..954
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
ATG CAT CAA AAT TCA CCC AAG CAG TGT CAC TTG ATA CGT GAA AGA ATA 48
15Met His Gln Asn Ser Pro Lys Gln Cys His Leu Ile Arg Glu Arg: Ile
5 10 15:
TGT GCC TGC GTG CTC GCG CTT GGC ATG CTG ACC GGT TTT ACG CAC GCA 96
Cys Ala Cyu Val Leu Ala Leu Gly Met Leu Thr Gly Phe Thr His Ala
20 25 30
20TTC GGT AGC AAG GAT GCC GCA GCG GAC GGG AAA CCC CTG GTT GTC ACC 144
Phe Gly Ser Lys Aap Ala Ala Ala A~p Gly Lys Pro Leu Val Val Thr
35 40 4s
ACC ATT GGC ATG ATA GCG GAT GCT GTC AAA AAC ATC GCT CAA GGT GAT 192
Thr Ile Gly Met Ile Ala A5p Ala Val Lys Asn Ile Ala Gln Gly A~p
25so ss 60
GTG CAT CTA AAG GGG TTG ATG GGT CCT GGT GTT GAC CCG CAC CTG TAC 240
Val His Leu Lys Gly Leu Met Gly Pro Gly Val Asp Pro E~is Leu Tyr
65 70 75 80
ACG GCT ACT GCG QGG GAT GTG GAA TGG CTC GGG AAT GCG GAT CTC. ATC 288
30Thr Ala Thr Ala Gly Asp Val Glu Trp Leu Gly A~n Ala A~p Leu Ile
WO 95/18632 2 1 ~ 0 4 2 7 P~ or i~o
49-
CTG TAC A~C r GG TTG CAC CTG GAA ACC AAG ATG GGC GAG GTG TTT TCC 3 3 6
Leu Tyr Acn Gly :I:eu Hi3 Leu Glu Thr Lys l~et Gly Glu Val Phe ger
100 105 110
AaA CTa CGC GGG A~- CGC TTG GTA GTT GCA GTT TCT GAG ACT ATT CCG 3 8 4
Lys Leu Arg Gly Ser Arg Leu Val Val Ala Val Ser Glu Thr Ile Pro
llS 120 125
GTG TCT cAa CGT CTT TCT CTT GAG GAA GCA GAG TTC GAT CCG CAT GTG 432
Val Ser Gln Arg Leu Ser Leu Glu Glu Ala Glu Phe A6p Pro His Val
130 135 - 140
0 TGG TTT GAT GTA A~G CTG TGG TCT TAT TCG GTG AAG GCA r,TG TAC GAa 480
Trp Phe ASp Val Lys Leu Trp Ser Tyr Ser Val Lys Ala Val Tyr Glu
145 lS0 lSS 160
AGC TTG TGC Aha CTG TTG CCG GGA AaA ACT CGC GAA TTT ACT CAA CGT 528
Ser Leu Cy6 Lys Leu Leu Pro Gly Lys Thr Arg Glu Phe Thr Gln Arg
165 170 175
TAT CAG GCG TAC CAG CAG CAG TTG GAT AAG CTT GAC GCG TAC GTT CGG 576
Tyr Gln Ala Tyr Gln Gln Gln Leu Asp Lys Leu A6p Ala Tyr Val Arg
180 185 190
CGC APG GCG CAG TCG CTG CCT GCT GAA AaG CGT GTG TTG GTG ACC GCT 624
Arg Ly6 Ala Gln Ser Leu Pro Ala Glu Arg Arg Val Leu Val Thr Ala
l9S 200 20s
CAT GAT GCG TTC GGC TAT TTT AaC CGT GCG TAT GGT TTT GAG GTG AAa 672
His Asp Ala Phe Gly Tyr Phe Ser Arg Ala Tyr Gly Phe Glu Val Lys
210 215 220
GGG TTG CAA GGG GTG AGC ACC GCT TCG GAA GCC AGT GCG CAT GAT ATG 720
Gly Leu Gln Gly Val Ser Thr Ala Ser Glu Ala Ser Ala His Asp Met
225 230 235 240
CAG GAA CTG GCA GCG TTT ATT GC~ CAG CGT AaA CTC CCT GCT ATC TTT 768
