Note: Descriptions are shown in the official language in which they were submitted.
11~15~89 17:~2 ~2~ ~82 ~031 S~d & BerrY ~ 7
2003~
HUI,-~IN~IN~ P~O'rEIN9
Tl~ p~ n~ lnv~n~lon r~lAte-~ to aQl~ular
~0 b~nding prot~n~, ~NA aequ~nc~e~ onc~o~in~ them, mathod~
~or th~l~ p~odu~t~on, an~ a~y E~y~ mploylng th~m.
MorQ ope~ifi~llv~ it rsl~ to bind~ng pro~n~ for
high-densi~y l'popro~in~ t~ . ~s~ay ~y~ u~ul for
l~n~lgy~ng high-densi~y l~pop~ot~ln snalo~;, and to ~he
25 ~n~log~ ~h~m~lves.
; ~
Ao~umulation of ~hol~t~rol in th~ va~aul~r
endothellum i~ b~l iaved t~ b~ c~y ~v~n~ ln th~
O development o~ at~e~o~cle~ro~ or re~iew j o~ Ro~,
~ . M@d~ .88-97, lg36) . ~arly ln the
developm~nt o~ Ath~roscleroti~ le~ion~, the artery walls
ar~ ~ene~rated by ch~ t~rol-cont~lni~g: ath~rsgen~G
partial~ h~s~ p~r~lal~ a~ b~ ved t~ ~e d~ri~d
35 ~rom low~ n~ty lipopro~ein~ ) or ~o b@ mitlo~
~ubpo~ulation~ of ~terol-~ich lipopr~'cein~. They ~e
~ecogni~ed a3 ~ore~gn by ~ croph~ges ~ w~iah pick up the
11/15~ 17:/d3 ~2~B B82 6~31 S~ed ~ BerrY
2g~33~
~.
par~lcle~, in~ra~e th~ ~:holR~t~rol, and th~roby boaom~
~oam cells . ~ccumulatlon o~ ~o~m cello 1~ ~h~ ~lr~t
~ta~ of Yatty ~tre~c forma~ion. The~ ~atty st~e~k~
~hen ~nlarge th~ou~h th~ gr~du~ um~llati~n of lipid-
S ~ontain~-n~ macroph~g~ and ~o~h mus~l~ c~ va~u-
~lly developing ln~o i~ibrous pla~ lah in ~mooth
r~u~cle cell~.
The s~x~Aat;uxe and aa~iv~ty o~ pl~ na lipo~
pr~tein~ hav~ b~on r~vl~w~d by ao~o ~ al. ~s~h~n
10 ~n~QLQ~ 3-41, ~ ). HlS~ en~l~y lipopr~t~in~
a~e a cla~s ~ lasm~ ~ lpoprot:~ain~ whlah aor~ t
0~ ~ole5terol t choles~e~yl ~ ter~, pho~pholipld~,
lyceride~, and apollpc~prot~in~ (~rin~pally apo~I
and apoA-I~). q~o ~ain ~ubclo.~s o~ ~DL ~ I.2 an
15 HD~3 ) ~n~y b~ ~epar~d on th~ ba~i~ o~ d~n~i~y. ~ third
subclas~, design~t~d }~DIIl or ~a~ cc>ntai~ apolipopro-
t~in E, ~ut no~ apoA . ~ h~v~ n impl ~a~d ln th~
tran~port of aholQY~e~ol ~prin~pal~y ln th~ ~or~ o~
ohol~st~ryl es~r~) ~r~m p6~ri~h~aral t~ ssu~ to th~
~0 l~ar, wher~ th~ chole~t~r~ t~bol~z~d or ~xo~6lt~d
(Glomset, ~idl~e~ g: l5~-~7, 19~ o~r ~ud1~
(~ram et al., ~Li~ ~,~ 21, 1983) h~0
shown th~t HDI~ car~ promo~ th~ aP~lux ~ ah~ erol
fxom c~ltur~d ~all~, pr~a~umab~ y ~y bln~ing ~o those
25 ~lls ~nd, through a ~e~30nd ~ ng~r ~}3on~e, ~ti~
latlng ~h~ t~n~er ~ ~ n~c~llula~ ~hO~t~l AC~0$~
th~ cell m~mbr~ne. Th~ shol~t~rc~ hen ~bso~b~d by
Hl)~ ox oth~ plasnla p~Ote~ nd i;ran~porte~ tç> th~
liver~ In addi~l~n, H~ hav~ b~en ~hown t~ }: ind ~o
30 st~roidogetlic tis~ue~ in r~t~ an~ ~r~ beli~Y~d to
paxticlpat~ in the d~live~y a~ ::hole~terol to ~he~e
t~ssues . ThU~, th~ir biolog~ c~l rol~ is compl~ac an~
app~a~s to 1 nvolvQ a ~r~r~ ~ty o~ ti~u~ e~i~ls ~f ~ct~ .
~ carry about on~-~hird o~ ~h~ tot~l s~r~ hola~t~3rol
35 in hum~n~.
HDL llav~ an ~hown to bind to exl:~hQ~atic
non~eroi~og~nl~ cell~ in cultu~o ~th high a$~inity in
~i ~ ~
S/8~ 17:~4 ~06 682 6~31 Seed & BerrY Ql~9
'
3~6
a ~atuxable mann~rr ~hi~ bin~ing i~ up-r~gul~t~d :by
lo~din~ c~ wi'ch un~t~ri~ri~d ch~ t~rolr Although
thl~ ~ugges~ that ~he ~IDr. lndu~ed ~lu~ o~ ahol~t~rol
~rorn aultured coll~ i~ m~diat~d th~ough bindin~ o~
5 l:o a ce~ ur~aae recept~, the ~xi~t~n~ of ~uah a
reaeptor i~ dieputed ln tho lit~ nd cor~lloting
dat~ hav~ b~n r~po~t~d . ~hon ~t al ~ ~l~h~
~,: 916~67, l~o) d~ onstrat~d that ra~ te~ hav~ a
spec:lfia high-a~ini~y bindin~ ~lte rO~ t~DI. which i~ not
10 ~n~ltive ~o proteolytia ~nzymo~ ggo6lting th~t tha
t~t~ and oth~r &t~roidog~nic ~ ues o~ ;~a~s obtain
chol~te~ol ~rom HDL by a ~llulas~ proc~ no~ involv1ng
r~c~pto~ pro~ein~. Gwynn~ and He~
~ 1087~-83, 19~0) ~ound ~ha~ rat ad~en~ ell~ poGses~
15 a sp~ci~ aturabl~ up~aX~ m~chanism ror HD~
~hol~sterol, bu~ ~he ~::ell birl~ln~ m~hanism o~ HDL ~a~
not ah2raot~rized. ~id~ ~t al. ~9
7 5~-65, 1g85 ) ~nd Fidg~ 2 65-
~8, 1~6) i~olat~d 78,0G0 Dalton ~4h-binding protein~
ZO ~or~ ~nem~r~ne~ of ~veral cell typ~ Taba~ ~nd Tall
s,~ s 13897~gO5, 1~4) ~tudied th~ ~8~
ciation of HDL3 w~th v~riou~ ~11 typQ~ ~n~ conclud~d
that this as~oci~tlc)n i not, m~di~ by ~p~al~ gand
and re~pt~ pro~ains, ~u~ in~olv~s lipid in~H~aa~:Lorls.
25 ~nael ~t al . (~ s l3l4n~ 8) tu~ied
the e~ects o~ ~dlA~ on ~n~ativa~lon o~ HD~-blnding
~Lt~s on human ~lbroblast~ ~nd ~onclladed tnat ~hey were
composed o~ p~ptide~ too ~mall ~ b~ ~onslder~d biolo~i~
~cep~ox~ .
3 ~ G~en ~he r~l~ o~ ~D~ in ~he remov~l o~
~hole~terol ~ro~ per~ p~ro.l aells, ~3J, ~ . agonists
hav~ a potent~ a~ appllaa~n ln th~ tre~men'c o~
A~ero ~leros;ls~ Ind~d, ther~ ~ a n~ative ~orr~
tion between ~ v~l~ an~ tCh~ inc:~d~nce c~ cor~n~ry
3~ ar~ery dis~se. ~ goni6t~ ~ou~Ld ~ u~ed to ~tl~nul~e
~he tran~lo~a~ion o~ ~hole~ter~ on~ tra~ ular
membr~nes to the cell &urf~c~e, wher~ 1~ would ba plc:kad
_ , ........ .. .
. .
11~15/~ 17:~5 ~P2~6 682 ~031 Seed & BerrY ~10
~2003~L6
d
Up by ~ L or other txan~por~ protein ~ B) ~ ~hrough ~he
u~e o~ ~n aBsay sys~em ~mploy~ng an R~ indln~ ~r~ln~
th~ a~nle.t~ a~n ~3~ idQnti~l~d. ~ tabl~ agonl~ts
lncl~de an~ bodl~ ~o ~h~ H~-bind.in~ prot~ n and o~her
5 p~ot~ln and nonpro~n mol~ul~.
In a~dition, a ~onad D~ ~rA~ment ~ncoding an
HDT~-bin~ing pre~ein aould ~ u~d ~o id~nti~y lndi~tidu-
al~ ~ rl~k ~or d~veloping aths~o~c~ro~ hrough
analysi~ a~ re~rl~tion fragment l~n~h ~lymorphi~
10 (~I.P~) or ~NA ~equ~n~, daic~nc~.~3s in H~L-binding
p~ot~in~ aould ~e ldenti~lad. ~th~rmor~, monoclonal
~ntl~odies ao~ld bR U5ed t;o scr~an blood ~ 3 ~r ~h~
~b~nc~ o~ ~un~tiorlal HD~bindln~ pro1;~1n
B~ie~ly ~t~ted, t.h~ ~r~3~n~ ~n~t~ntlon ,.
dl~clo~s ~ub3t~nt~ ally ~ur~ ma~allAn pr~teln~ haYing
l;he ~oll~wing 4har~c~t~rl~1as~ ~a) molGculAr w~ht o~
approximately ~.O,OOo ~lton~ (b) ~lnd II~I~3; t) bin~
~0 ~po~~I proteolipos~m~ ~nd a~oA-I~ protooli~o~ome3t and
~d) cont~in approxlmAtoly l~$ o~r~ohy8ratR by w~i~h~.
~h~ proteins in a~rtain embo~imQn~3 ~A~y al~o b~ aharac~
t~rized as h~v~n~ ~n i~oelectric~ polr~ tw~n about 5 .
and 6.0, and may ~ d~r~ved ~om bovino o~ hum~n c~115.
~5 W~thin a relat~d a~p~t, the ~ nt in~nt~on
dis¢lo3~3 arl i~olat~d ~NA ~ol~oul~ ~n~od~ng a p~o~eir~
h~lng ~h~ ~oll~wing ~haxaat~ ic~: ~ a~ c-llar
we~ght o~ ~pproxir~at~ly 100, 000 D~lton exclusi~rQ o~
carbohy~rate; (b) blnds HD~3 and ~ inds ~poA I ~n~
3 O ApoA-II p~oteol ~po~om~3 . Wi~hin on~ di~n~nt, thR DNP,
~equenc~ erlc~ode~ th~ amin~ a~d sl~uenc~ ~own in F~ r~
3 ~rom glycin~, am~no aaid num~r 2 ~ tO glu'camio ac~d,
~mino acid num~sr l~O ~
Within ano~e~ ~bo~ nt, the ~NA G~quence
3 5 encode~ ~h~ amint) ~ ;:id ~ena~ 5h~n in ~igure~ 4 ~r~m
m~thionine, am~no ac::id nutnb~r l, ~o g~ut~m~ acld, ~nino
as;:id n~m~r 119~ ithlrl y~ ~no~h~r e~od~mel~, th~
`'. ~, ~
. ~:
11/15~ 17:~6 ~20~ ~8~ ~31 Seed ~ B~rry
2003~
.~
~N~ secauen~e ~ncod~ the amino ~c~d ~e~anae 3hown ln
Fi~ur~ 5 ~r~m methionin~, ~mlno acid num~ar 1, to
i~oleuc~n~, amino aaid numbf.~r 12~2.
Th~ pr~on~ v~ntion ~ di~los~ ~ethod~
~or ;1 ~olating ~n HD~3~binding pXo~in, ¢omprising
olubilizing mamm~l.lan cell m~brana pro~ln~ t~
prod~ce an ~nrich~ r~otion; ~b) oX~ing ~h~ richQd
~rac~ion to an antlbody that birld~ ~o 8 prot~in ~hat
blnd~ ~DI.3, to p~oduce ~ bo~nd rr3.ctlon and a~ ~mbourld
lO ~a~tio~s ~c) r~coYe~ g ~h~ ~und ~raG~on: and
td) ls~latin~ ~h~ ~DI-3 bindi~g p~o~in ~om thQ bound
f~a~tion. ~h~ m~thod may ~l~o inc~ ho ~p ~ ad-
ing the ~ lian C~118 wi~h ~hol~ ol p~:ic,r to the
g~gp o~ 3~lubillzlng.
Xn anothar asp~ o~ t.he pr~n~ in~ren~i~n, an
isola~ed D~a~ molecul~a ~n~:o~lng a pro~in hav~n~ t~e
followin~ cha~ateri~ic~ is d~clo~e~ ) a mol~qul~r
w~ ht ~X approximately 3B, 000 ~alton~ S (b) bind~ ~D~
and ~c) ~ontaln~ app~oxim~toly 331 ~mino a~id rosidu~
20 With~n on~ ~mbodim~nt, th~ prot~i~ oompr~ th~ amino
seqll~na~ ~hown in Figure 1 f~om glycin~ ~mlno ~c~d
2, to glut~m~c aal~, a~ino a~id 331.
Wi~in a r~l~t~d ao~o~ of ~he proson~
inven~ion, a ~u~s~an~l~lly ~rs prote~n i~; d~lo~2d
2 5 h~ving t~ ~ollowirlg charaot~ a ~ oular
~eigh~ o~ approximat~ly 3~,000 ~alt~n~J (13) ~inds ~ 3;
and (a) c~r~tain~ appxoxima~ly 3~1 a~ino ~cid rt~s~du8~;.
Wi~hin ~rt~in ~bodi~n~nt~/ ~he prot~in re~ w~th
poly~lon~1 ~n~i~era to ~ 110, 00~-D~lton }~D~3 ~aindlng
30 ~o~ein i~rom bovln~ aox~c endo~heli~
C~ ran~fect~d o~ ~r~n~ormed with a~
~xpre~aion ~rec~o~ ~om~ lng a ~omote~ operably linked
to a DNA ~equ~nc~ ~nc:o~ing 2 pro~in a~ de~ib~d above
~r~ 50 di~clo~d.
~n addi~ion, ~nt~30~ tha~ hind to t~e
protein~ d~s~ e~l ~bove are ~l~o di~clo~ed~
11/15~8g 17 ~7 ~ B82 G~31 Seed ~ BerrY _ _ 00~2
26~03~
In yet ~noth~r fl~p~Clt o~? the pre3~nt
invention, mQthod~ ~o~ do~cting a g~n~ic de~a~ ;ln ~n
HD~ ind~ng protoin in ~ pati~nt ~re di~c:lo~3d. The
mathod~ ~fln~r~lly ::o~pris~: ~a) i~olating DN~ ~rom a
5 l?~tlQnt J tb) cl ~ving ~h-3 ~NP ~ith a r~t~lotion endo
nu~lR~8~ to p~odu~e DN~ ~raç~ment~ ~ ~a) hybrldl~in~ ~h~
~NA ~r~ nts ~o a ONA m~leaul~ com~p~rinlng a portlon Or
~hs nucloo~id~ n~q o~ ~igu~ 1, th~ nucl~okl~e
se~uonoe o~ Flgure 3, t~ nu~l~ot~qQ ~ ce o~ u~
10 4, the nual~otide ~;e;~uenc~ o~ F1~ 9, 5~ 0~ ~ompl~montary
t4 a poxti~n OI on~ o~ th~s~ ~e~uenc~, whereln ~h~ 1: N~
molecule iB ~ lea~ a~out ~8 nu~l~8tlde~ i~ length5 and
~d) deteoting ~ p~t~rn ~ h~bridi~ti~n ~ the DNP~
~r~gment~ and th~ D~A molacul~, whar~in th~ p~tt~n i~ ~.
15 dia~no~ti~ ~or ~h~ pr~aence or ~senc~ o~ a gene~i~
~e~e~t ln ~n ~ bindlng pro~ain .
Th~ e a~d oth~r a~peat~ o~ th~4 p~e~en~
nvon~lon will become ~vident upon re~Qrf3no~ ~o ~he
~llowing de~ailed deEwrlp~ion and att~ch~d drawing~. These
drawings are not to be construed as limiting the scope of
the invention.
Figure 1 illust~te~ thR nucl ~o~ quence~
of the co~ing ~rand~ o$ ~wo ol)NA ~lc7ne~ en~oqlng
~DL-bir~ding prot~in, toge~h~r wi~h ~h~ ~n~rred ~mino
2~ ~cld ~RqUenC~ of on~ o~ the t:l4r~ Nu~ber~ ~ove the
lines re~r t~ nu~leo~ Ide po~itlonæ~ ~un~ers b~low ~h~
;L lne~ re~e~ ~o amino a~id po~itlon~ .
~l~ure ~ illu~t~a~es th~ con~t~c ~on of~ the
pl~mid Zem217.
3 0 Fig~lre 3 lllu~1:rat~ tka~ nu~leotid~ s~quen~
o~ a ~loned cDNA ~noo~ing ;~ po~tic7n o~ ~ h~an t~D~
blnding pro~ein, together w~th ~h~ d~dUC~d ~ino a~id
se~uen~e. ¢~rt~in ~ iC~i4n æ~y~e git~ a~e ~own.
The tQrmin~l Eco RI ~lt~ a~e th~ re~ult o~ ~he ~lo~in~
: 35 pro~edure. Numb~r~ ~t the end~ o~ linos ~ 0
nucleo~l~e po5~tion~ Number~ ~lo~ the lin~ ~e~er to
, ,,__. . .. .. .. . _. _ .. _ _ _ . ~ . .. . .....
11/15/89 17 ~8 ~B B82 B~31 Seed & Berry 1~ 3
2~
amino ~cld po~itior~. ,Th~ ar~ow india~ th~ poln~ oP
div~rgen~ ln th~ Eleclu-anaola o~ olon~ ~2a-1 and ~IEI. 13.
El~ur~ 4 illu~t~r~ tho nuolooti~ qu~n~
o~ ~ ~lon~cl DNA, de~i~nated H~ 13, ~3naoding a human
5 ~ inding p.ro~ n ~ogeth~r wi~h the dedu~d amno a~ld
~quenc~ . Carta~n re~r;l ~ n ~t~ ar~ ~hown. Number~
at th~ ~nd~ o~ the~ lln~ r~ o nucl~o~lda po3~ion.
N~mb~rj below ~h~ lin~ r~fe~r to ~mino ~d po~ition.
Flgure 5 illustrate~ the nualoot~de ~quanc~
10 oS a cloned DNA, ~ nated H~I. 13/8 ~pllc~, ~n~ding a
human ~ blndiny ~ro~ in, ~og~h~ wlth the d~duc~d
a~4lno aald ~quenc~ C~rt~in r~stric~lon ~ite~ are
~hown. Numbe~ a~ th~ en~ th~ lin~ reS~r ~o
nucl~o~ld~ po~ition. Num~ers ~elow ~he line~ re~3r to
15 amlrlo aaid po~ition~
Figur~ G ~hows p~;rti~l r~t~lotion map~ o~
human H~L-~inding prot~in cDN~ clon~s. ~s~rlotior
~it~ ar~ d~ign~ted a~; RI, E~c~ RI ~ P~ P~t
Bam HI; G, Bg1 IIS N, Nar IS A, APa IJ ~, H1nd II~7 V,
20 ~vU II~ S, S~t I7 X, Xma I- Th~ min~1 E~ it~5
ar~ th~ re~U1t O~ the O1Oni~ Pr~ Ur~.
F1gUXe 7 ~ Btr~te~ ~e ~n~ruatlon o~ ~he
v~cto~ Z~m22~F~ and ZMB4. SylTbols u~ed are: DHFR, mouse
dlhydrofol~t~ r~du~ e g~n~s SV40p, S~140 p~o~t~5
~5 sv40~, sv4~ te~lna~; MT~l, mou~e met~ iorlein
pr~mo~er; MII~, adeno~irus 2 ~or l~ke p~omo~e~5 and S~;,
~pli~ng ~i~n~
3~ Pri~ to ~et~in~ for~h the lnYen~e:ion~ {~ m~y
l~e h~lprul to an und~rs~anding ther~o~ to sot forth
de~initions o~ certain i:~3~15 ~Q b~:u~e~ ~r~ln.
~ t~ A DNA molecule or
s~quenc~ which h~s ~en en~ymatlo~lly ~yn~h2~ized fr~m
35 the se~u~nc~s present in ~n m~NA ~ernplat~, or a clone o~
such a molecule.
........... _ ,.... ~ ......... ;
11/15/~ 17 ~9 2~2~)6 6~2 6~31 S~ed ~ ~errY 1~1~14
3~6
Ex~e~ion Y~ A ONA molec:u~ or a clon~3
o~ ~u~h a molecul~ whlch ha~ b~en mo~l~led th~u~h human
~s~terv~ntlon ~o con~in ~egments o~ D~A whl~h ~r~
~m~in~d and ~uxtapo~d in a mann~r whiah ~ould not
5 o~ho~ e ~x1~t in n~t.~re and which aontAin~ at l~a~t
on~ D~A ~equanc~ to be exp~ d in ~ ho~t aall and
s~quence~ whlch :~aailitat~a f3uah expr~iont ln~ludin~ a
t~anscription p~omot~r and t~minator. Repli~At~orl ~
an e7cpre~sion v~:at~r in a ho~ oell ~o~nonly oacs~rs
10 autonomously, du~ to ~he prQsence ~ a repll~a~ion
origin on ~he veator, or may occur ~y in~g~ on into
~h~ ho~t ahromo~ome.
The p~ent ln~ten~ion pro~rides HR~-binding
prot~ins and methods o~ m~Xln~ thase prot~ins, a~ well
15 as u~e~ul polypeptide ~ragment~ o~ thelse ~rot~ ~s . Th~
inding ~ro~ein~ and p~lype~t~es ~re i~olElted ~rom
cell~ which naturally produce ~h~m, o~, pr~er~bly, ~re
~nade ~y eXpre~ing cl~n~d ~ ~quenaes ~n recoml~in~nt
~ell~. ~h~ ~nven1:lc)n provid~ t~e3~ protein~ ~n
20 substan~ially p~re ~orm, that i~, ~t lea~ ~Q~6 ~ur~, and
in one embodimen~, free of other pro~aine o~ ma~nalian
origin. lrh~ L-~indin~ prot~ and polyp~p~de~ ~hu~
prod~ ad are ~s~ul 1YI te~t ~y~tem~ ~or ~c:resn~n~ poten-
tlal HD~ analogs, $ncludlng a~oni~ts and ~nt~goni3tæ O:e
25 H~h~ In anothex embodimen~ o~ the pre3~nt inVen~ion~
th~ binding pro~eirss and polyp~p~iào ~r~m~n~ ~hereo~
ar~ u~ed as an~1 g~ns ~o generat~3 ~n~b~di~e~ whic:h may be
u~e~ D~ analog~ analog~, in~ludin~ ~ti1~ot~ e~,
other pro~ine ~ peptide~ an~ nonpr~t~in c~ompound~ y
3 0 ~e u~ed thex~peutl¢;~lly in th~ ea~men~ hy~erc:hol~-
~ero~emia snd atherosc~ ~rosls, HDI. a~ g~ may ~1BO be
U~d ~o ~reen pa~ent~ ~o~ da~e~ or defi~i~ncies in
HDI.-bin~llng protein~ and. t~ I. rec~pkor pathway.
Th~ pre ~nt invsn~lor~ al~ p~ovid~s ~NA
35 sequence~ enco~lng H~L-b;indin~ pro~ein~. The3~
sequen~:e~ are u~ ul in producing ~e HD~ binding
prot~ns as not~d a~ov~, and aXe ~lso uY~ul in the
'~.
11/15/g~ 17 1~ ~2~6 ~82 ~31 S~J & Berry ~ 15
26~0~31~i
diagnosls o~ ~n~tla d~fa~s in ~L-b~ndln~ prot~in
produ~tion, s~ch d~ t~, whlOh m~y ~ ocl~d with
a hi~h risk ~or ~h~ de~elopm~nk o~ atheroocl~ro~i~, may
~a d~tect~d by using tho ~NA ~que~aes ae probe~ ~or the
pr~ence ~f r~tr~c~ion ~ragm~nt length polymorphi~ms
~F~Ps). Thn~e polymorphi~m0 ar~ th~ r~sult o~ ~hange~
ln DNA ~e~uehae~ and c~n b~ detoct~d as di~erena~ in
the 8~ZRS of ~ra~m~nt~ gen~ra~d ~fter d~ge~ion of D~A
wit~ re~t~i~tion ~ndonucl~ase~. ThU~ a c~NA ~ncoding
an HDL-bin~in~ pro~cin or a ~ragmen~ o~ ~uch a c~NA aan
u~e~ to probe ~or RFLP~ in dige~t~ o~ genomic DNA
i~ola~ed ~ro~n, for ex~lnpl~, bloo~ l~uXocyteu. ~:~ner-
ally, ~rob~ w~ll be at least a~ou~ 14, pr~fe~ably at
16~Ast 18, ~lUC180~ n length. Hulnan ~ub~eat~ wi'ch
15 variou~ ~orm~ o~ hyp~r- arld hypo-lipideml~ are ~ar~ened
to iden~i~y particular }~LP~ ~hat are dia~n~tio ~or
sorders in lipoprot~in ~e~aboli~m An~ predi~p~sition
~o ~th~ro~ol~roslR . ~F~,Ps ahow Mend~l lAn lnheritano~
pa~ternF~ and ~an be u~ed t~ study the segr~g~iorl o~
2 0 genes: ln :Ea~nily E;tudi~s . ~ethods ~or carrying out ~FI,P
analyses are generally }~own in th~3 art and are
desoribed, ~or ~xample, by ~3 et al. (~_9~t.
76:10~0-g5, 1~5). Al~c~rn~ively, the ~NA ~an~s
provlded by the pre~nt inven~on ca~ be us~d a~
25 ~pecl~i~ probes or ~rime~s in ~thl3r co~lY~ntional
sc:rsening ~skem~.
~ rie~ly, H~ bin~ing pro~ins ~re i~olat~d
î~om mammalian cell ~y ~o~iliz tion o~ membr~n~
Pollowed by a combination of ~nvention~l ~olumn 2nd
30 sla}: ~el chr~mato~ap~y techni~uQs, ln~ ding ar~l nity
chroma~ogr~phy ~Qing H~L apoprote~ ovalently }30und to
aL sui~al~le in~c:lu~ ma~rix (e.g~, Sepha~o~e, Pharma~ia,
Pisc~ataway, ~ he ~ ln~ns~ p~cot~ns m~y also b~
purl~ied by immunoa~f~ni~ ~hronl~to~r~phy ~ing an~i-
3 5 bodi~ ~r iln~isera px~par~d a~ ~eE;cr~ ~ed ~ereina~te~.Monoclonal anti~odle~ ar~ parti~:ul~rly ~r~ferr~.
~r~ red ~ourc:~s o~ ~L~binding pxo~eins Ln~lud~ ao~t ~ c
,. .: '' ''''` ~ ' '
, : ~
1~1 01
11/15/~ 17 11 ~2~B 6f~ 31 See~ & Berry
~033~6
~o
endc)thelial ~ells and fibroblas~3. c~ells are pre~erably
lo~ded wlth c}~ol~trol by incubdtion in c2l01e3t~rol~ or
I:DL-con~ainin~ dium prior ~o protq~in i~olatiorl.
~lt~rnati~e~ly, ~h~3 ~n~ indin~ protQirl~ Ihay be
5 p~odu~ed by expres~ing olon~d DNA ~eS~Qnce~s ln reco~nbi-
nant cell~ pr~arrecl ~o u~ loned ~DNA, because
~he u~e o~ intron~cont~ining gerl~mia ~NA o~n result in
~h~rant expr~s~ n, particul~rly in ~ roorgan~ s~ oe~t
cells~ Suit~ DNA ~qu~n~3s m~y ~ o be ~yn~h~izad
10 aacording tc) con~ention~l m~thod~ (t3Q~, ~0~ exampl~,
Caruth~s et al~, U.S. Patent No. 4,458,06~ Ita~cura, in
~ ~, E1B~ r Bio~hemi~al
P~e~ 82) ba~ed on th~ amine aaid ~e~uence 4~ an
i~ola~ed ~DL-bin~ln~ protein, Two ~uch amlno ~cid
sequRnce~ ar~ disclosed her~in.
Im~unog~nlc polypept~d~ Pr~an~ may b~
prep~red ~rom H~L-bin~lng protein~ ~y ~ ion ~ith
proteolytic enzym~s or ~her ~g~n~ .g~, ~yan~æn
bromide~. Alternatively, p~p~id~s m~y ~ pr~du~ed ~y
~o ex~res~ion o~ ~loned ~NA ~eqUQna~ or by ~tandard
ch~mical syn~he~i~ t~chnique3.
For expressl4n ln ~ecombinant a~ , a DNA
~q~enae encoding ~n H~ inding prot~in 1~ a~ted
into a ~ull;able ~xpre~aien v~ator, whia~ ih ~urn i~
25 in~e~t:ed ~ n~o appropria~e ho~ he m~thod o~
~nsertion Will depen~ upon ~h~ p~t~culax }~oE~I: c~
~hos~ll. M~thods fo~ tr~n~ect~ng 2n~mrnal1an c~alls ~nd
tran~orming bacteria ancl ~ungi wit:h ol~d DN~ ~e well
known in the art. ~ t~ble eXpxqa~io~ ve~t~
3~ co~p~ise ~ ~romo~e~F whic~h i~ ~ap~ble of dir~c~iny the
t~ansa~ipt~.on o~ ~ cloned ~NA ~quon~3 in a ~30~ cell
and ~ runational ~r~ns~;:ription termination ~taO In
~m~ in~t~nces, i~ i~ pr~er~d t~at ~xpr~ ion ve~tor~
~urth6~r co~nprise an ~rigin ~ reF)li~ion, ~uenaes
35 ~hi~h regula~e ~d/o~ ~nh~nc~ ~3xpre~3sion l~vels, and one
or more sel~c~ble n~ark~r3, ~ep~n~ing on th~ ho~t c~ll
selected. Suitable ~xpr~ ion v~tor~ y b~ d~riv~d
'
,
11/15/~g 17 12 ~B 682 6031 .',e~d ~ Berry ~17
2~ 3~6
11
~rom plA~mi~, RNA and DNA vl~u~, or c~311ular DNA
sequence3, or may contain ~l~m~n~ o~ 6~ac~h.
Pr~err~d prokaryotlo ho~t~ ~or u~e in
~arryiny out the present lnv~n~lon ~r~ ~train~ o~ the
5 bActeria ~Dh~1~ S~ , althou~h ~L~I~ an~ other
gonera are also w~ul. ~hn~ or tran~ortnln~
the~e ho~t~ and for eXpr~Sing ~or~iyn PNA a~quenc-3~
aloned in thelP ~ro wel 1 ~cnown in th~ ar~ ( s~e, ~or
2xample, Maniatis et al. (ad~. ~, ~1
10 l ~o~at~rv ~n~ , Cold Spring Har):~or ~a~ora~or~ Cold
Spri~g ~ar~or, N.Y., 1~2). tra~tor~ ~ad ror e~xpre~ing
~eign ~NA in ba¢terial hos~ wlll g~n~rally contaln a
~eleo~abl~ m~rke~, ~u~h ~a a ~eno ~or ~n~i10lo~1~ ra~
tance, a~d a promote~ whlch fun~tlon~ ~n th~ ho~t cell~.
15 Appr~pria~ prc~mo~er~ include ~ ~ tNiahol~ and
Yano~cy, ~q~ ~ , 155~ 3) ~
(~asadab~n e~ al ~, J BAQ~ 71~0 , 1980~, ~AC
(Ru~all et al ,, Ç~ ~Q : 2~1~4~ , 198~ n~ ph~e
pr4mot~r~. Pla~mid~ usef~l for trAn~f~r~ning baateria
~0 include pBR~22 ~Boli~ar et al., ~a a:~5-113, 1~77) ~
the ptJC pl~sm~d ~M~s1n~, M~h~in ~ SLY LQ~ ~~
q7, 1~83~ and V~eixa 2nd ~ sing, ~ 25~-~8, 1~3~),
p~Q~t2 (~ue~ lo , 1983 ), ~nd
der~va~ives t~reo~.
Eukaryot;i~ roorge~ ms, s~:h ~ ~he yeast
5Z~Ç~!i~ or ~ilam~n~ou~ fungl, ~nclud~ng
Asp~gillu~, m~y Alsc7 be used ~s ho~t a~lls. Partic~-
larly prefe~ed ~pecie i; of A~per~illus include
30 Teohnlqu~ r trarls~orming y~a~st ~r~ d~aribe~ Py Begg~
~ 27~:~U4-0~, ls7~) and Hlnn~n e~ al. ~Q~
Ag~S~ 1740~ 84). Expres~lon v~t~r~
~or u~e in yea6t inclllde Y~p7 ~Btruhl ~t al~, 1
~ Z~,S 103~-3~ 7~), YEpl3 (Broa~h
35 ~t ~1~, ~ 8 s 121-33 J lg7~), p3~B2~ at~d pJDB21g
~B~gg~, ibid~ ), and ~riva~iv~ t~n~reof~ Slloh vec~ors
w~ 11 ge~nerally compris~ a ~el~ a~le Darker, su~h as ~he
11/15/8~ 17 14 ~06 682 ~031 _ Se~d & B~rrY
~01~33~6
12
~3erectlve ~le~table Inark~r E~ X~wJ~ ki nnd hell
EP 171,14~ MUrray ~t dl~,~, U.S. P~tant ~0.4,7~,O7~), or
a mltritlonal mark~r, ~uoh a~ the I,~ ~ he, P~a~erred
pr~motar~ ~or u~ ln yda3t eXp~eO~Oh v~c~or~ ln~lude
5 ~prolnoter~ ~rom y~ col~lo S~on~a ~tltoanlAn e~
ao7~-~o~ l9~0J Albar ~nd K~waoaki,
;L~L~o~ 41~-34, l~na~ X~w~aki, U S
pa~eht No. 4,59~,311~ o~ als~ohol d~hyqro~ana~pe gen~s
~Y~uny ~ al ., ln Jiollaolld~ ot al . (n~. ~, E~l~
lo ~ ~ ~, Now Yorlt: Ploh~4nl, lg~,
p, 33~ ~nd ~m~Qr, ~y~ ~U.sl92-~ol,
~3~ a~ilit4t~ pllrl~ica~lon ol~ r~ign prDtoin
~roduc~d in A ye~t trans~ormant ~nd to obt~ln, ~hon
neoa~a~a~y, prop~r di~ul~ orl~ ~or~natlon, ~ signal
15 ~e~u~nao ~rom a y~A~t gene enooding ~ ~oa~st~d protoin
i~ ~olnetl in thQ propo~ reading rr~ o tho ~eqUan~Q
on~odlng tl~e ror~n prot~$ll. Par~ ula~ prer~rr~d
oi~n~l ~e~eh~s ihqlude tho~3 onoocling ~h6~ pr~-pro
re~ n oP ~he ~E~l ge~n~ ~Xur~n ~n~ Horoxowltz~ SQ;~.
~0~933-q~, lsaa; slngh~ EP 123,54~t and Xurl~n a~ al.,
U,~, Patorlt No. 4~s4~oa2) Ahd th~ ~oor~ry p~ptld~
portlon o~ tho Oh~ ~MaoRay et al., U. ~ . P~!ltelht llo
~ ,572~ ~aoKay, EP 2~0~68Dt ~aaK~y ~t Rl., U.S
P~ten~ ~pplla~tion 07/Z70,93~.
~rah~orm~d coll~ ~r~ a~l~urad ln appropriate
~edla contalnlng oar~on ~nd ni~rog~h ~o~o~ 3 w~ll a~
oth~r nu~ nt~ or s~lea~lve a~entd whl~h ~ay b~
r~guit~ by th~ partlaul~r ~train. y~s~ ho~t a~
tran~or~d ~i~h pla~ld~ aontaihing th~ 1 o~lea~able
marker m~y be ~41tured i~ ao~pl~x ~qi~ aOh~ining
gl4coo~ a~ a ~a~on ~oura~. ~
H~gher ~uX~ryotlq aell~ ~y alr~o s~r~ a~ ~or~t
qell~ ln oar~ying out the preseht ~hve~ion.~ ~ultu~
mAmr~ an ~lla, sUah A~3 th~ ~X, CJI~, Ns-l~ sp~/o~ H~60
an~ J658L o~ ine~, ~re prè~e~r~d. Th~se an~ othe~`
~itabl~ 0911 lIne~ a~e ~v~ ro~ a ~ar~ety of
aou~a~ u~h a~ tha Am~3rla~n ~ypq Cu~ 3 c~l~ w ~lon,
a : .
-
' ,~
.~
11/15~89 17 14 ~2~6 ~8~ B~31 Se~d ~ Berry E~
~ 33~6
~3
~oc:kvlll~, Md. ~hH ~k~ K a~ll lln~ tk-t~ Wa~ t~ar
~nd Ba~rg~, prQ~ . _Natl . A~ad . S~ 7~ ~1106-lO,
19~2) i~ ~ pa~xtla~ rly Fr~r~rr~d. ndhe~nt ~ell line.
Expr~lon v~a~or~ r~r ~t~ m~mn~ian c~ wlll
5 ~omlprl~ a promot~r a~p~bl~ OL~ dlr~a~ln~ th~ tran5arlp-
tlon oP a oloned gen~ or cD~tA. Pu~icUlarly pr~er~d
promot~r~ inalud~ ths SV40 promotor ~ubram~ni Gt ~
~5Ql,5~1~ ;L: 354-~4 ~ 19~), thO m~tnllothlon~in
qqn~ -l) promot6~ tPal~nit~r ~t al. . ~QlQn~a iL~L~8o9
10 14~ 1933J Palt~lt61r ~t nl.~ U.S. P~tl~nt No. 4,57~,~21)~
the 1llDU5El ketpp~t ~ens p;ro~otsr ~o~an o
N~:l. ~c~d. ~c~ U~ ~,:70~1-4~, 1984), and t~.te
ad~noviru~ ta~or lat~ promot~r ~B~rkn~r ~tn~ Aharp, ~,
~ a4l~57, lga5~ 0 aDntain~d is~ tha
15 oxpr~ts~iort v~or~t io a tran~a~pti~n te~ntlnator,
l~oat~d down~ aT~ Or ~ho lnoe~tio~ ~lt~ ~or tho 1: NA
~t~qu~no~ to b~ exp~o~d ~ A p~ore~r~d tqrltt~ nator i~ the
hu~ttan growth ho~non~a ~hGH~ ~ene t~r~lna~or ~D~Noto
~t al., ~u~ Aq~a~ 1~ 9~3719-30~ 19el) . Sn ~ddition,
2D veotor~ will pr~:erably ont~in ~nh~na~r ~equenaa~
approp~iat~ to th~ particular ho~t c~ll lin~.
For ~xpras~tlon in ~ultured mnmn~llan aE~ t,
expra~olon v~to~ aon~aining clonRd ~ quanc~ ~r0
int~odua~d into t~ cRlls by ~ppro~riate t~ns~c~ion
25 t~chnlque~ ~Uoh as ~al~Lum phoelpha~o~ t~ t~ s~-
tl3n l~or ex~ plo, a~ m an~ n dcr ~b~ ~y
~s456-67, 1~73, a~ modi~led by Wigl~ ~t ~
,, ~, BCi,~ 3~67-70, 1~8~) or ~l~atropora-
tion ~M~umann e~ al. ~Q~. 1:84L-45~ 1932). ;5a~ ~
30 typical tr~n~ e~tic~ a D~lA-oal~iu~ phC~ph~t~ p~oalpi~
~a~ lz i~or~ and ~hen appl i~d ~¢o the 0~ 9 ln ~h~
pre~no~ o~ mAdluEn contalning ahloroquin~ ~100 ,~1). The
ro lrlcub~te~ 3eor g~r hour~ ~rith t~ ~ro~ipi~te,
rollow~d l~y a ~wo~inute, l5% gly~e~ol ~ioolc, ~ por~i~n
35 o~ tho 01~113 ~ake ~p t;he ~N~ ~n~ nt:~ln it in~ the
c~ or sev~ral day~ m~ll r~a~ion Or ~h~ ~ell~
lnt~qrat~ th~ U~A lnt~ ~he g~no=~ ma~7~t~in 'c~ NP. in
~,. :
`, ' ~ , ~
"~
`` J
.. . . .
. .
~ ..
il~l5/8~ 17 15 ~2~6 682 6~31 Seed ~ Berr~ !~02~
~33~
~4
nonshromo~o~ ucl~Ar ~truo~ur~- The~e tr~n~otonts
can ba 1~nti~1o~ by cotran~oction wlth ~ ~ane that
confor~ a ~alectable phonotype ~a ~Hl~a~ablo mark~r).
Pro~orrod ~loot~lo marker~ inalud~ tho DHF~ ~eno,
5 whlqh l~np~rt~ oollul~r r~a~tano~ to h~othotrexa~ tMTX~
An lnhibitor o~ nucloo~ yntha31~1 or thn b~at~riAl
nuo~oin r~ noc ~no, ~hlOh o~nr~ro rooi~kanco ~o
~ho dru~ a~4~ n i~hlbitor o~ prot~in ~yntho~
A~tqr the hoet ooll~ h~v~ ta~en up th~ ~NA, dru~ ~el~a-
tion i~ appll~d to ~fll~ct ~ar a pop~lation o~ c~ that
are expre~lng 'che ~ o~ablc ma~X~r ~ 3ve~18 hi~h
~nough to ~on~r r~31~t~noe.
Trnn~ t~d mammall~n oelle are ~ùl~u~ed ~n
~orum Qontainlng or ~ru~-~re~ m~di~ ~on~lning appro-
p~latn nutri~nt and antibiotlG s~ppl~nts. sult~bl~mod~a ara ~vail~la ~ro~ aomm~ralal ~ppl~er6 or m~y b~
prepare~ aqoo~dln~ te publi~hed ~oai~D (50H,
catalo~ o~ th~ Am~rican ~yp~ ~ul~ur~ ~oll~otion).
Coampllfl~atlon can b~ u~d to inar~a~a
~0 nXP~ iQn 10Y~1~ ln eell5 tran~oot~d wi~h thn ~IF~
n~ar~er. ~lgh ~ona~n~tlon~ 0~ M~X are add~d t~ the
~ultur~ medium at the ti~n Or lnltl~l ~olQc~lon~ ~ m~y
~e added by s~uenti~lly inGr~a~in~ th~ ~onc~ntration of
M$X in ~he mQdlum, ~ollowe~ by ~apeated olonlny by dilu
tian o~ th~ d~ug-r~ tan~ ~ell lln~ a~te~ ~aah ampli-
floatlon ~tep. ~ wh~ah expr~3~ th~ PHPR m~ke~
~hen 3elected and ~¢reened ~or pro~u~tion o~ tha ~L-
binding pro~oLn.
~r~ening o~ n~Pa~ted oells may b~ don~ ~y
the liyand blott~ng teahniq~4 ~G~h~ ~nd Ora~ ~. Biol.
Çh~m~ 7439-42, l98~ ia~ly~ o~ atlons or
a~lture mèdia ar~ ~p~q~d ~y ~ rophor~ on
polya~rylam~da ~lab gel~. ~ho ~ r~tod pro~ln~ are
thRn tran~es~ed to nl~ro~e~lulo~e ~y ~lectrop~or~
~he nitroaellu1o~o blo~ ockad ts prev~nt
non~peol~1~ blndlng, then in~ubat~ ~it~ ~h~ la~ d
lig~n~ o~ Ln~ere~t (~.g., HDL~. Alc~rnativ~ly, ~
-: .
.'' ' " :, ,.~ "
llr15/~9 17 16 ~p206 632 6031 Seed ~ BerrY ~ ~21
~33~
~y be &a~e~ned by a ~unct.ion~ ~s~y ~ha~ mes~ure~
~lux o~ la~ d chol~t~rol ~ro~ o~ in the pr0~na~
o~ ~D~. ~rl~ly, tr~n~eot~d o~lla a~ lncubat~ in
ot~cl-rloh m~dl~m to le~ o~ with ~holo~rol, an~
intrAohllul~ pool~ o~ uxoes~ ohnlentorol ar~ radio-
l~eled with ~ ~ynthutla proaur~or ~o.g., 3H-mev~lono-
la~tone). Hor,-mod~tod t~n~loca~ion ot intraaoll~l~r
3H-~hol~s~rol ~o tho a~ u~o~ ~nd l~to t~ m~lum
i~ ln~iaatlv~ o~ HDL roaeptor ~tl~ity ~lu~e e~
~ ol~ Çh~m. æ~ ago4-so7~ 1~87).
~ not~d abov~, ~ntlbodie~ to HDL-binding
protein~ ~ay b~ u~ed ~ gonist~ ~nd a~tagonl~ts and
rO~ tn~ ldRn~ifl~lon and purifl~a~lon o~ ~$-~indlng
p~at~in~, Nonoolonal an~i~odi~ Ar# ~n~ally pre~err~d
15 ~or th~s~ purposo~ . HD~-bindlng p~o~ ar r~ n~
th~reo~ A~o ua~d to make mo~oclon~l ~ntlbodle~ acco~lng
to m~thod~ g~er~lly d~s~ribqd ~n tho ll~e~ature.
P~ein ~r~gment~ u8~ul ln lmmunlz~tlon wlll ~ o~
su~lalent ~iZ~ ~ be immunogenlo, pr~orA~ly at le~t
~lx ~ino aold~ in length, moro p~ ra~ly about fi~t~on
Amino aoido ln length. Pre~e~od ~ub~ct ~nim~l~ ar6!
~ic~ o~ rat~, with ~A~B~4 mlca bqi~ ularly
pro~rrod~ ~h~ appropriAt~ anlm~l~ aro lmmuniz~d wl~h A
pr~paratlon o~ the HD~-blnding pr~ln, pr~r~bly a
pu~ or p~rtially pu~ prepA~lon. ~r~rab~y, th~
anlmale a~o lmmunlz~d wlth ae lea~t 140 n~ ~oh o~ ~he
prot~in pr~pa~a~lon, mo~t p~er~bly gre~tor than 500 n~
e~oh. For immuniz~tien, th~ teln 1~ pre~era~ly
adsorb~d to a ~olld-phasa ~trlx, p~e~ably to nitro-
o~llulo~e pape~. The matrl~ h~ lnt~odua~d lnto ~hoa~mal. ~ahni~ue# ~or $ntroduatlon ~ t~ ~djo~4ed
prot~in include lmpl~ntatihn ~U.8. ~Aten~ No. 4,6s~2~0
or ~ billzation ~r the ~olid ph~ a,nd lnjeckion o~
~h~ ~olubiliz~d m~ ial ~Xnuda~n, An~l" Ri~dh~.
3$ ~:z:2as ~8~ l9a~ h~ a~lld-phJ~e m~trlx may be ~olu-
bill~ed in An appropria~ organiG ffolv~n~ ~o.~., hM5s)
and sither ~lxed with ad~u~nt or ~lln~, or in~eoted
'
~22
11/15/8~ 17:17 ~2~6 G~2 ~31 ______See~ ~ BerrY
20~33~6
lt
~lroctly. Altcrnatlv~ly, ~e protein m~y be ln~eatad ln
the d~na~ o~ ~ soli~ matrix and/or a~uvah~. ~n~nc
tlon or implanta~ion may b~ lntrAporlton~Dl, ~ub~u~-
noou~, inkramu~cul~r or lntrn~cno-~, pro~ra~ly
5 lntr~p0~itono~1. Th~ anl~ln may al~o b0 ~n~oat~d wlth
~uv~nt, ~uo~ ~s ~reund'~ ~diuvan~. 31ng.lo or m~ltipl~
boont~ i~nuniz~tionD ar~ U3~ a~an ono And 4~v~n
d~y~ prlo~ to kh~ ~u~ion d~to, pro~rAbly on day~ on~
bhrough rour, lntraveno~ in~ctlonH o~ th~ HDL-blnding
1~ prot~ln may b~ ql~n dally. W~9~ u~lng ~m~ll p~ptld~
a~ l~nmunogen~ it 1~ Pre~or~ed to qon~u~a~o th~ p~ptide~
to ~ hapten, ~u~h a~ koyhol~ limpat h~mooyanln~ Mathod~
~r ao~u~atlon a~a well ~no~n in tho art.
Betwa~n Oh~ ~hd ~o~n d~y~, pr~rer~bly ~aur
d~y~, a~a~ the adminl~tratlQn a~ the flnal b~o~t~r
~ nlz~tion, ~pl~Qns or po~tion~ th~oo~ ara ha~Y~t~d
rrOm ~ha lmmunlzod ~nlm~l~. A~ thl~ tim~, th~ lymph
nad~0 ~y al~a be haxva~t~d ~nd ino~udod in th~ o~ll
~rap~tion. ~ho harves~d org~no ~3 mlnc~d u~in~
tdc~ni~uo~ whicb di~up~ the s~ructuro o~ tb~ or~n but
~lch ~r~ not dot~lm~ntal ~o the lymphocy~a~. ~h~
o~an~ Are p~o~ably minaed ~ th B~isdo~s ~ pr~s~ed
through A me~h ~roun, and ~lx~d ~ith growth m~ql~m te
enxi~h thU pr~paration ~or lymphoayt~s~ ThR ~inc~d ~nd
~train~d tia~ue ls h~rves~d by ~an~rl~u~ation, th~n
mix~d ~ith ~ro~th ~qdlu~ to ~o~m a c~Il su~pen~lon. ~he
red bloo~ a~ may ~e ly~d ~y ~d~lng ~ ~ypo~nlo or
hyp~rtanl~ ~olu~lon to tb~ uq~n~lon. ~ pref~r~ed
m~thad ~er aell ly~ 5 to fl~d di~llRd ~ter to tho
~usp~n~ion~ ~nd quiaXly return thQ ~u ~nuio~ to an
190toni~ ~tat~ ~ith ~odiu~ ahlorid~. Any rem~lnlng:
ti~u~ may b~ r~oYed by ~lt~tloa through ~u2e.
~ ha harv~t~ oell s~pe~slQn ls th~n mix~d
wlt~ ~ my~loma ~ell; line, pr~ra~ly ~n~ ~hiuh Is
~yngenela wit~ tha immunl2~d anl~ y~lo~ a~ll lln
~o~ vn~iouo spe~le~ a~ wid~ly ~v&ila~l~ throu~h, ~or
~xampl~, Ame~ n Type Culture C~ t~on, Rc~kvll1e,
, . .........
_........... . .... ~
.
'~
, .~
. , ~ .
,:~
11/15/8~ 17 18 ~2~6 ff82 6~31 Seed ~ ~err~ 23
33~
17
Mcl. Mya~om~ o~ll lin~6 oommonly ut~od inalude Po563Ag8
(A~CC TIB 9), 8Pa/O-A~14 ~A~C~ C~ 15Ql)~ PO (ATCC CR~
~646), ~nd 2l0-RCY-A~l ~Galrre at ~l., ~ ~,1 ~31,
1~7i). A pr~err~ o~ll llne i~ p3~91~ g4 1 ~A~CC
5 ~rI:~ la), hR~RlnA~er r~rretl to l~a N~-l. 'rhe N~-
~~Qll~ ~r~ pra~rably tont-d to dt~tormlno the olonln~
e~lo~onoy o~ ~ho ~tr~in. ~hi~ may te don~ by olonlng
~u~ the N9-1 ~t~ln by llmltLng dll~tion and carrying
out tu~t fu~lon~ wi~h the lndlviduAl N8-1 olones to ~lnd
1~ cAndid~te~ wlth tho hlyhest ~usion ~ n41~- The
myolomn cell~ ~re oultur~ in An ~pprcprlate mAmmallAn
c~ ow~h m~dlum~ A vArloty 0~ w~oh ~r~ y~nar~
known ln the urt nn~ .avnil~bl~ ~rom oomnor~ urc~.
Mammall~n ~ell llnos are routlnoly ~rown b~tw~en 36 c
and 40'~ und~r aondltlon~ wb~ch maintaln ~n optlmal p~
b~w~en 6.0 nnd ~.0, pre~erably a~out p~ 7.Z. ~he pH o~
the cul~u~e m~y be main~lnud throu~h ~h~ ~8~ o~ ~
~arlety Or bur~0~ ~ys~m~ known ln ~ha ~rt. A pr~ d
bu~r oy~t~m lnvolvu~ ~rowln~ th~ ~lls ln ~ bl~a~bon-
ate bu~ ln A humldi~l~d lno~ba~or ~ont~inlng ~2pre~rably about 74 C02,
Th~ ~u~ion bs~w~n ~h3 lymphO~yt~ rrO~ t~
lmmunlzed ~nlm~l ~hd tho myeloma oell~ mAy b~ carrled
out by a varioty o~ m2thod~ de~ri~o~ in the lltq~tu~e.
The~e methods includ~ th~ us~ o~ ~oly~hylens gly~ol
Ea) (~rown ~t ~1., JL~ L_ a~ 9ao~83, 1980)
and ~l~otro~u~lon (Zlmmerman nnd Vl~X~n, L~ UOhL-
~Q1L *7~ Zt an ale~tro~u~lon g~n~ o~
mh~r¢lal~y avail~ rom B~ot~chnologles ~n~
3~ Exp~rlmen~al R~earoh, ~no" ~n ~le~o, C~lir.~.
Followlng th~ fu~ion, th~ o~ are platedl~o multi-well aulture pla~, pra~erA~ly ~6-Well
. A reagent which ~el~o~lvely ~llows ~e~ th~
~growth o~ the ~u~d myoloma ~ell~ ov~r th~ un~U~e~ oell~
-:3s i~ a~e~ to th~ cultux~ mod~um. ~ pr~rr~d ~el~tlon
t~hnlg~e u~e~ HA~ ~hypoxanthin-, a~inop~erin,
, ~ I
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ll/15r8g 17 19 ~20~ 6~2 ~)31 Seed ~ BerrY ~ 024
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thymldln~) ~el~c~lon. Othe~ ~la~tlon ~chnLque~ may
Also bo us~d, d~ponding on tho my~lom~ aoll lLna uqed.
Alt~rnAtive ~q~hod~ Or ~rod~oln~ mo~oelon~l
~tibodio~ u~llLz~ invit~ l~uni~tlon toohn1quH~.
Lympha~yta~ m~y be harva~tod ~rom ly~phold or~ns, ouoh
Ae ~pl~un~ or lymph noda~, or ~rom whole blood aB
p~riphor~l blood lymphooyto~. ~ho ly~pb~oyto~ a~a p~t
lnto aultur~ in ~h~ pr~a~nac o~ the ~D~-b~ndin~ Protoln.
O~cn immunoSt1mulatory polyp4~tld~ will bo add~d to
~he culture ~dium oonou~r~ntly. At v3r~0U3 ~lmo~
followlng the oultu~in~ o~ tho lym~hooyt2~ In vi~, th~
lymphoayt0~ ar~ hArve~tod and PU~e~ wlth th~ my~loma
o~ll lins a~ d~arib~ abovo.
Othe~ ~eohnlqu~ ~or pr~dualn~ ~nd maintalnln~
~ntibody-searstln~ lymph~ay~e c~ aul~ure
lnalude viral tran3~tlon o~ th~ ly~phoa~t~s to prcduoo
tranfl~ox~d a~ll line~ WhiQh will do~tinu~ to graW in
cultura. Ep~eln-B~rr vir~ ~E~V) hA~ ~-nn used ~or
thi~ teohniqu~ tr~n~orm~d a~ do not r~quire
fuslon wi~h myeloma oollff to allow 4Qntinuad ~row~h ln
c~lturo.
Thymocyt~ ~tay bo u~ed a~ A ~eeder l~y~r to
&ondltion th~ mediu~ ~or th~ ~uc~d ~ t. Th~,tncyt~
m~y be prep~red from ~uvenlla mLo~ lo~ ~han sigh~ wo~s
old. ~ha thymus ~land~ ~r~ ~rvest~d, and ~ u~p~n-
~ions aro pr~parcd a~ d~crl~eP a~ov~t. Al~h~1v~ly,
periton~al ~a~ropha~os or ncttlm~une sple~n ~Ctl~ may
used as ~ ~eAer layer. ~nothnr #~orn~lv~ 1~ to us~
~ondltion~d modium ~ro~ thymooytus o~ ~orophaqe~.
~t ~n ~ppr~pria~ tl m~ fDllo~ing the ~u~on
dayl the fu~d ~ells (by~rldomas~ ~o t~en an~lyz~d ~o~
thç produotion Or ~nti~ody ag~ eha ~n~ en of
~hol~e, Thl~ ean ~q don~ by ~ wl~e ~Ariety o~ t~oh
n~qu~s, l~olu~ln~ We~t~rn ~l~t, EL~AI lmmunopre~lPl~a~
tion, lnflu~no~ on ~lolo~le~l aa~ity ~ays, and
lmmuno~ytoch~ l atainin~ h~s~ teohni~u~s And
others arra well de~3ribed in t~ tcra~ure. ~s~, fo~
., ' ~
., ~; .
., ,, ~
.
. ~.
'~ ~
, ll/15/$~ 17 2~ ~S2~6 682 6~31 Seed & BerrY ~ ~25
2~ 3~6
19
example, ~. G. R. Hurroll (ed.~,
A~i~Qsli~: ,"T~ DO~ }~aton,
~la.:C~C P~ o., 1~8a.) Uybrldoma~ w~h ~eo~
~ntibodles ~ in~a~t ~r~ maintainoa ~n oultur~. Tho
~all~ ~ro axp~nd~d ln cul~ur~ und, ~t th~ Dam~ tlm~, inaybe clona~ ln su~h n mann~r ~ to obtain oolonlo~
orl~lnAtin~ ~rai~ ~lngla oall~. ~hi~ ~rovld~ ~o~ ~h~
monoalonallty or tho nntlbcdla~ obtaln~d ~rom ~ho
hybr,idonua. A wl~ loty o~ t~hhlquo3 oxl~t ~or
~lonlng ~ , lnoluding llmltlng dllu~on, BD~t n~a~
olDnin~, and ~luore~c~no~-~otlvated o~ iorting.
ono~ olanuo o~ o~ ara ~btain~d, they ar~
r~a~yed ~or th~ produ~tion o~ th~ ~ntlbody ~
intere~. Th~aa cell~ ar~ th~n ~xpand~d in aultur~ to
~illow ~or th~ produa~ion Or larger ~ount~ o~ the
~ntlbody. M~thods for ~xpan~lon o~ th~ ludo
m~intaining th~ c~113 ln ou~tu~a, plaoo~nt o~ th~ ~all~
in a biorRaotor or o~ha~ typ~ Or lurge-scal~
aul~uro environmont, or oulturln~ the o~ u3in~
~ari~u~ a~r cr g~latin o~rrl~ ma~rio~. Antibodl~
ar~ th~n isolat~d from ~ho cell cultute modla.
A pre~err~d method ~or p~odu~in~ l~r~ amount6
o~ ~ntib~lo~ involva~ ~rowln~ ~ha hy~rid~m~ cRll~ ln
a~oit~ flul~. ~he hybridomas aro pre~r~bly i~ol~ad
from tha ~ultur~ media by o~ntri~ga~lon ~n~ wa~hed wl~h
an i30-03mo~ic aol~tion, pr~fQrably ph~p~a~o-bu~r~d
salina. ~h~ o~ are th~n ~a~u~penfl~d ln an i~c-
o~otlo ~olution and in~otad into the ~orieon~al cAvi~y
Qf ~n ~pproprl~te hoet ~imal, pr~r~bly ~ mo~o~, ~nd
allowed to q~ow wlthln tho h~st Anim~ h~ ~o~ animal
~ay r~c~ivo a prein~e~ion ~ p~iJt~n~ l~,6,10,14~
tetr~m2~hylp~nt~deoane) prior to the i~e~tlon oP ~h0
hybrldoma 4O11e, pre~r~ly ~eysn to thl~y day3 pri~r
to the lnt~o~uotlon o~:th~ hyb~idom~ ollowing ~r~wth
oP th~ oells in th~ peritoneal c~vity, a~aite~ flUi~,
~ontain~ng the antlbody o~ int~r4~ 15 coll~oted.
;
. .. , .: . -
~.
.,
11~15w29 17 2~ B82 G~31 Seed ~ ~errY . ~
133~6
Ahtlbodi~s m~y be puri~l~d ~rom conditlonad
m~dla or ~olt~ fluid ~y ~ ~siaty Or ~o~hod~ known ln
th~ art- ~h~ m~tbada lncluda ~mmJnlu~ ~ul~Ato praQ
ltation, ion-oxchnngo ehramato~n~by ~a~ ~urrQ
lbicl~), nnd hlgh-pr~a~ur~ llquld ~h~o~q~ogrAph~ uBing ~
hy~roxylap~tit~ ~upport tytan~or ~t ~1. . Ll_;U~Ui~L
MQ~Q~a ~157, lg85~. A pro~errad ma~hod ~r pu~1ryin7
antibodi~ Pro~ ~anditionqd m~dla or a~aita~ ~luld
U~lliz~ ~ oommorQially nvAllA~l~ Prot~ln ~-8epha~os~
CL-4B aolu~n ~Ph~rmac1a, ~ t~way, N.~.~ Sl~ma~ ~t.
Loul~, Mo.). An~lbod~a~ may be puri~iH~ with th~Q
calumns u31ng condition~ ~ugg~tæd ~y ~h~ manu~aotUrar,
~yplo~lly, th~ aon~ltloned mec~lum or agcit~ ~lUld i~
m~x~d w~th ~n ~u~l volum~ Or ~NEN ~0 WM ~rl~-~a~ l pH
8.0, lO0 ~M ~aCl, 1 .nN ED~A, 0.94 NP-40) a~d ~pplled to
th~ aolumn. ~he an~lbo~ ara ~luto~ uaing ~ p~
~radie~. Pr~rably, th~ ~lutlon ~ or comp~i~os 0~1
N sodiu~ aLtr~to.
A~tib~Les may al~ ~e pro~ua~d by ~0combin~n~
~thod~. Antibody g~ne o~qu~na4~ o~ ln~r~st aro
l~olat~d ~om antlbody-pr~d~alng coll~ ~nd ar~ aloned
and ~xpre~ed aca~dln~ to Xnawn ~r3codure~. Recombl-
n~nt m~thad~ ~acllltata tho produa~lon o~ human mono-
~lonal antibodie~ ~hd ~h~m~ri ~ntibodioa aontninLng
human con3tant rogi4n~. M~thod~ ~or produain~ r~com~1-
~ant ~ntlbodl~ a~e dl~olo~od by~ ~o~ ex~pla, No~b~r~ar
~t al. (WO 86/01533~ and Win~er ~publi~hed E~rop~n
pat0nt appllo~tion ~P 23~,400).
Antibodi~s and oth~r pot~ntl~ goni~t~
ar~ ~a~ned in a ~ul~i~tap ~o~ay smploying tho i~olate~
bLndlng protein and/or recombinan~ ~oll8 whiah expre~
the proto~h~ Po~en~lal a~onlsts aro rL~t or~ened for
t~e~r a~lllty to bind to th~ ~D~,blh~ing prot~ih by the
llgand bl~tlng ~ech~ique. m o~ ao~psund~ whl~h a~e
~ount to bind are ~h~n oareen~d ~o~ thglr abll~ty ~0
~tl~ulato ~ran&port ~ aholes~erol r~Om ~all~ e~en-
tially A~ desorlbed ab~v~. B~ ly, ~b~ ar oholu~-
: ~
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11/~5/~9 17 21 2~2~6 682 6~31 Seed ~ Ber~y~ 7
33~
Zl
~erol pool6 ~r~ labeled and th~ c~ ro inoub~ted ln
the pr~nc~ Or tho o~mpou~d to b~ tootc~ Total llpld~
~r~ th~n ~xtr~otod ~rom the ~dium, ~ap~r~t~d by thln-
~ r ohromato~r~phy, and A~sayed ~or th~ preoen~ o~
5 laboled oholo~tarol.
Prat~ln~, inolu~in~ antlbodl~, th~t bind ta
I~DL-~in~iny protoln~ Ar~ uoe~ul wlthln din~nostla aompo-
~itlono tor ld~nt.l~ying d~ato in the ~yntho~ nd
praa~ooinq Or HDL-~lnding proteino ln a p~tlent ~nd ~o~
lo d~t~otin~ struatural deieot~ in ~D~-blndln~ protein~.
Given the rolo th~t ~DL 1~ b~ vad ta play ln aholoa-
terol ~ransport ~nd the preventlon o~ ~tharosclerosl~.
dlagno~ie oi ouah de~eot~ ~ay b~ use~ tQ identlf~
i~dl~idu~16 at rlok ~or ath~rcr~ol~ro~i~ or to ~lagno~e
tho p~rticular oondltion rosponsible ~or ~th~rogone~
To æ4reen for gunet~c de~at~ in HDL-bindlng
p~otein~, c~lls n~e ~tained irom ~ p4tiont ~nd ~naly~ed
~or th~ pre~ena~ and 9trUCtUrH or th~ p~oteln~-
Pre~xred aell~ in thi~ ~eg~d inolu~e ~lbro~la~t~ ~nd
leukocy~e~. Fib~obl~ot~ may b~ o~tnl~ from ~kln
~bmples nnd a~ reAdily GUltU~d in V~VO. ~ko~yte~ may
be obtaln~d ~rom ~lood campl~ by ~tRn~d p~ocodur~.
Analyoi~ o~ cell membr~n~ oo~pan~nt~ by ~o~ern blot~iny
(~owbin e~ al.~ Pr~ 1. AQ~dl Sci~ a ~4350~4,
~5 1~) with antlbodi~ to ~D~-hlnding p~otoin~ i~ u~d to
datect ~tructur~l dqfe~t~ or d~iclena~o~. Tb~ use e~ ~
panel o~ ~n~lbo~lo~ thAt reoognlz~ dl~er~nt ~pl~op~ on
a protein i8 parti~ularly ~dv~ntdg~ous ~r det~atih~
~trua~ural d~ects~ Q~netla de~eots ~ay al~ b~
dotucte~ by pr~lng o~ lAr D~A rro~ 8 ~tient ~ith
~oned NA ~eguenoe~ ~naoding H~ indln~ p~ot~ins or
fragmon~ ~her&of ~nd an~ in~ tho hybrldis~tl~n
pattern, whi~h i~ lndlca~l~e o~ th~ ~re~e~ er ~b~ena~
~ a ~utatlon.
~L agoni~t-~ ~ay ~e u~d to ~tl~ula~ ole~r-
anoe Or chole~t~rol lh p~ti~nt~ wlth ~QW l~vel~ L
or defect~ in apopr~toin ~tr~atur~. P~pt~d~ R~o~l~t~
,: . .
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11/15~ag 17 22 ~2~6 682 ~1 Seed & aerrY ~ 2~
33~
22
wlll g~nerally b~ oombin~d wlth a phy~ologlc~lly
aaa~ptabl~ o~rrl~ or dllu~nt and admlni~t~rud by ln~oa-
tlon, although small peptlde~ may also bo ~dmini~tored
by '1ngQ~tl~n o~ inhal~ n u~ aonvuntional ~ormu~a-
tlon~. chemio~ onia~o wll~ pr~orably b~ ~dmini~-
tar~d by ln~o~tlon,
~n~L
iLQ
LDL and H~L3 wr~ od e~om tho pl~sma 0
h~althy ~ting ~ubj~at~ by ~qu~tl~l qontrl~ùg~tlon,
~o d~orlb~d by deL~ and GO~m~n (
~ nnl~ 1:45~-7B, ~5~ h~ N~3 ~r~ction wa~ OUb~t~d
to h~p~in~Rpharo~e a~inlty chrom~tQ~rn~hy t~ x~movo
apo~-~o~t~lning partlol~ ~B1~sbrOe~k Qt ~1 " a~ LLnL
rnvQQ~L 711525-39 1983). Llpoprotain~ wa~o 1~dln~tod by
~ od1~ication o~ th~ ~ono~h10rldo proo~dur~ ~ilhel~or
~t ~ 15S~U~L ~ E~Y~h~ a~Qs21~21, 1~7~).
~rot~olipoD~m~ w~ro prhpared u~ln~ ~g~ loo1t~in hnd
puri~led apopro~oln ~-X, ~ or ~ b~ tho ~ol~t~
dlalyql~ m~hod ~Ch~n and Alber~ 0-
gl, 1~8a) ~t ~ lnolar ratio o~ lth~ to apopro~eln
300:1,
~ultur~d h~an a~ln fibrobla~t~, bovlnq Aortio
cndoth~ l c~ , swi~ 3~3 ~ibrobla~t~, ænd
tran6~0rm~d maarophag~-llk~ 401l linea (~g- ! m~u~e J~74
~acropha~a~ and human ~EL aell~ e plat~d intD 1,~0 ~m
oultu~e di$hes ~n Dul~e~ao'~ modl~i~d Eagle~ meilum
~o auppl~ented with 10~ ~Qt~l bov~n~ oe~ nd wer~ g~own
tc con~lu~ncy. C~ re loa~ed wi~h a4Ole~erol by
inoubatlng them ~or z4 to 4S hour~ With ~e~um rr~e
medlum c~n~ainin~ 0.2~ b~vine ~erum ~lbu~i~ p~u~ elther
nonlipoproteln ahole~terol ~ ~dded ln ~th~n~l ~r
aao~y~ated LDL ~Or~ 45-59,~
198
11/15/8g 17~3 ~20~ 6~2 ~31 Seod & BerrY 1~l~2~
2~)~3~6
23
Cell membr~ne~ we~ p~par~d ~om oh~le~l;erol-
tro~d a~lls e~ntl~lly a~ da~orlbad by aa~u ot ~1.
lol. ~hq~. ;~.: 38~2-5~, 1978) . t~ll monolayer~
w~ra w~hed three tima~ Wi~h Pa~ ~lU~ O.~t ~ d~ room
5 tomp~r~tux~ nd th~n chlll~d ~o 4'C ~n~ ~arap~d with A
nylon pollc1~m~n lnt~ ~.0 nM Triu-NCl, 3~H 7.4, oc;ntAin~ng
150 ~ tA~ banzar41dln~ 0~9 mM ~DTA~ And 1 DM
ph~nylrao~hyl-~ul~ortyl ~luoridq di~olYua in di~thyl-
~ul~oxido ~ ell~ w~ra ~entri~uy~l at 200 x g ~or
10 S ~inut~3 at 4'~, re~u~p~nded in T85, pooled,
homog~nl~ed with two 5-~eaond p~ of ~ Polytron
homog~nlzer ~T~km~r ~o " cln~lnn~t~ Ohlo), ~nd
~entri~u~ed a~ ~00 x ~ ~e~ 10 minutos at 4'C. ~h0 ~00
x g ~up~rnat~nt W~s ~entrl~uyod ~ ~00.000 X g ~or 60
~inute~ at 4'C, and th~ mumbrano ~llut wa~ ~tored at
-70'C. In ~ ~ploa~ prepar~tlon, th~ total mem~r~n~
~rotein yi~ qu~ntlfled ~y tho mo~hod o~ ~,owry
Qt al. ~J. ~iQl. chQ~ a~-73, 1~ om 3~ 130-m~
di~h~ WaH approxima~æly ~ M~
M~mbran~ p~llue~ Werc ~olublll~e~ by ne~dl~
~plratlon in ~letrophore~ia 4~mpl~ bu~er ~nd ~ollod
~or 3 ~lnu~ ln th~ pr~n~ o~ 104 (v~ 0-moraAp-
to~hAnol~ ~r~lons ~ontainlng ~lAl amount~ o~
pro~oin ~ner~ 500 ~9) p~r lan~ ~r~ ~leot~ophor~d
on 7~ polyA~rylaml~o ~ g~l~ aocor~lng ~o Laem~l~
~n~ 2~680-85~ 1970). Soparat~l prot~ns w~3r~
tran~orre~ onto nl~rooellulo~e ~Soh1~1Ohe~ ~nd 9chuell,
5 ~m~ ~y ~leotxopho~e~ Towbln et a~ 5YJ ~lt
~ CA~ . US~ 4350-54, aQ79), 0~1~ we~e ~ t~d
u~in~ pro~tain~ or iodinat~d ~Dl~oular w~ight
ndDrd8,
To a~say protein bindin~ ~at~vity, nitro~llu-
.~o e ~trlp~ we~e lnoub~t~ f~ 2 ~our~ wlth blockin~
bu~rer (1~ ~M ~ris-~Cl/ p~ 7~4, o~n~lning lSo ~M~Cl~
35 1~ w~v h9~ w/v Carn~tion n~n~ iRd ~ilk, 0,Dl~ i
v/v antl~oam A, 50 ~g/ml LPL~, ~ollo~d by a further ~- !
hour inoubatiDn in t~ ~a=e bu~r~r oon~ai~n~ ~ ~gJml
I
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,:
., ' , ~ .
. 1
11~15~8g 17 24 g~206 682 6~31 Seed ~ herry ~ 030
~33~L~
24
l~5I-~DL3. stri~ wer~ wa~hed once rapidly and than
flv~ tlm~3 ~or lS minut~ with wa~hing bu~P~r ~blc~kin~
buf~r withaut ~ lottin~ Inouba~ion~ ~nd
wa~A~ w~r3 a~ roo~P ~au~parn~u~e, ~f~r drying, proteln
b~nd~ w~ vl~uallzed Py æutoradlogr~hy~ ~u~orndlo
graphs ruv~Al~d ~h~ tho 125I-HD~3 bound to a m~or
m~mb~no protein h~vin~ ~n appar~nt m~looular mn~ o~
approxlmat~ly 110,000 Dalton~ in all th~ ~o~omention~d
~qll typ~ The relatlv~ migr~tlon ~nd bin~ing actlvity
of thi~ protoin worR not ~lt~rhd by bollln~ oP the
~olubill~d m~mbr~nu3 o~ by pr~reatmont Wlth 1~ v)
~-~erc~ptoathanol. A~ oo~p3red to chol~t~rol-lo~de~
~113, 0~116 incubated with ~hol~st~rol-~ra~ m~dlum ha~
low~r or undotea~Able ~mount~ o~ the 110 kDA ~rot~in,
~u~ ting that it~ binding ~tlvl~y w~ ~p-reg~l~t~d by
chol~tero~ lo~ding o~ o~ . Pr~in~u~Atlon o~ hu~an
arteri~l ~mooth m~s~le c~ wlth ino~u~ aona~ntra-
tl~ns of ~hol~tqrol pr~uc~d B doae-do~nd~nt ln~rease
in th~ binding ~otlvlty o~ ~h~ 110 ~Pa pro~eln.
ao ~ryp~l~ treatment ~f tho ~n~th~lial aell
~onol~y~r a~olish~d th~ a~tiVity o~ tho HDL~blnd~n~
pr4teln, indloating ~hat 1~ o~ted in the pla~a
m~mbran~ ~nd i~ ~ho~fore avail~ h~ o~ ur~a~
for l~t~a4~ion ~lth ~DL. Und~r th~e a~ndl~ion~, ~hc
aell monalayer r~main~d intao~i ~nd ~bll ~orphology, a~
a~s~ d by llght ~iaroa~opy, wa~ ~nA~r~ot~d.
Troat~ont o~ the 110 k~a HP~blndlng protei~
wlth endo~lyoo~l~as~ H ~0114~ed ~y ~ lea~opho~
o~ th~ tr~a~o~ prot~ln lndlaat~ ~ o~rbo~ydrato oo~tent
o~ appraxl~atoly 1~ by w~ight.
~x~pl~ 2 - Purifiçati~n ~ L~
Th~ HD~-binding ~rot~in d~ribod 1~ ~x~mple 1
i3 pr~p~red Pro~ mi~o~o~al m~F~n~ pr~p~ed ~rom
~ul~ured bo~ine endo~h311al ~Rll~ by ~oluhili~ation in
1~ Tri~on X-100. gol~bili~d prot~ina ar~ p~s~æ~
throu~h a wh~at g~rm ~glutti~in ool~mn. ~he ad~or~ed
I
'
. ..
.
. r~
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., ' `,': ~
11/15/~g 17:25 ~2~ 6~2 6~31 Ssed ~ B~rry 1~1~31
~0~33~6
ylycop~ot~in i~ alutad ln th~ p~e~or~oo 0~ exae~
N-~c~tylgluc:o~mlnu, dlaly~d, ~nd ~u1b~Qtad t~ l~o~l~c-
tric focuDln~ ct~Dphor~si.~ ln tll~ pr~noe O 4
ure~ . Protalh~ havlng th~ iH~oloctrla point o~ th~
blndln~ protoln tpH 5 . o to pH 6 . o~ ar~ l~olatHd nnd
puri~iHd by ~D~-polya~ryla~ a ~al tlla~ophor~oi~. Th~
po~ition o~ the ~DL-bln~ing ~rc'c~in on tho ~ol 1SD
ldantlrled by ~llver atAlnlng and ~on~irmod ~ the
r,d blot ~a~ay. ~h~ H~-binding p~ot~ln 1~ ~han
10 ~lHtro~lut~d ~rDm thH gHl. Pro~e~n pllrJ rlod by thi~
mathod 1~ a~ l~R~t ~09~ pure~ Al~ detu~qnln~d by ~llv~r
staining ~ poly~a~.~yl~mld~ 51o~ h~ prot~in me~y b~
~urth~r puri~i~d by UPL~ ale n~a~ssa~y. Al~erna'cl~ol~t,
the HDL-bindlng protuin msy bH puri~ied by lmmuno-
lS ~lnlty ohrom~ogr~phy o~ ~lubili~ed m~mb~an~ ~slng
n3noclonal ~ntib~die~ pro~uoed ~ de~o~ib~d h~r~in. ~ho
purl~led ~re~eln i~ us~d for antib~dy ~ropar~tlon ~nd
prot~ln oharac~erizatlon~
~g~
. Pr~para~l.on 0~ m~ 9~LL_~ItL olL L 2 -~Y
Fi~rQ~L~t ~ RNA
~otal ~NA wae pr~p~x~d fr~ b~man flb~obla~t
~SK-5) c~ cultu~d 1~ the ~r~nce o~ lo~ a~ru~ by
~h~ gu~nldlne l~othlooyanat~ m~thod t~hir~win et al.,
t5Z-94, 197~ POly tA)~ RNA WA~
obtain~d by two round~ o~ a~init~ chromat~raphy on
~ o (~ U10B~ ~Manlatl~ et ~ " lbid~ t
and ~econd ~trAnd ~N~ wa~ ~yn~h~lzad ~ ~ith~r ollqo
~d~) or random pri~in~ riF~ rand ~ynthssl~
lFri8~h~r ~t ~1., S~ .771017-28, 138~) ~rom 5 ug o~ 2
X ~elected poly ~A)~ 2N~ de~orlbed by aubler ~nd
~D~m~n ~Ç~n~ ~s263-69, 1~83) a~d ~a~n b~ X~
Natl. A~d. ~ d ~s241~-16, 19~6~, Th~ ~dou~le-
strAnd~d a~A w~s m~hylAted With E~0 X~ ~ethyl~e
'~
.~
,:
11/15/$9 17 25 ~2~ 632 6~31 Se~d & BerrY 1~1~32
;~o~363~gi
~M~niat1q et nl ., ibid. ), Paaa~ Ver a S~Ph2~rC~ CL-¢3
~PhaL~ P1DC~UW~Y~ N3. ) oolu~ luntlld wlth 1'4
~A pol~r~o ~Mnn1~t1~ ~t A1~ ih~d.), li~7~ted to
~Sco'~I lln~cc2r~, fli~ d W1t~h ECO R~ pUI!~3/~S OV~
5 S~PI]~r~ CI,_2~ tYh~aC1n) ~O1U~I~ Th~ Cl:)NA Waa
ll~tt~d tc~ ~n E~o F~-dig~tod, phoaph~ta~d l~mbd~ a
v~ctor ~YoUn~ and ~avlEI~ ~"~;lq~
.QQ~1194-~, 1983) and p~cXnqad in l~mbda p~ak~ln~
~xtr~ot ~S~r~tag~n~, ~nc., ~a ,Joll~, CA11~ ~ . Plht~
10 lysat~ libl~rle~ w~re propar M~ni~atl~ et ~ ibld. 3
u~ing ~ i. YlO~ a~ t hu ho4~ ~train. Th~s~ plate
ly~at~ llbrari~a werfl ~torod ov~r ~hl~ro~o~n ~ 4'C
un~ll usod ~or l:Lgand ~or~n~ng. ~h~ ra~om-prim~d oDNA
ll~rary was oompo~d of approx~ 'co~y 1~, millioJ;
15 i~olltes, and th~ ollgo (dT)-pr~mud ll~r~ry w~ aompo~ed
af approxlmat~ly ~3 r~llllon l~ola~
Thlrty platoa (lS0 mm) s~ N2~ r waro e~h
2~ ovorl~y~d with 10 r~l o~ NZY 0. 7~ a~aro~ aon~alning
nll YlOP0 c~ ad~or~d wlth app~oxl~a~ly SC~00
~ll that aontalnod ollgo ~r)-pr~ nd random~prlm~d
human ~l~robla~t ~NA in~er~d at th~ E~o RI ~ rh~
platos were incubated at 37'~ ~or 3 ho~lro, overl~yed
a5 wlth 137-mm nltrooellule~e ~l~t~r~ ~chlelohe~ ~nd
S~uell~ pravlou~ly sonk~3d in 1~ m~ I~G ti~pl~opy~ D~
thioqRlaCltOpyr~lnO:~Ildli~ lh c:homical 00" ~t. LouiF~,
Mo. ), ~nd ln-~ub~ed at ~7'C overnight. me fllt~rs W~
w~ hea twi~e fo~ 15 mlnu~ ch ~t roo~ t~mpor~tur~ ln
30 ~0 mM ~ris-~l, p~ 8.5, lgO mM NaCl, and w~hod ~or a
hour~ ht rooln t~mp~r~tUre in li~and~^~lottin~ l; loc:king
bu~er. l~he ~ilters wo~e trans~e~r~d 'e~ ~re~h ~locking
b~f~r oont~lnlng ~ ~y~ BI-HDL3 and i~cub~od for 2
2~4U~Y ht ~04m temp~tu~e. ~e ~i1t~ ere ~hoa
thr~o ~ ror 15 mlnutlss ebch at. 10-~ in wa~hing
b~rzffr, two t;im~ fo~ lS ~ninut~ ~aa~ ~ lo~ in 50 ~M
Tri3-~SCl, p~ 8.S, lS0 m~S N~l, 0~2~ ~$A, and or,~ ~or 5
,~ .
'~ ~
.,
,' " ' ~ .
" , ~ . ,
11/15/~g~ 17:~6 ~2~6 ~ 31 Seed ~ BerrY ~33
Z~ 391 6
27
minu~s at lO'C ln 50 mM ~rls-llCl, pll B.5, l50 mM II~Cl.
Th~ ~ilt~r~ we~ air drlod and oxp~od to X-r~y ~ilm at
roo~n tomp6lr~tur~D for lO dsys. Po~ lv~ ~glons ~n th~
plal~ ro plok~d, t:lt~r~d, An~ r~plat~d ~ qlva lo, ooo
S and 50,000 pla~uo~ ~aoh on ~ 5~ , YlO~o cnlls ~Ic~;Jrdlng
~o Atandar~ proaod~ (M~nla~i~ et ~1., lbld. ~, ~a-
~o~d ~ qrs w~r~ ov~rl~yqd and ~c~e~ned a~ d~orib~d
abovo . Po~itiv~ pl~ w~ro plok~d and r~plat~d to
glva 500 plag,ue~ e~oh on ~ L Y1~9C~ aell~ G-
~oakqd $11ter~ wRr~ ov~rla~ed And slo~e~nad 8:}~ de~rlb~dabov~. Pl~te lys~te ~took~ War~ pro~nred a~ d~o~ribod
by Naniati~ et al. ~ibld.). ~hag~ ~N~ Wa~ prep~r~ by
ln~u~nting ~ ODbo~ unlt~ o~ i YlO~0 o~ in~cted
with 5 x 1a6 phhg~ in 500 ml c~ N2Y broth flt 37-C ~or 3
15 hou~ he phag~ were r~aov0r~d by pr~alpitn~ion with ~
NaCl and la~ ~wfv) polyethyl~ne glyaol 3000 at 4'C and
aentrl~ug~ion at ~000 rpm in a ~orvall ~3 rot~r or 15
mlnute~. ~he pha~e pr~olpltnt~ w~re ea~h dl~olv~d ~n
5 ml Or p~l.50 ~mfml ~3Cl in lQ mM ~rl~-~el, pH 7.4~ ~00
2a ~ NaCl, 10 ~M MgsO4, snd ~ntrl~g~ ln ~ 8~vall 8W55
roto~ ~ 35~ ooo rpm ov~rnlgh~. Th~ O~Cl-bnnd~d phage
w~r~ reoev~r~d hy 4th~01 prealyltAtlon~ ~n4 DNA wa~
uxt~aot~d by r~su~pending th~ ~ p~llet in ~50 ~l o~ 50 mM
Tris-HC~, pH ~ adding 100 ~1 o~ lO0 m~ ~rl~ ~Cl, pl~
~5 8.0, 100 mM E~TA, 0.1~ 9DS and ~0 ~1 o~ 1 mg~ml
prot~ina~e X ~ollow~d by inaubat10n ~or 1 ho~r ~ ~7'C.
~he DNA W~s ~xt.raot~d twico with phonolJc~l3:l~on~
al~ohol (~4~ PCI), once ~lth CRal3tl~oa~yl alaoho~
(24~ , and pre~lpitate~ wl~h A~OniUm ~tate ~nd
eth~nol. Th~ puri~led a~N~ W~ u~p~n~e~ ln 75 ~1
o~ 10 mN Trl~ p~ ~.0, 1 mM EDTA, ~ig~ d with ~co R~
at 37-~, and the oDNA ins~t~ per~ re~ove~e~ by
eleatrop~ore~is ~n O.a% ag~o~ gels. The o~ fraq~nt
~rom a posi~l~e phag~ alone ~ro~ tho ran~om-prlmed
3S libra~y (designa~e~ 17~1~3) wa~ ~aovered on ~A45
(~ohlaiahs~ an~ S~huell~ ~n~.~ X~n~, N.~.~, elut~d ln
1,5 ~ N~l, 10 ~M ~ria-~l, pH ~.0, 1 ~ TA ~t ~5C
':, ~ ' ''~ ' '
'` '''~
}~k
.
., . , `' ~
. ~,;
il/15/~ 17:27 ~206 6~2 6031 Seed ~ ~errY ~034
2(~33~
~or ~O minut~, PC~ and CI ~xtract~d ~nd ~th~nol pr9cip-
ita'~ed. Th~ oDNA ~ag~Qnt wa~ ln~rt~d at the ~oo RI
slt~ of M13mpl~ ~n~ pUCl~, and th~ V~tA soq~lenoe wa~
d~te~lr~d by ~h~ thod ~ sange~ k al . ~L
5 ~ggl s~ .U~54~3-67, 1~77). P~ult~ d$a'ce~
tha~ thi~ ~lon~ ~on~inod nuol~ctldo~ 257-8a8 o~ th~
~quon~e ~hown in ~igur~ h~a pU~ a~N~ cl~no wa~
d~ n~te~ pM~O-1.
A ~ kb human ~lb~obla~t ctD~tA w~ ol~t~d by
~r~enln~ thR human ~ibrobla~t ~gtll DN~ llbxsry With
th~ p~tlal cDNA ~om pl~mid pM2~0 l. All bne~crln
ph~g~ platln~, plaquo ~url~icatlonG, and DNA p~ri~ica-
tion~ were p~r~ormed n~ described abo~o. ~hR 2 kb c~NA
wa~ ~xoi~sd ~ro~ ~ cl~no 1~ and lneo~ed in~o th~
E~o Rl ~lte o~ pUCls to constru~ umid pM253-3. Tho
aDNA WA6 ~l~o in~ert~d into Ml~m~18 a~d Ml~mpl~, nnd the
DNA ~uenc~ Was d~termlned by the me~he~ o~ sanq~r
c~ lb~d.~. Th~ nucleotide s~queno~ o~ th~ olon~d ~
kb cDNA and th~ inferred amino a~ld ooquuno~ are ~hown
~0 in Flgur~ 1. Whlle ~he amino ~ld s~qu~noo ln ~l~uro
1~ ~hown With an ~mlno~rmin~l methl~nine ~id~e,
oellul~r proce~lng ~ay re~ult in ~ ~rot~ln hnvin~ an
~mlno-t~rmlnal ~lyain~.
C. ~onst~u~ o~L_p~_g_~an C~mid ~14r~x~
The Ye~tor 8~217 wan pr~p~d ~ro~ pJB8 ~ Lau
8nd X~n, _~v~ 5l~ ~ ~L~ C~ Q:~22~-2~ 3)
~n~ pgV2~n~ ~Sou~hR~n and BRr9, L _ILaL.~ eL ~en
~53a7-4~ 8~) by oomblnln~ th~ 3.4 kb ~ind III
~r~gment o~ p~B~, wh~h c~nta~ne th~ ~V~O prom~*ex and
~OS reglon, wlth th~ 2.65 kb ~ind III~Eoo ~ ~ra~m~n~ o~
pSV2-nso, ~hich cun~ins ~h~ neomyo~n r~ tan~e ~en~
~nd SV40 t~rmln~tor, lnto t~e ~l~nd ~X~ and E~O ~I ~itH~
o~ pUCl3 ~ee Figu~ 2).
Hlgh mole~ul~r woigh~ ~NA w~ olatea ~om
~he hu~n acut~ lympho~l~Yti~ leu~eml~ c~ n~ MO~T-4
M4nowa~a ~t al., I ~U~ =U~ 91~5, 19~;
..
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11/15/89 17:2$ ~2~ ~82 ~1 Seed & BerrY
)33~
.
as
A~CC ~R~ 1582). ~he 4N~ waA ~arti~lly d1~e~ted with ~a~
3A and ~1zo-~r4at10rl~t~q on ar~ NaCl gr~cl1~t ~3
desc~ibod by Dill~ln And Wo~ ,a, 1 4, lg~
DNA rraatlorl~ oont~inin~ 30 ~o ~0 k~ g~n~s wore
5 11g~t:o~i .tnto t~o ~am ~ o~ Zo~21~, ~nd th~ lig~tion
ron~t~ion wa~ fn vl'~o paokas~od into ph~ ptlrtlolo~ ~u~lng
paok~ing Xi~ obtnln~d ~rom 5t~at~geno, ~no., ~4
~olla, ~ali~.) wh10h were u~o~ to ~rAn~oct ~",~, 1~1.
~ ho proc~urR ~ave rloo ~o ~pproxl~tely ~.~ x
10 lO6 ~mplc~illih-ritsl~tant ~oloni~ whioll wore hAn,~eeted
~ro~n plate~, ~lxed tog6~the~, allquoti~d, and ~ro~esl ~t
-70-C. StAphyloa~acal g~ow~h wa~ prev~nt~ by th~ adcll-
tior of v~noomyain-HCl tLilly) at Approxim~tely 3
~g~ er to A~ploillin~ con~ainin~ madla .
15 ~h~ h~man cos~nld llbr~ a~ ~c~re~n3d witb
3~P~ eled pM253-3 2-Xb C~N~. ~wen~ pl~t~ ~150 ~m)
of ~B ~edla con~a~,nlng 50 ~g per ~ r o~ neomyaln woro
lnoaul~ted with approxllR~tely S0, 000 aoloniql~ oAah Or
th~ hu~h~n ~oslnld 114 a~y and inaub~t~d o~nlght ~t
~0 37'C. Nitroacllulo~e ~ er llft~ or ~he platn~ wq~s~u
pr~par~d and hybridl~ed wlth the p~53-3 oDl~A, and po~l-
tlve oolonie~ wero pu~lfiod a~oordlns~ to at~nd~d
~nathod~ (~7anlatls et al . ~ ibld . ) . q~h~ thrae po~itivo
~lone~ o~tain~d WH~ signathd pM~0-16, pM380-~7 ~nd
~5 pM:~80-20.
Polyalonal ~ntl~tra weri3 pooled ~rc~ w~ H.
whioh had boen immunized witb a pa~tially p~2rl~i~d
30 pxop~tion ~ tl~o bo~rinR llO,000 Dall~an ~lrJL~ di~g
p~otoin. ~ha posled ~r~ wbr~ d~ ted o~ aht
anti~odieR b~ ~roma~4gr~ph~ on ~N~ ati~at~ ~eph~lros~
~pharmAci~ Pi~ taw~f, N.~. ~ oonju~ted t~ ar~ extraot
o~ a. 5~ Y10~0~ ~wunty plate~ ~130 mln~ O~ NZY a~r
35 ~ re oaoh o~erl~y~ with 10 wl ~ N2Y 0. 7~ ig~ros~
~ntaining ~., çQ;Li ~lO90 Cly~ d~o~d with npproxi-
matel5~ 50, QOO ~gtll ph~ o~n~aining r~ndoh-p~lm~d
.
.
,
;~
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5~9 17 2g ~2~6 ~2 6~31 Seed ~ ~e~
)33~
human fi4roblaRt oDN~ in5ertQd at t~ Eao ~I sit~. The
platea w~re inoub~t~d at 37'~ ~or ~ hour3, thRn over
l~yHd With 137 ~m nitro~llulo~ ~llt~r~, whinh h~d b3fln
pr~vlou~ly ~oA~fl in 10 mM IPTG, a"d inOUbntUd ~t 37'C
ovornlqht. The filtura wqr~ W~h~d at room tbmp~r~t~r~
ln 50 mN ~r ~ C1, P}~ 8.5, 1~0 h~M NQ~1 ~er 1~ m1nUt~8~
1n 50 m~ C1, PH B,5, 130 ~t Na~1, 3~ 83A ~O~ 1
heur; ~nd ov~rnight lh So m~ Trl~-~Cl~ p}~ 8.5~ 1~0 ~M
Na~1, 3g ~SA nontAlninu th~ pool~d, d~ t~d an~is~
o dllut~d approxi~na~ly 1 ~ ~oo . ~h~ ~llter6 w~r~ then
W~shafl ~ ro~m tomp~r~tur~ in 50 mN ~ris-HCl, pH 0.5,
150 m~ NaCl ~or lo ~inute~ ln 50 m~ ~rls-~Cl, pH 8.5,
150 mM ~aCl, 0.1~ NP~40 ~or lQ mln~t~s~ ~wl~a In 50 ~Y
~rls-~Cl, p~ 8.5, 150 m~ NaCl ~or 10 ml~u~es e~ah~ ~nd
in 50 mM Tr1S-~C1, Pll 8.~ 0 mM NaCl, 3~ ~8~ ~o~ lO
mlnute~. T~o Yllt~r~ wer6l th~n inoubated ~02~ a . S hou~o
ln 50 mM ~ri~-N~l, pH ~.5, 1~0 ~M NRC1, 3~ P~A With
app~ox1~atQly 1,000,000 op~ per ~ilt~r o~ 125I-l~b~led
aYflnlty-pu~i~ied rAb~i~ anti-~ou~ Ig~ ~O~anon
~0 T~nik~-CAp~ll, Malv~rn~ P~ he ~ ers w~re th~n
~ashed at r~om te)nper~ture in 30 m~ Trl~-H~l~ p~ 8.5,
150 m~ Na~ r 10 mlnutess in 50 ~M ~rl3-HOl, pH ~
150 ~ N~Cl, D.l~ NP-40 ~or 10 mlnute~) and twl~ ln 50
rnM ~rl~-HCl, p~ a.5, 150 n~ NA~1 ~0~ 10 mlnute~ ~ah.
~h~ ~ilt~rs Wer~ Air drl~d an~ a~tor~d~gr~phed at -70'C
with ~n lnten~l~ying ~ar~nu PosltiVe r~glon~ on th~
pl~t~ wG~r~ plaque-puri~led as d~a~i~od ~b~v~. ~b~ on~
positlve c:l~no obt~ln~d W~E~ de~:ign~t~d la~-l. P~ DN~
w~ puri~sd ~rorn alon~ l~B-l as d~Aarib~d abov~.
Plhgu~ la~-l wa~a u~30d to lyqo~ni~e ~
Ylo~s~ l~he lnr~cted ~a~toria W~r~ pl~te~ on ~
ampicillin media at 30'~, thah r~pllsa plo1~ed onto ~-
amplcillln and lnaubat~d at 30'C an~ d2tC. ~10~ 5 th~t
~iled to grow at 42'G ~r~ lysogeniæed by ir,~a~lon
35 with IP~, and the ~0-g~lacto~ld~ rwlOn p~otQln W~5
pu~lfioà ~y ihmUno-afrihit~ ~romatogr~phy ~u~ing
Proto$o~b l~a~ ~d~rb~3nt ~obtAiri~d ~roln pro~sTR Blot~
.. ._. . ,~ :
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11~15/89 17 30 ~2~ 2 6031 Se~ & ~erry, , ~ ~37
33~6
~.
31
Madlson, wi~., and used a~ ~pooi~i~d by the
manufactu~r).
ThR puri~lq~ ~u~io~ protoin w~ dl~ly~od
ngal~t 50 mM AmmonLum bio~rbonnte, lyophlllz~d,
rr~u~pQndod in Pas ~nd ll~od t~ prnp~ro p~lyolon~l
antl~rn, Tho protain ~l.0 ~g~l) Wn~ orbod ~o two
nitrocallulo~ dl~oc using lO ~l po~ dl~o. ~h~ dloac
were dri~d at 37'C ror 3-4 hour~, tb~n uaoh ~18~ wa~
dis~olvad ih 100 ~1 DM90. ~h~ ro~ul~iny ~olutlon WAB
aombln~d wl~h Pr3und'o ad~u~nt ~nd ln~eat~d intr~p~rl-
ton~nlly into m~ nlb~c ~ioe ~ob~ln~d rrom ~imon~an
~b, Ina., Gilroy~ Call~ e miao w~ro i~munlz~d
w~th an lniti~l in~ation of 2 ~ o~ protoln p~r mouss,
~oll~wad by ~ov~n boo3tos ln~otion~ o~ 2 o~ 4 ~g ~aoh
~5 at ~- to 3-WeQk int~rYal~. To obtain ~ntls~, blood
was ooll~cted frcm tho miae by retro-orbltal bleQdO and
a11Owqd to alot, and tho s~rum wa~ r~ov~d.
For ~e~uena~ ana1yel3, ph4g~ DNA ~ao r~oover~
from the ~2B-l r~eambinant a~d th~ aPNA w~s excla~d by
~o Eoo RI dlg~tlon. Eco ~I ~ra~ment~ a~ 261 an~ 339 bp
w~rs ln~rt~d lnto the Eco ~I aitR or p~C19, and ~h~
r~ltant plasmld~ w~ dae~qnatod pM283~ n~
pN2a3-lo~ pectively. Tho ~ra~ment~ ~are al~
lnYorte~ lnto Ml3mplS ahd ~e~enaod b~ tho m~thod Of
Sang2r e~ al. (ibid). Th~ DN~ ~qu~nce o~ th~ l2~-1
c~NA is ~hown in ~lgurQ 3. The 600 bp a~A hR~ ~n ~pen
readlng ~ram~ ~hat ox~en~ ~or ~t~ ehtl~e l~ngth,
suggestiny t~at it encode~ an int~rnal tra~m~nt O~ ~h~
HD~-blnding protoin. No slgnifioan~ matohc~ w~a ~ound
3~ to th~ ~DNA sequeh~ or th* aorrespo~dln~ ~lx pot~n~
tran~lation p~oducto using the ~AS~A and ~FA~TA ~Pe~rson
an~ Llttmah~ Pr ~. N~l, Ao~d, sqi, ~A ~2~ 4~
1988) Gomputer ~2ar~has of th~ 6en~ank, EM3~ ~nd ~R
d atabP~es .
~ho in~ivid~al Eao RI fragman~ o~ the 12~-l
phaga ol~ne were 32P-1abel~d u~n~ ~ndo~ p~
(A~ersham ~orp., Arlington H~ight~, I11.) *nd u~d ~o
~.
- ;.
': , ' '
" ' ' ' ' . ~:
~1 ~
11/15/8917 30 ~2~6 68~ ~031 S~d & BarrY ~3~
.
2~
32 .
p~b~ North~rn blot~ (Thomd~ Pxoa.,_~tl ~ ~ar~i, ~al. U~A
7.7 ~ 5~01, l~0) o.e poly t~) ~ RNA ~Chlr~wln ~It
~hr~ 52P4, 1979~ Avlv 6 L~dor, ProQ. N~tl.
~.i~ ~.2.:1408, 1~7~ om tha ~ow~ m~oropha~
5 oRll lln~ J77~ ~ whloh had bo~n oult~r~d ln tho ab6l~noo
~nd prus~no~ of oxogonou~ ao~tyl~ltod L~ ~or~m ot ~l .,
;L~l~Sh~ ~IZ~0~-lO, l~7~. ~d~n~lo~ blot
pattorns w~r~3 oht~ d, lndloa~in~ thAt t~a o~N~ snoom-
p~b~S ~ n~tur~l ~cv nI ~i~o. ~oth ~N~ pr~p~ratlonn
aont~ln~d hybrldlzln~ m~NA~ o~ 4.4 o~ ~6 ~b, but ~ho~
~NA.s w~r~ npproxL~ately ~ou~Pold ~o~e ~bun~a~t ln th~
L~ own ¢~ . Eq~A1 ~m~unta of R~ r~ ~how~ to b~
pr~ont ln hoth ~m~ by ~t~ipplng t~o p~obo ~r~ tha
blot and rehyb~idlzin~ th~ blot wlth ~ ~at gly~er~ldo-
15 byd~-3-pho~phate d~hydrog~na~ ~DNA proba. ~be 12~-l
fibro~la~t ~PNA prob~ yield~d ldantl~al ~NA blot
pa~terh~ in humah ~p~2 li~r oar~lnoma, bl~dd~r
oar~lnoma, rhabdomyo~arcom~, and nor~ mononual~ar
~118~ indl~atlng that ~ P~-bindlng p~o~ein ~on~ 1G
~xp~ cd in ~ny o~ll typ~.
ona ~illloh r~omblnant ~nmbd~ ~tll plaqu~s
aontalning oll~o td~)-p~lmod hum~n e~ytbroleuX~mia ~H~)
aell ~NA ~L~p4z ~t ~l., PI~Q~_ Na~lL~ ai. U~a
~t5~ l9, 19~7) w~re ~re~n~d ~o~ hybr~ atio~l ~o the
25 12~-l f ~ ~robla~ cDNA. Nitroc~llulo~e ~llt#rs, ~o~
aontainihg approximately 50,000 plngue~ ro hybridl2ed
with ~h~ ~2~1ab~1~d o~NA in ~0~ forma~ido, 5x
D~nh~rdt's (l mg~ml Fiaoll, 1 mg~l p~yvinylpryroli-
don~, 1 mg~ml ~SA), 6x s9P~ 20X ~;5PE ~ 3 . ~ N ~lac~ 40
~M Na~aPo4, 20 mM ~A, pH ~.~), O~lt S~S and loO ~g~ml
dqnA~u~d ~lmon ~pa~ ~NA (M~nlAti~ ~t ~
Mol~~l~L _~lar!~nc~r A ~L~Çxal,t~y M~nU~l, C~.d pring
Narbox L~o~atur~, C~l~ sprl~ rb~r, ~.Y., ls~a, pp.
326-2~ h~ ~ilter6 w~r~ wa~h~d~ ~w¦~ in ~x ~9C, O.lt
~DS ~t room temp~aturæ for 30 mlnu~ p~r wa~h~ ~wlae
ln 0.2x 3$C~ O.l~ ~DS at 650 ~0~ 3~ teo p~ wa~h;
ln O.lx 98C, 0.1~ SDg at 55'C ~or 90 ~inu~os~ in Q.l~
''.' ~' ~ ~' '
~: ,""., , . ' 'i' ~.i i
.
.
11/15/89 17 31 ~206 6~2 6031 SeetJ ~ BerrY ~ 39
2~30~3~
SSC, 0.1~ S~S, 50& ~or!n~mi~ A~ ~'C ~Qr D0 mlnuto~ ~nd
in 0. lx S9C, 0.14 ~D5 at 65-C for 2 h~ur~. TWo ~o~iti~r~
plaqu~s~ d~ n~ted I~EL 13 and HEL ~ ~ w~re pur~ d,
pha~ç DNA Wll~ obt~ino~, and th~ ODNAsl w~ro ~xcis~d by
5 ~oo E~I dl~tlon. Clon~ HE~ 13 ~ontAir,çd thron E~o RI
iragmHnt~, da~l~n~ d 13A, l~ dnd ~a~ who~ç approxi-
m~e o~zo~ wçro do~e~min~d na 0.6~ o.a nnd 3.~ kb,
Qo~iv~aly . ~ach ~ the H~L ~3 ~co RI rrag~m~nth w~3
ln~rt~d .tnto ~he ~o RI ~lte ~ pUCl~, yi~ldlng
plazmld~ pMa~a (13~), p~72ag ~13B) ~Ah~l pM2~0 ~l~C~-
ClOne ~EL B cont~ln~d two ~oo ~I ~ra~nt~, d~Nignat~d
and 8~, whoa~ ~pproxi~ate ~lZ8~ We~ t~to~n~d as
0.~ and 1.~ kb, r~upa~ ly. ~ho ~ E~o RI
~ra~mRnt6 were oAch ina~rtod i~o ~hc Eco RI ~lt~ o~
pUC19, yi~ldlng pl~m~ds pN2~ ) and ~292 18~ h~
lntact ~EL 13 a~NA and rlanXlng L~b~R ~1 DN~ w~
~x~ d by dig~ion with Rpn I And S~t I, ahd wa~
lns~rt~d into pUCl9 to ~on~t~ua~ plas~id ~M293. DN~
hy~rldi~ti~n analy~ia demon~tr~t~d th~t th~ pN~8~-10
~0 cDNA hybridlz~d to th~ 13A and 8~ ~raym~nts, whor~ the
pM28~-11 cDN~ hybridisad t~ ~he l~C and 88 ~ragm~nt~.
Re6trla~1On andonuol~a m~pA ~re prepa~ed for the 4~NA
frag~ent~ ln pl~miq~ pM2~ to pN293 . Th~ ~pe lndl
eat~d that the l~L 1~ ~DNA ha~ a mucn longe~ 5' end ~nd
25 dift`~rs ~r~m tha ItEL B And 1~-1 cDNA~ ~t th~ n~ by
an ~ppar~nt al~ernatlv~ spllc:ing ~v~nt. ~h~ c~mparatlve
r~riction msp~ are ohown ln FigurY ~ . Ans.l~r~ls ol~ the
~E~ 13 DN~ B~qUenc~ ~howed thR pr~enc~ o~ ~oU~teen
in~erna~ r~p~4t~. Each r~peat wa~ ~hown ta oont~in an
3 O lhte~rnal ~ons~n~U~ ~e~u0nae bo~nd~d ~n ~ither ~ld~ by ~n
~mphipathic hslix. A oomp~ on betw~en the ~ 8 In~
13~ NA Y~ na~6 sug~eEIted ths~ 12B-1 repre~nt~ ~n
lnt~rn~l ~ragmen~ Or REI. ~ . s~quQno~ ~na ~ r ~hs
MEL 13 cl)NA tFigur~ ~) al~o lndioatod th~ pr~3~n~e oY
35 stop COdOh ~Pter nuol~otid~ 3~1 ~Fig~ra 3).
A oompari~n of the ~EL 13 ~nd ~E~ ~ DNA
~quenc~s ~q~ted th~t ~E~ 13 m~y ~ 2 vari~nt ~r HEL
'~
.
`~
11/16/89 17:32 ~2~6 6~2 6031 _ Seed ~ BerrY
33~ ~i
~.
34
. A computer genorated DNA se~on4a ~howlng the RE~
~qqu~nco ~olned ~o th~ C-t~nnlnal DNA llqquor~oe or ~EL P,
dAsignated ~fEL 13/8 ~plioe, i3 ~hown ln ~l~ura 5. ~ DNA
soquano~ enooding tl~o ~fEL ~a npli~ gan~r~t6~d a~
5 ~ollow~. Pl~mId pN2~0 i~ dig~s~tad tl~h Eao R~ ~nd ,~t
I~ to ioolato ~ 3 . s5 Jcb fxA~ nt oompri~in~ tll- s '
oodin~ ~Hqunn~e iexom H~L 13. Pl~mid pM~05, ~ompri~ln~
th ~DN~ ~r~ment oP ~fs~ 8 ~ubolorlad ~a ~ p~rtial E~ R~
fr~ nt into pUCl~ dige~tod wlth 1~ and ~ol I
1~ to iool~t~ tl7e ~pp2~0ximataly 0. ~ kb Xrsg~hQnt. Pla~mld
p~a91 ie dlge~ted wlth Bal ~ ~nd Eoo ~I to i~ol~ta the
~pproxlm~tely 0.39 k~ ~r~ament cnoodin~ the 3' oodis~
region o~ ~EL 8. q`he Eqo RI - ~t ~I pM2Y0 ~ras~nen~,
the ~at 1~ - sol I pM305 ~ra~ nt ~nd ~he ~o~ ~ - Eco RI
15 pMasl ~ragm~n~ nrR joined w~t~ E~o lU-~ln~i2~d ZM~4
(Ex~mple 4) to ~onotruo~ a ~4mm~1ian axpt~s~n Iv~tor.
~he in~arts ,~ra oh~raate~lzed ~cr orldnta~loh ~th
r~pRat to the pro~ote~. A plasmld ~ont~1nlng the ~EL
l3/a ~pl loa D~A ~qu~nc~el in ~he oo~r~t o~i~nt~tion ln
20 th~ otor ZN~4 i~ d~slqn~te~l H~L ?3~8 oPll~o~ZMF4.
The ve~o~ 8eh~a9R w~ con~rllotod ao ~hown ln
~5 Flgura ~ ~rom ~m2~3. Plasmi~ ~em~29 is ~ pUCl8-4~d
expr~sion veotor a~ntalniny a unlqu~ E~am ~ lt~ ~a~
ins~iDn o~ olonQ~l DNA ~e~w~en t~e ~OoU~ etallo-
t~llonein~l promotor and SV40 t~n~crlp~on te~mln~to~
ahd an ~xpr~lon unit oontainlrlg ~h~ SV40 ~rly
30 prom~ter, ~ou~e dibydro~olate reduat~ n~ ~nd S~740
terhinato~. Z0~229 W~B modlrl~d tO ~ ata the two Eoo
it~ by par~lal d~6re~lon ~itb ~ao ~2, bltln~ing with
DNA pol~ r~se S (xlon4w fraf~meh'c) ~nd ~P~, and re-
li~ation. Pl~estlon o~ the re:~ultin~ pla~ml~ wlth ~2
3S tiI ollow~ b~ llgation o~ thq linsari~d pla~nid ~
~am Hl-Eoo R~ ~dapt~rs ~eeul~ce~ ln ~ un~ ~u~ Eao Rl
.
.
11/15/~g 17:33 ~0~ ~8~ ~31 Se~d ~_~erry ~41
33~ ~
3S
cloning ~ h~ res~lltant pla~mld wa~ d~ nut~d
z~22sr~ .
~ hfl axpreg!~lon ~,rea~or ZM~ wa~ ~on~truoted
.~ro~ Z~ R ~nd pDX ~agen et nl ., U. ~ . Pat~n~ No .
4, 784, ~50~ o~n in ~lguro 7 . 2~P12~R ~ dl~e~tod
witb l~lnd ~II and Foo RI, and thl~ 920 bp ~aSrm~nt
~ont~itling tho ~40 nnd M~-l promotorlt wa~ rolno~#d~ ~h~
lar~a ~rbgment Or Z~m2~ wa~ thon ~oinud te tho ^'llDO bp
Hind I~-Eao RI ~r~mont olr pDX, whl~h aon~alnel the SV~O
10 p~moter~nh4no~ th~ adonovlrua ma~or late promot~r,
and a ~ o~ ~plloin igt~nls.
~ he HE~ 1~ oDNA w~J ln~ert~J ln~o th~ E~o RI
~lt~s or th~ ~nammalian cell ~xpr~slon v~otor3 ZN~4 and
Z~mzz~ by ligation o~ tho ~ollowin~ ~ra~cnt3~ ~o Rl -
~ rom pMZ~o~ X - Xma ~ r~om pM2~4, ~mA I
- ~oo ~ ~ro~ p~2~9-s and Eco RI-diq~tod v~o~or. ThR
in~er~ w~ro ~har~atarlsod ~r orlcnt~tion Wit21 re~p~
to th~ pro~otHr~ A pl~id ~ont~inih~ th~ oPN~ in ~h~
3~n~ ori~ntatlon ln æMa~ wa~ ~uelqnat~d ~M2~6-10.
(Pl~mid pM2~6-lo h~ been d~poGlt~d wlth A~riOqn ~ypO
~ulture ~ollaation, ~ook~ , Md., as hn ~L ~Qll H~101
trahsrorm~nt. ~ A pl~mid containi~g ~e ~D~A ln th~
~nt1~enaR o~lo~ation ln Z~4 ~a~ d~signated pM2~S-7.
plasmld ¢ontAlning thc aDNA in tha oen~e ori~ntatl~n ln
~5 ~m2~R w~ dRcignat~d p~297~4, ~nd a pl~6mi~ ~ont~ining
the a~NA ln th~ ~n~ ns~ ori~ntatlon w~a d~ignato~
pM2~7-2 (Ta~l~ 1).
Baby h4m~ter k$dnq~ t~X) c~ ttk-t~3~ in
growth m~lum ~ulb~cc~s minimu~ entlal ~di4m
~DNE~] c~ntainlng 10~ fet~l b~vlne e~rum, 2
glutamln~, 1 mM ~sdium py~uvat~, 50 ~g~m1 ~enicil~ln, 50
~s~ml ~trept~m~cin, And 10 ~g~ml neomyc~) w~r~ ~e~de~
into 10 c~2 tis~u~ ~ulture di~h~ and ~r~wn o~rnlght a~
37~ wqre trah~ect~d wlt2~ pl~mid ~N~ tlsih~ th
35 ~alolum p~c~ph~te preU~plta~ieh hi~thod ~ aham 211d V~
~er E~, $bid). Tbre~ huhdr~d ~1 of 10 mM Trl~-~Cl, 1 ~M
ED~A, pH ~.0, cont~lning 0.5 M c~ , lo ~g pl~8-1d DNA
.- ..
.
:'",' ' ', ' ' ''
:''
::
:, : ~
,~
11/15/~9 17 34 ~2~G ~82 ~31 Seed & BerrY [~l~t4~
2~033~
~6
~nd 12 . 5 ,ug ~z~lmon aparm DNA ~ dd~d dropwi~e, wlth
~or"exing, to 300 ,1~l o~ 2x HB9 ~2~0 ~ NaCl, ~0 ~M
Hepe6, 1.5 mM Na2~1P04, pH 7.15) . ~be illixhlro ~a~ t
at raom tomperatu~a ~or 30 mlnut~ t~ ~lloo~ proeipltat~
~ormatlon. six hund1-~d pl o~ prQ~olpit~to U~ ~dcled to
~a~h 10 qm2 dish o~ ~HX qnll~, und tb~ dl~h~ wor-l
lnoubatod ov~rnlght at 3 )'¢. Th~ aultur~ m~ium and
pr~olpit~te wlare a~plrat~-d :rrc~m tha dloh~ nd 5 ml 0
15~ glyaerol in ~MEN W~ added ~or 3 mlnuto~ . ~he
10 dioh~ w~rH wa~h~d ono~ wlth DMEM, ~nd 10 ml o~ Slrowth
mudium waa addod. A~tar 24 hour~ ths cul~ur0 mcclium w~
ahang~d to suleatlon medium ~DMEM aon~alning 10~ ~t~l
~ovln~ ~erum ahd 250 nM m~thotr~Xat~). Sub~Qqu~ntlY,
~ne ~luct~on mediu~ wa~ ~hahge~ eV~ry 3-4 day~.
~olonl~ o~ ~ran~atQd e~ Drmea wlthln 10-14 d~y3
a~t~r ~r~n~feation. ~rAn~pQ~ant aol~nle~ Were
t~yp~lniz~d and ~eplnted ln ~el~o~ion ~edlu~ Por
~ontinued p~ag~ o~ th~ tr~n~otant p~ol~. ~nu pool~
were assign~d the de~lgnation~ ohown ~n ~b~e 1.
~1
; 2-22A pM2~6-7 ~An~i~4n~)
2-~2~ pM~96-10 (~enae)
2-22C ZM3~ Vooto~ aontrol
3-14~ pM297-a ~n~l~o~e)
3-14~ pM297-~ ~aen~)
3-14~ 2~a~9~ veCto~ ~sntrol
Cell mem~ran~s fro~ ~ul~u~ed tran~P~a~ant
pool 2~22A, 2-22~, and 2-22C wer~ ~re~Ar~d ~P~ llgand
blot ~n~ly~l~ (G~aham ~nd Or~, Z~7439-
42, 19~7~. Me~b~A~e pro~eln~ w~re ~x~l~e~ ~h
po~y~aryl&mld~ ~15 an~ transbl4t~ed to nl~roa~llulo~
~bran~. Inoubation o~ nl~roc~llulP~ ~t~ip~ Wlth
75I-~DL3 ravealed ~he pr~a~a~ of ~ 110 XDa p~tein in
. ''~'
,, ~ .
. .
' ' .
`~
11~16/8~ 17 35 ~2~6 682 6~)31 S~ed ~ Berry ~ 043
21~033~6
37
me~b~ane~ from ~11 thr~a tran~Sect~ht~. ~r~n~Yeata~t
2 22B ~n~) mambr~ne~ con~Aln~d thr~a tlmas more oY
thl9 p~ot~ln than ~lth~r 2-2~A ~anti-~eh~o) o~ 2-2ac
~c~n~rol) membrane~.
Mo~olay~r~ o~ ~ran~roct~n~ 2~a~ 2-22B~ ana
2-~ac wor~ r~hlll~d ~o O'C and inaubAtad with oith~
12~I-HUL3 or 125I-LDL to te~ ~or c~ urraoe ~indlng
u~ the~u lipoprotoins. Roeult~ ind10~tod th~t
tran~ROtast a-2a~ ~s~n~) bcun~ two ~c ~hro~ time~ more
1~5I H~I.3 t.han el~hHr tran~roctant 2-22A or 2-22c,
whor~a3 blndlng o~ 1~5I-LbL w~ tho ~me ~or ~ll thr~
trsn~c4tant~
~ult~r~d monolay~ro of trAn0~e~t~n~ 2-22A,
2-22~, and 2-a2C were sterol-load~d ~y prelncuba~lon
wi~ m~lum enrlche~ with ~holssterol, th~ pulse~ ~or
three hour~ wl~h 31~-mav~lonolad~one to bio~yn~h~ ally
radiola~el lntra~ellul~r poGls o~ ~terol. C~ w~ro
then àha~d for rour ~our~ wi~h m~dlum ~ontalning ~ith~r
nAtiv~ HD~3 or HDL3 prevlo~ly tr~ated with t~yp~in to
~0 dig~t apoprot~ins~ and th~ app~ar~na~ o~ radiolabeled
~t~rol 1~ th~ m~lum wa~ m~ r~d. Trypoin~tYo~tod HDL3
was u~d a~ A aontrol ~o monlt~r r~o~pt~r indop~nd~nt
~t~rol ef~lux~ sino~ th~e p~rticles do no~ lntera~t
w~tn o~ll r~ceptor~. ~e~ul~s indioat~d th~ natlve H~L3
promo~d grea~er 31~-st~rol ~lUX f~o~ tr2n~fe~tan~
2-Z2~ than dld trypeln trea~ed ~3~ wher~s b~th
tr~tHd and untroated HDL3 r~moved ~hc Pame ~mount o~
3H-~te~ol rro~ tr~n~reotant~ ~-aaA snd ~22C.
Cultured ~n~fectant~ 2~ , 2-2~, and 2-~2C
were in~ub~ed with hD~ containing 3H-ohol~teryl o~t~r6
to radiolabel intrA~ellular sterol pool~ vi~ a r~o~ptor-
~dl~ted ly~o~omal pathway~ C811~ w~e the~ ~h~qd ~r
~our ho~r~ ~$th medlum ~ont~inln~ ~lth~r no llpop~oteln
or B~h3, And th~ app~an~ o~ 3~ h~ me~lum
3~ wa~ ~a~ure~. In tho Prese~ae ~ 3, ~rano~actant
2-228 ~cr~te~ ~wo- to th~f~ld mora 3H~t~r~l than
~$ther tran5~eotant 2-2~A or 2-22~.
''
, , ~.
.' ' ~
'' ~
.
,' -:,~ .
11~15/8~ 17 35 ~206 682 6~31 Seed & Be~r~ ~ 044
201D3~6
.
Cholestorol-loa~d monolay~rs o~ t~hs~eotants
2-22A! 2-22B, and ~-22C wer~ lnaubat~d wltb ~edium
contRinlng ~lther no llpoprot~n or ~D~I- A~ter ~ix
hour~, cell~ wer~ pul~od ono hour wlt~ l4C~ololc ~old~
~nd inoorporation o~ ro~iola~4l lnt~ c~lular
ahole~teryl ~t~rA w~ dete~mln~d. ~hla ao~y m~ouro~
~h~ r~la~lva deg~ou o~ d~plRtlon o~ lntr~c~llul~r pool~
of ~ree chal~t~rol ~o~g avail~bl~ ub~t~atq ~or the
~loro~om~l ~st~rl~icatlon ~nzy~. HD~3 Ohu~od a two- ~o
~o thr~e~old great~r dopletlon of thi~ oubotr~t~ po~1 ln
tran~f~ctant 2-~3 than ln ~lth~r t~an~qt~nt ~-2~A or
2-2a~.
~mP~ P~o ~ ~lon ~ A~
~n"N~- ~ r~
SQl~ yloag ~llo w~r~ lyoo~nized wi~h
phage clon~ 17~I-E3 es~entially as d3sorib~d ~bDvR. The
h~brld ~-~al~otosid~-HDL-bindlng p~ot~ln~ wero ~PrG-
lnduo~d and rc~olv~d by paly~crylRmld~ la~rophore-
ao ~ Youn~ ~nd ~avis~ ibid. t SlXel~ and ~hn, ~Qg~~n~l- A~. SQ~I US~ 303~-42~ 1387). ~he h~b~ld
protein WAO partlally purlfi~d u$ing a ~-q~lact~ol~
a~inlty ~ub~trat~ column (51gma Chamlcal C~, 9t.
~oul~, ~o.) o puri~iQd ~sin~ ~n ~ Q~ g~laeto~ldasG
~S antibody col~mn (Pro~g~ Bioteoh, ~dlson, Wia.) as
s~ec~iQd by tho ~up~ ho par~lal~y puri~
p~otuin w~ usod s~ an Rnti~en for ~ntibo~y produatlon.
~ alb~o mloe.were irm#niz~ ~ultiple tim~ wlth
th~ hyb~ld ~-~alaato~ida~ blnding pru~oln ~ol~ule.
Th~ purl~i~d solubl3 ~ol~aul~ wa~ blot~ed ontc n~t~o-
oollulo3~ (s~hleio~r ~ S~h~oll~ 0.45 Pm). The ~lot~
w~r~ dri~d at 371~, and ~ho nltrooQllulo~ w~a ~ olved
With D~S0. ~e s~mpl~ wa~ then mix~d ~ith Fr~und'Y
adjuv~nt ~n~ in~eo~ed intc~ 3 p~r~tone~ vit~ o~
the ~l~e. ~o obtAin poly¢lo~ ntI~a~ blood W~
remoYod from th~ mi~e ~y retro-o~b~t~ ds ~nd
allow~d to olot, and tho ~e~um w~0 rR~ov~r~d~
.. ~.. ,. .~ .
- ~'.. ' ' '
.. , ~.
~ ~, ~
llrl5/~ 17:3~ ~0~ ~82 6031 Seed & BerrY ~045
33~
39
A ~oond ~ro~lp ~ mic~ w~ i~munlzad with
p~r~Lally puriri~d m~mb~ne~ o~ boYlna ondothali~l
C~118. coll mem~r~n~ is~ln~ed ~rom bovino endotholi~l
~Hll~ wero ~QpArAtRd by 9D~-PA~, th~h oloatrophor~tl-
onlly tran~Hrr~d ~o nltrooollulo~u. ~ho nitro~ollulos~
~huo~ W~rQ lnaubuta~ with ~25I-lubal~ HD~. A r~lun
~ th~ nit~OURllU10~ ~huot oor~os~on~ln~ to tho lOO ~Da
rango ~ tho ~D~ ~Ql Wn~ ~ound to bind the radlolab~lod
~ h~ ro~lon or th~ nitrOQ~llUlo~o ~huet abrr~pond-
10 lng ta thi~ ~r~a W218 th~n cut out ~nfi U~d r~r lmmuniz~-
tlon o~ the mioe. Tho nitroaollulo~o ~h~ot WA~ oolubl-
ll~ed ln DM90, ~hon mlxod with F~Rund~ ad~uv~nt. Thls
mlxtur~ wa~ injea~ed lnto th~ porl~on~l oa~ 6 a~ ~ho
~ice. ~h~ procRdur~ wafi rapoated ~Ultipl4 ~i~e~.
1~ ~olyolonal antiser~ w~r~ obtained ~y bl~diD~ the ml~
a~ ~xtraatin~ th~ sorum u~ing ~onv~ntion~l m~thod~.
Ilybrldom~ ~11 line~ w~re g~no~ted by ~uoln~
ly~pho~yt~ Prom ~ mouGR imm~ni~ad Wlth th~ i~olA~ed
me~or~"e~ o~ bovine an~o~h~lial aoll~ Wlth th~ N~-l
mous~ my~lo~ o~ll line ~A~C~ a].
~ o p~op~r~ $or th~ Pu~ion b~tw~n the
immuni~d m~u~o aHll~ s~d the mou~e my~lo~a ~11 llno~
~ho ~ploen~a and lymph nod~l; o~ th~ i~muniz~fl ml~e wor~
r~movod and mina~d wl~h ~cisl~or~ ~n top o~ rln~-me~h
25 stainleq~ ntoel ~cr~r,s. ~he minaed tis~ue~ w~rq w~h~d
through 'c~e ~lne wr~n~ in~o potri ~i~hel~ with 10 :ml
R~PI 1640 (~RP~SI, GIBC0, I.aw~no~, ~a~o. ) q~he~ re~aind~r
o~ the minc~d ~ Ub~ wa~ pr~ed th~ough th~ ~cr~nfi
w~th a ~patul~, ~n~ the ~craen# w~Pr~ wa~h~d wlth 5 ml
30 ~PMI. To remo-~a ~ny remaining o~ t~rlal, the
bottom~ ~ the s~r~an~ were ~crap~d and tha
m~terl~l was add~d to the p~t~i ~iohes.
~ h~ atrainad ti~Rue~ wal ~ transf~rr~d to 5~ ml
oentr~fuge tubes, The p~tri di~has w~e wa~h~d with lO
35 ml RPMI to r~r~ov~ any ~m2llning mat&rl~l~. 'rhi~ ce~l:
su~p~n~l4ns w~re c~n~rl~uged ~ 10 ml~u~ at ~00 x ~
~he sup~rn~ahto w~r~ ~lacard~d, ~nd ~h~ p~ t~ we~s
'^~~~~ ' ~
-
.~ .
'',' ' ~
i
ll/15/~3 17 37 ~2~6 ~2 6~31 Seed & BerrY ~4~
331~
~su~pQndRd ln 4 ml RPMI. A~tQr resuopen~i~n, 1 ~1
f~tal eal~ ~ærum ~lo~ r~on, Ca11~.~ w~ ad~'led t~
e~h tubs.
Th~ red blood c~11 oonte~minant~ ~r~ ly~d by
5 ~dding ~ t~ril~ ditltill~d watHr ~o th3 a~ u~n-
~lon~. ~ho m1xturo~ Wor~ ~wirll~d qul~kly, Dnd S ~1
4 . 25~ N~1 wa~ add~Pd to ~aoh tUb~. ~!hb ~oll eU~p~n~ n~
wb~e q~rtrlru~d ~or 10 minut~ at 200 x g. 'rho uup~r-
nU~unts Werb dl~aardod, ~n~ tho ce11 ~ t9 ~a~usp~ndod
10 in 10 ml RPMI .
r~mov~ Any remainln~ tl~s~;uo ~te-rlal ~ th~
~u~p~n~ions wer~ rilt~r~d through two 1~yer~ o~ at0ri1e
gnuza lnto 50 ml tu~e~ . ~h~ centrl~uq~ ~ub~ an~ ~au
w~:re rin~d with an addi~iona1 10 ml R~ . Dil~tion~ o~
th~ x~3ultaht o~ u6~nelon~ w~re ~oun~d wi~h
h~macy~m~t~r to d~termine the yleld Or ~ymphocyte~.
Tho prep~red c~ we~e k~pt at ~oom ~mp~r~t~re ~or
appro~i~ately one hour until r~dy ~or u~u.
~he N~-l mDu~ my~loma csll llne ~os u~d ~or
20 th~ ruqlOn. ~o opt~mi~R tho ftl~lon procdur~, th~ I~S-l
line wa~ ~loned out to l~ol~t~ a clone wl~h A hi~h
~u~lon e~lolency. Th~ N~-~ aoll~ wcro olonod out by
llmltlnsl dilutlon ln~o ~-well microtiter plates at An
~veraye of ~lv~ and ~n a~lls p~r w~ll in N~ edlum
~abl0 2) + 2.5 x 1o6 thymooytos/ml ~p~parQd a9
de~orl~d below). ~h~ pl~Q9 w~r~ in~ub~tod ~ 37'C, 7
~2 ~ en dayg,
~A~LE Z
3~ e,~um
P~r a 590 ~1 ~olution:
5 ml 10 ~M ~on~ential amino aci~
t~IB00, Lawr~nce, Ma~
5 ~1 100 mM ~odium pyruvate
35 ~IrV1ne, Santa An~, ~A~
5 m~ 200 mM ~-glutamin~ ~O~B~0)
5 m~ x P~nl~llllnJ5~r~pto~ycin~N~omy~ln ~ O)
, i~.
~' ' ,,"' , . ~
11/15/~ 17:38 ~2~ B82 ~3i Seed & B~rrY ~ 47
X1~33~L~
41
75 ml lna~tlvat~d ~et~l c)alr ~ru~
t 91 c~C~ C`~ O~1~ C~
1 9~ N~HCo3
Add RPMI lG40 ~G~CO, :CRwr~tno4~ Ma~a. ) to a totnl
volumo o~ 500 ml.
~rllls~n hy ~lltrqtion through a 0.2Z ~ llt~r.
~I~Q2L~ ,StQ~
0 38 . $ m~ th!fmi~ine
136.10 mg hypoxanth~n~ ~lgma, ~t. Lo~3, Mo.)
Dl~olve ~h~ tbyra~n~ an~ hypox~ntlne in di~tillltd
H;~O ~nd 1~in~ volum~ up ~o lOO nll . Wa~ tn~ ~ol~tl~n to
60'-70'C to dls~olv~ th~ lld~. Att~r ~h~ l3011~ h~ve
dl~olvod, r~n~u~t t~ volum~a to 1~0 ~1. St~lllz~ by
~lltration through a ~.aa ,um ~lltor. 8tcre ~roz~n ~t
-~O'C~
2 0 ~9~Q~
17.~ nç~ aminopterin
~ dd ~terll~ di~ d wnt~r t~ th~ aminopt~rln an~
bring th~ volumo to so ml. Add 1 N NaOH dro~wl~a until
25 ~ch~ amlnopt~in di~olYea. Bring tha ~lhal voluln~ to
100 ml with dl~tilled ~2~ 8t~rl~ lzo b~ ratlon
t}lrouyh n o. 2a ,~ llt~. 9~:ore ~ro2~n at -20'C.
50 hl 100~
s ml 1000X A stook
4s ml di~tillod El20
stsrili~e th~ ~olu~lon by fil~ratiq~n througt~ ~ O . ~2
35 ,~m ~ilt~r. S~r~ ~oz~n ~t -~O~C.
~ .
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' : ,
ll/15/~ 17 39 ~208 88~ ~031 Se~ err~ 4~
3331 6
7 ml N~-l m~dium
2 ml ~Htal ~alf aorulD
~ ml DMSO
Mix th~ lrlgrodiQnt~ ~nd m~co ~rc~h ~or e~ah
f r~q~ln~ ~
On cl~y 10~ tho ~qllo w~ro ~x~lned ml4ro~00pi-
1~ o~ and ~rQQned ~or w~ oont;~inln~ sln~l~ oolonl~o.
4n th~ s~m~ d~y~ lOO ~l o~ rr~s~ iUIl~ conta~nln~
2 . ~ x 1o6 thy~oytu~/ml W~B addod to ~a~h ~ ll . on th~
~ourt~nth day, ~ight Or ~lle mo~t vlgorou~ly ~rowing
~ingl~ ~ol~rllo~ wer~ ahoo~n to ex~and ~o~ ~usion.
~5 ~l~o ~i~ht oandidat~ a~lon~a~ ~r~ ~r~o~r~rr~d
to indlvldu~l 2q-w~ll plat-as oont~lnlng l.~-2 ~l NS-l
~edlu~ + 2.5 x 10~ thymo~yt~well. ~4e~e pl~te~ w~re
~alntainad a~ 3?'~ 7% ~2 and ~h~ c~ pllt ~t ~prb-
priatQ int~vAl~ by bXpandin~ t~ ooll~ to ~ra~h ~llc
oP thQ plate and addln~ ~r~h NS-l me~ nia prooe-
dure was ~ep~ated uht~ th~a ~a~ a ~ur~lci~nt nu~b~ o~
c~ to ~xp~nd ln~o a 7~ om2 t~o~ua oul~u~ ~la~k.
thl~ point, 1 X 107 a~ rom th~ growing aulture~ Wo~
u~ed to ino~ulat~ n 75 a~2 ti~ua oultu~fl ~la~k ~ontain-
2~ lhg 50 ml N~-l modium. ~hR ~1~8X~ w0~ lnc~bat~d A~
37'C, ~ Co~, untll the c~ r~ach~d ~ d~hsity o~ at
le~t ~ x 105 aR1~ h~ 0~118 wer~ ~hen harv~ot~d
by ~en~rl~u~Ation and dilu~ed t~ ~ con~ntr~tlon o~
ap~roximately 5 x 10~ with ~r~eElng me~
(~a~lo 2~ ~h~ oel~s wero divL~3d into 1 ~1 ~liquot~
a~d fros~n ~epwl~ Pl~t at -~0-0 and th~n at -130'C.
~ o ~ y th4 alon~ o~ NS-~ c~ o~ ~usi~n
e~ficieno~, one vial for ~oh clon~ was ~uiakly ~haw~
~n wa~r held at 37-C. ~he aell~ were lno4ul~t~d ~n~o
~ Xs c~nt~lnln~ N5-1 ~dlUm ~o ~ cona~tration o~ ~ x
~05 aells~l. The a~ wer~ g~own ~t 37-~ ~ with 7S ~2
The a~ w~re cut ~ck to 2 x 105 aell~ml ~ally. on~
" ' ' "' '. ,
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11/15/~g 17 39 ~2~16 G82 ~3~)31 S~ed ~ BerrY ~1~4~
2~)33~6
43
day b~ora fu~ion, two 75 om2 ~lnsk~ contRlnlhq 50 ~tl o~
call culture w~r~ s~t up. ~aoh o~ th~ aan~ldAto ~$-1
olon~ W4~ iXI~ wltkt lmmuni~ d mou~o ~le~n o~ nnd
Yu~d a~ d~rl~od ~low.
~h~ r~auLt~ o~ the ~usion~ oh~wod tl-~t on~ af
tho sln~ oolony iAolnto~ showod ar~ aren~od ~uclon
Q~1aionay . Tbls alono W~o do~i~nat-d c:lono ~ and w~
~ubse~uontly 115~d ~ tho my~l~rna a~ll linc ln
~urch~r ~u~ion~.
~hymu~ ~lAnd~ ob~Pinad ~rom baby miao wor~ tho
sourao of th~ ~hyrn~ayte c which w~ uood ~ P ~edar
l~ye~ to aondition tho oul~uro ~din rOr th~
~u~ion~. ~hymu~ ~lnnds were obtalhod ~rom 3- to 4-w~X-
old ~alb/e mioe. ~he ~hym~ glsnd~ wera rih~Dd wlth N-~-
l$ ~ m~dium and mlno~d on n ~ine-mo~h, ~lnl~u~ ~tfl~l
screen W~th ~ls~o~ hu min~ed ~ u~ were rln~0d
throù~h thR ~cr~n with 10 ml N~-l medlum lnto a pqtri
dl~h. Thq thymu3 ti~sue wa~ th~n pre~d through th~
~sr~n with a ~patul~ lnto ~h~ pbtri dl~h. Tho ~ar~n
wa~ then wa~hed wlth ~0 ml N~-l m~dium. ~he ~o~ O~
thR s~e~ wn~ sGr~ped to r~mov~ any a~ho~ ti~au~, and
th~ tl~u~ w~u pool~d in thu petrl di~h. Tho ~tr~lned
ti~u~ was trah~ss~ed to a 50 ~1 ~entXi~u~o tube
throu~h ~W0 l~yers o~ ater~le srauz~. ~h~ p~3trl ~l~h ~nd
25 gau~u wer~ rln~ed wi~h arl ~dditional 10 ~1 N9-1 m~dium.
~he oel~ were ~ntrl~uged ~c~X 10 Ininutn~ ~t 200 x g.
The eupernatan~ ~er~ dilsc~rde,d ~nd ~he p6~
re~u~p~n~ed ln lo ml Na~l m~dlum. Oilu~ion~ o~ thc oell
~u~pf~n~ion~ w~r~ count~d ~ ln~ a bomA4ytomotor~ Th~
30 y~ld ~Xom tw~ tbym~ glandsl wa~ routin#~y ~bo~t 400
mlll ion cell~ Thl3 ~ell~ Were ~to~ed &t ~o~m te~hp~r~-
tur~a unt~ 1 r~ady ~or u~e.
To p~epAr~ Por fu~ion, t~0 NS~ lon~3 F
a~ r~wn a~ de~c:ribed ahclve~ wc~ ~rAn~ r~d to 50
35 ml a~ntriru~e t~lb~ nd oen~xl~uged ~or lo mlnuteo ~
200 x 9. The ~up~rn~t~nt~ w~o ~i~aa~d~, aJ~d t~ aell
pt3~ let~ w~re re~usp~nded ~nd ao~ln~d $n 10 ~1 R~
' :
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11/15/~g 17 4~ ~2~ 2 6~31 .SQ~ & BorrY ~ 05~
~0(1 331t^j
44
The c~lL ~uep4n~1~n wn~ 04unt~d with a h~lRacyto~ot~3r to
moni~or th~ oell vlA~llity ~E~ d~t~Min~d ~ trypan blus
exclu~ion. q~he a~ llity Wa~ dot~nin2d to b~
qreata~ than ~5~.
For ~u~lon, ~.5 x 107 NS-l clon~ U11B wero
addad to tha p~epared l~ nl~d moua~ ~oll~ ~pYepar~d ao
de~ori4a~ ~buve) . ~rh~ mixed o~ W~rl~ aentri~ug~d ~o~
lo minuk~a at aoo x g. ~he sUperP~tant; w~o ru~oved by
~pir~tlon w1th ~ Pa~teur pipo~ attn.4hod ~o a vaouu~
~0 11n~. ~h~ ~oll pell~t w~ ru~u3p~nd~a in lO0
and warmod ln a wat~ ~ath At 37^~-
one ml 50% ~olysthyl~na glycol (p~a-~s
~ulu~ion in P~P~II, W~R y~l Ad~ust~d wlthin th~ r~nge o~ p~
7.0 to pH 8~0 Wlth 20 ~ul 1% ~o~ium blcar~orlata. ~he ~G
~s solutiol~ waa added to the a~ u~penslon ovor q perl~d
o~ ~no mir~ute wit~ ~ehtl~ ~tlrring. ~ho ~ ponilon w~
~tirr~d for an addltion~l minUt~. on~ ml o~ NS-l m~:dlulP
wa~ add~d oYor a pericd o;~ Ohe r~ u~ h gontl~
8tirrln~. ov~r an ~ddltle~n~l p~rl~cl o~ one ~inute, 1 ml
20 o~ l medium was added to thl3 5u~p~n~ion with g~nt~o
stlr~ g. Ei~h~ ml N~-l mad~um was addod over a p~rlod
o~ t~o ~inut~s. Tho ~u~p~Dsion was oentrlrug~ t 12~
x g nt ~oo~n t~mp~r~ur~ ~o~ 10 minUto~. ~ho supernatant
wa~ di~onrd~d, nnd 23 ~l NS~ dium wa~ addod to gontly
25 re3uspond ~h~ pelle~. ~h~ aells war~ harve~ed ~en~ly
lnto ~ plp~t qnd tran~rred ~ a 175 c~m2 rl~k~
Four hundred million thy3nooyt~ ~prepAra~ a~
~o~ w~e adaed to th~ rlask. Th~3 vol~ a w~ ad~ustqtd
ta 160 n~l with N9-1 m~dium, and ~he mlx~ rra wa~
30 ln~:ub~d n~ 37'C, 7t CO;! J~or tw~ to ~ur ho~r~.
A~t~r lnaubA~lon, 3.2 ~nl of 50x ~A~ ~ab~Q 2
wa~ addad. Th~ oell ~usp~n~lon wa~ tr~n~rlar~d to ~i~h~
96-w~ll cul~ur~ pl~tes a~ 200 ~l p6~r w~,l, u8ihg plp~t
tip~ with ~ch~ ehds ~ut o~ to ~vo~ rln~ ror~es.
35 ~he plat~ w~3r~ lncubated at 37'C ir~ a ~umid inoub~tor
with 7~c ¢C~2~ me plate~ w~re examir~ed r~1~ro~c~piq#11y
aft~ ~hr2e d~ys tv d~t~rmin~ fu~ion ef~ n~y, w~th
., :.... ~5
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Ij~ 051
11/1~8~ 17 41 ~7~3~ 6~2 6~31 Seecl & Berr~
20033~L~
th~ oxpecthtlon o~ ~pproxlmately ~ive hybrldoma aolonl~o
p~r well. ~he oell~ w~r~ ~3d r~ r ~eYsn d~y~ by
r~P].ACing 100 111 ai! t~le mSltiiUm Wit~ r~oh N~-l m~dlus~
con~ainlng 1 x ~A~ and ~ . g x 106 ~h~ocy~e~l p~ ho
5 hy~ridom~ w~r~ te~ed betwo~n d~y~ 9 ~nd 14 ~or tha
p2~0duotion of 4p~0ifio ar~tibodl~.
Thc hybridom~ o~ w~rl3 ~arc1~h~ by We~tern
blot (T~w~ln et ~ ibid. ~ for tho produatian aP
an~lbodl~o to ~ha b~vlnu ~ndotheli~l ca~l moh~rane~.
10 E~ovln~ Rhdoth~ mt\r~nca w~ 0pa~atHd by 9r)S-
PAaE, ela~trophor0~1~ally tr~nsrerred ~o nltroodlulose,
and ~hR hi~roa~llulo~ w~ in~lbated ~n bl~klng bu~er
to pr~v~nt non~p~ io blndlng l~y ~b~ ~ntibodi~.
Seot1Qn~ o~ ~h~ nltroc~ ose ~heet w~rOE thon inoub~te~
~5 witl~ ~onditian~d media P~aln ~h~ h~brldema o~ll llne~,
and ~h~ prel34na~ o~ ~paoirio antibody w~u d~t~hnined
u3lh~ A~C-peroxida~e ~V~to~ ~borAte~tle~, ~urll~ g~no,
Call~. ~ a~ desorlhed by tho m~u~ U~Qr. 8~rer~l
hybridc~mA aells ~eor~tln~ antlbody ~eo ~ndotheliAl
2 0 mQmbranas w~re det~t~d .
~ h~ ~ntlbodi~o ar~ clharactRri~e~ detectlPrl
oP ~he llO )cl)a ~DL-bindinç~ prot~in ~nd ~or th~l r~co~ni-
tlon o~ ~he reao~blnant ~L-blndln~ pr4t~in.
~ h0 hyb~idoma c~ll lln~ produclng ~ntlbodlos
o~ lntere3t aro ~lon~d 4y limltlng ~l~utl~n to obt~ln
coloni~a o~ a~ orl~inatinq ~rom sln~la ~ . Th~Ae
~olon~a a~e oxpand~d in CUl~U~ ~or ~nar~tlon o~ lar~
amoun~ of Antibo~y.
~ .6 - H ~
Antl-E~DL ankil~odie~ ~r~ ~re~ 3dl f~r ~0~9i~1e
antaqoni~tia ~nd agonl~ p~opertl~ pXe~inq
~DL xeceptor in A ~on~tltutive m~nne~ o~ln~
aor~lQ endot~eli~l a~ or a~lls tr~n~reat~d wlth an
~xpr~ioh v~ator en~odin~ ~n H~L-bindinq pro~in are
p~ated on miorQtlte~ pla~es ~nd~ ~xpo~d to t~e anti-
bodle~. Th~ oalls ar~ theh in~u~at~ with l~5T-HD~ to
~ '
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` 11/15/~ 17~4~ 25'2~6 B82 6~31 _ Seed ~ Berry ~ ~5~
~0~3~6
~6
lden~l~y antibodle~ that ao~pet~ r~ HDL bindlng to
lntact ael 1~,
Parallal cultur~ Or c~lla ~r~ u3ed ~or tho
prep~atlon Or m~b~na pro~einn rOr llg~nd blot
immunobl~t analy~ia. M~m4~an~ protulh~ nre bound to
nl~roo~llulo~ ~trlp~, And the 8t~ipJ n~ lnoubhtod wlt~
anti~ody, th~n ~xpo~d ~o lZ~I-H~ tD det~rmlno whloh
~ntlbodie~ bloa~ HD~ binding to th~ ~aolnted ~roteln~.
Anti~odie~ thdt blnd t~ lsol~tod HD~rb~n~n~ protolno
~ithout bloc~lng M~L hind~n~ are d~l~n~ed a~ non~
blocking antibodl~ oth ~looking Anq non~blooklhg
Antibodl~s ar~ u~ in ~U~4tl0nQl a~ayn to a~ th~lr
ablliti~s to aat ~ ~D~ ~gonl~t~ o~ ~t~gDnlet~. For
the ~unctional a~Ay, ~ultu~ed c~ [o.~ " ~lbrobla~t~,
lS ~nd~t~olial cells, or ~o~opha~) hr~ ~r~Ated ~lt~ 3H-
m~ onolautonR to r~diol~b~l lnt~Aacllul~ pool~ o~
oholestorol. ~bo o~lls are then e~po~d to ~edl~
cantalnlng antlbodi4~ plu~ ~lth~ nati~ or oth~r
ohol~t~rol aoceptor partlcle~. O~h~r ec~ptor
p~r~icle~ lnoludo ~h~ia~lly tr~at~ HD~ thst no lon~Qr
lnt~r~s~ wlth HD~ rocoptor~ b~t ~o Rbl~ to ~ct
chol~storol aocePtors ~rlnton ~e al.,
6a:4~-503, 19~6). ~ho a~o~ o~ an~ibodi~ on th~
x o~ labolod ~erol indiaat~ ~oni~tlu and
~S ~nt~gon~tla prop~rtio~. ~bOB~ antlbo~le~ ~hat ~lo~
tbo o~lux-~timUl~tlng ~P~at~ o~ ~D~ ar~ ~DL
aAtagohlsts~ tho~o tha~ st$~ulAte ~o~ol ~ x ln th~
pre~en~o o~ ~ st~ol acaop~o~ th~t ~oa~ n4t ~i~d HD~
rea~ptors qre HDL a~ohl~t~. ~h~ 0¢~0~ o~ th~
anti~odi~s on tran~locatlon of ~Idiulab~lQ~ ~t~ro~ f~o~
ln~r~a~ it~ to the c~ urraoe ~n t~:p~e~enoe
~ ~b~en~e o~ ~ative H~ ar~ ~ea~ur~ by d~to~lni~
t~ei~ Abillti~s to ~ti~ul~t~ the a~no~ o~ ~t~rol
t~om et~r~l-lad~n ma~roph~g~ roph~g~ lo~g~d wlth
3s a~etylated L~-dRriv~d ohole~e~ol ~re :$nQ~'o~t~d ~h
~di~m oontainlng ~hs te~t anti~ody plu~ eitne~ ~a~ive
or che~lc~lly modl~isd ~D~ pa~ticla~, And the ~o o~
. ~, ~ ..
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11/15/8~ 17 43 ~208 B.~2 60~1 Se~d ~ Berry ~ ~53
20~33~L6
4J
cJisapp~aran~c Or O~cQ~ intr~aQllUlar ~torol i~ da~t3r-
mill 1d U3 U fllnotlon Or timt~. q~ogq ~tudla~ p~ovida an
ln~icat:lon oP tht~ t~lernpoutla potcn~lali o~ individunl
nqonlst ant~ dl~ in promotin~ th~ clourAno~ o~ exco~
sterol rnonl ~onm ooll~ o~ tho ~rtt~ry wnll.
From th~ I~oro~oin~, it will ba approt~lated
tl~At, ~ltho~h apooirl~ ~m~ot~imont~ o~ ~h~ inventio
hAVe b~en doaori~od horoin ror purptia~ o~' lLlu~trnt~on,
v,lrlous modl~ioAtloll~ m4y bo m~do ~lthout dovi~tlng ~rom
10 tha spirit and ooopa o~ tha invontion. ~a~ordl~gly, tho
inv~ntion i~ not li~ni~t~d oX~Qpt ~9 by the appended
elaims.
3~5
~tl~/v~
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