Gln Glu Leu Ala Ala Phe Ile Ala Gln Arg Lys Leu Pro Ala Ile Phe
245 250 255
ATT GAG AGT TCT ATT CCG CAC AaA AAC GTT GAa GCG TTA AaG GAT GCG 816
Ile Glu Ser Ser Ile Pro His Lys Asn Val Glu Ala Leu Arg A6p Ala
260 265 270
WO95118632 2 1 804~7 F.~ [iJ.I
-50-
GTG CAG GCA AGA GGG CAC GTA GTG CAG ATT GGA GGC GAG TTG TTT TCT 864
Val Gln Ala Arg Gly His Val Val Gln Ile Gly Gly Glu Leu Phe Ser
275 230 285
GAT GCG ATG GGG GAT GCG GGT ACG AGC GAG GGT ACC TAC GTA GGG ~ATG 912
Asp Ala Met Gly Asp Ala Gly Thr Ser Glu Gly Thr Tyr Val Gly Met
290 29s 300
GTA ACA CAC AAT ATC GAT ACG ATC GTT GCT GCG TTG GCT CGC ~ 954
Val Thr Hi3 Asn Ile Asp Thr Ile Val Ala Ala Leu Ala Arg
30s 310 315
TAG 957
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 318 amino acids
(S) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQIJENCE ~S~n~ J~: SEQ ID NO:2:
et His Gln Asn Ser Pro Lys Gln Cys niS Leu Ile Arg Glu Arg :Ile
5 10 15
Cys Ala Cys Val Leu Ala Leu Gly Met Leu Thr Gly Phe Thr His Ala
20 25 30
Phe Gly Ser Lys Asp Ala Ala Ala Asp Gly Lys Pro Leu Val Val Thr
35 40 4s
Thr Ile Gly Met Ile Ala Asp Ala Val Lys Asn Ile Ala G'ln Gly A~p
so ss 60
Val ~is Leu LYL Gly Leu Met Gly Pro Gly Val Asp Pro His Leu Tyr
65 70 75 80
Thr Ala Thr Ala Gly Asp Val Glu Trp Leu Gly Asn Ala Asp Leu Ile
WO gS118632 2 ~ ~ ~ 4 2 7 P~ u,. /~ 0
-51 -
Leu Tyr A~n Gly Leu ~is Leu Glu Thr Lys Met Gly Glu Val Phe Ser
100 105 110
Ly6 Leu Arg Gly Ser Arg Leu Val Val Ala Val Ser Glu Thr Ile Pro
115 120 125
Val Ser Gln Arg Leu Ser Leu Glu Glu Ala Glu Phe ARP Pro His Val
130 135 140
Trp Phe Asp Val Lys Leu Trp Ser Tyr Ser Val Lys Ala Val Tyr Glu
145 150 155 160
Ser Leu Cys Ly~ Leu Leu Pro Gly Lys Thr Arg Glu Phe Thr Gln Arg
0 165 170 175
Tyr Gln Ala Tyr Gln Gln Gln Leu Asp Lys Leu A~p Ala Tyr Val Arg
180 185 190
Arg Lys Ala Gln Ser ~eu Pro Ala Glu Arg Arg Val Leu Val Thr Ala
195 :~00 205
Xis Asp Ala Phe Gly Tyr Phe Ser Arg Ala Tyr Gly Phe Glu Val Lys
210 215 alO
Gly Leu Gln Gly Val Ser Thr Ala Ser Glu Ala Ser Ala l~i~ Asp Met
225 230 235 240
Gln Glu Leu Ala Ala Phe Ile Ala Gln Arg Lys Leu Pro Ala Ile Phe
245 250 255
Ile Glu Ser 8er Ile Pro His Ly~ Asn Val Glu Ala Leu Arg Asp Ala
260 265 270
Val Gln Ala Arg Gly E~is Val Val Gln Ile Gly Gly Glu Leu Phe Ser
275 280 a8s
Asp Ala Met Gly A~p Ala Gly Thr Ser Glu Gly Thr Tyr val Gly Met
2go 295 300
Val Thr His Asn Ile Asp Thr Ile Val Ala Ala Leu Ala Arg
305 310 315
