CA2019665A1 - Hybrid proteins between an extracytoplasmic enzyme and at least another protein, processes for preparing them and their applications - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The present invention provides hybrid poroteins between a fragment of an extracytoplasmic protein Pl, a protein P2 and an extracytoplasmic enzyme, the protein P2 being, in particular, an antigen or a single-chain variable frag-ment (Fv) of an antiboby, to a process for preparing them and also to their application, in particular, as a diagnostic agent and in the screening of a nucleic acid library or in the selection of recombinant clones. The nucleic acid sequence, consists of a hybrid sequence comprising successively the leader sequence of the structural gene for a suitable protein ?1 exported or secreted by a microorganism, in particular chosen from the group which comprises bacteria and yeasts, a nucleic acid sequence coding for a NH2 terminal fragment or the mature protein P1, a nucleic acid sequence coding for a protein P2 and then a fragment coding for at least one functional fragment of the mature sequence of a suitable extracyto-plasmic enzyme, the assembly of these fragments being in a single reading frame and coding for a hybrid protein possessing simultaneously the properties of the enzyme and some properties of the protein P2, in particular that of interacting specifically with an antibody, an antigen or a receptor.

Description

-1- 2~96~
~he ~rs~ent iAnven~ion rel~tes t~ hvbri~ pro~ei~s between ~ fragment of ~n ~x~r~cyto?l~s~c p~otein Pl! ~
pro~in ~2 ~ tra~l ~ lc ~ yme, ~ ~ ~æin 22 Deing, Ln par'icul~r, ~n ~nt~gen or ~ ~ngle-cha~n v~r~bl~ f-~g-ment (Fv) of an anti~ody, ~o ~ proce~Cor p-e?ar~ng the~
and ~lso to ~he~r 2?plications, in ?~r~icul2r~ as dl~gnostic agen~ and _n the screenlng of a nuc!eic ~c~d l~b-s~v or in the se!ection of -eco~oir.ant clone~.
~.e pre~ent Lnvent~on aiso rel~tes to ~,e DNA
se~uence~ codLng tO_ the hybrid proteins, to ex?re~sion vec~or~ contalnlng the sequence~, to ~ra~ns of b~cteria or of yea~t~ trans~ormed by the vecto-~ ~nd also to their app11cation ~or ~he produc.ior. and ~cre-~ion of the hybrld proteins.
In the llterature, ~ number of hybrid proteLn~
have been desc~lbed, ~s well as a method for prep~rLng ~em.
Ther~ may be mentioned, ln particula-, US P~tent~
4,411,994 and 4,338,3g7, which dQscrl~e ~he use of the penic~11inase gene ~n pl~mid pB~322 to produce fus',on protein8, whlch are transported to the periplasmic ~p~ce by the penlc~ nase "lesder~ 8equence~ u81ng c~NA
sequences ~oding for preproinsulin inserted ~t the P~t~
s~te o~ pl~B~id pBR322.
Th~re m~y ~180 b~ mentionet Europe~n P~tent Appl~c~tion 1~6,864, which descri~es tho use o ~e ~lkaline oho~phato.sQ. " l eoder" ~equence follow~d, downat~eAm ~nd in the re~din~ ~ssune, by ~he sa~uence cod~ ng fo~ the de~ir~d prote ~ n .
The system fcr e~p~ssiQrl of the r~ ar.ce ~ a s~ ~le pm~ (for e.~ple ~ pro~oter of ~c~ ?'~'~ lSn~;e~
opor~ lonally to th~ hybrid codlng ~ uenc~ cnd, do~-st-e~m ~ro~ the ~equenco, ~ suit~ble te~Ln~o~, ~ n part ~ cul~r ~he pho~ ter;aln~to- .
Thls Europe~n Patent .~ppl~cation 196,86~ dS~-clo~ea th~t only 60-called 8u8cepti~1e prote1ns can be ~used ~o ~he N-termin~l portion of al~callne pho~phata~o 20196~ j ~nd exported; it i3~ ~n addltlon, ~t~t~d tha~ a protein 15 con~idered to be ~u~ceptible when it contain~ ~n ~min~
acld sequence which interact~ with the ~lkaline pho~ph~-tase le~der ssquence ~nd qiveB rise to the secretlon of S said sequ~nce.
It 8 ~l~o stated that the susceptlb~lity of the protein, doubtless due ~o the n~ture of it~ ~mino ~cid sequence which permLt~ thi~ inter~ctlon bet~een the two ~equence~, i8 not at pre~ent under~tood. It iB ~ howeve~, cle~r that ~ome heterolo~ous protelns are cap~ble of intersctlng wlth the ~ignal sequence derived ~rom a number o~ bacterial "leader~r, wherea~ other protein~ are inc~pable of thL~ lnteraction.
Thls Europe~n ~pplication d~cribes, more e~pe-ci~lly, hG~ and TNF ~ 8uscQptible proteinB, where~B I~-2 i~ con~Ldered to ~8 an in~u~ceptible protein, th~t i~ to say not exported. ~he hybrld protein8 de~cribed ln thL~
~pplic~t~on are de8igned to prod~ce, ~n parti~ul~r, hGH
or TN~ in indu~trial quantities ~y a genetic recombin~-tion method, th~ alkaline pho3phat~ce 8ign~1 sequonceserving exclu~ively for tran~port of th~ foreign proteln.
Europe~n ~atent Application 242,2~3 de8cribe~ ~
hybrld p~otein compri6ing an enzymo cont~lning an epi-tope, in~erted in 8uch a way into the pQptlde ch~in of tho enz~me th~t the epitop~ i~ exposod 80 th~t, when the hybrid protein i~ ln imm~nologi~l cont~ct wlth ~n~-~odie~ directed toward~ the epitopo, ~ complQx 1~ fonmed betweon aAid hybrld prot~in And B~ ~ d antibodie~ and ~he onzym~tic ~ctlvity oÇ ~ald ~nzym~ pre~orved whcther in the uncompl~xed ~t~te or in the compl~xed ~tAte. ~he hybrld pro~oln descrlbed ln th~s Europe~n appli~tion l~
tucked into ~he outer m~mbrane of ~ b~cterium in ~uc~
w~y th~t the cho~ell epitopo 18 expos~d ~t the ~urf~ce of the b~c~erlum; E. colL ~train~ carrylng at their outer surf~ce modlfled p~age lambda receptors contalning the hybr~d proteln6 defined above expo~ed at the surf~ce are al60 descri~ed i~ t~is appl~catlon. The hybrLd proteinJ
described ~n this appl~cat~on c~n, where appropr~ate, comprise nlkaline phosphat~se, poroxLd~se or ``-3-- 2019~6~

~3--9dl~CtO~ idas~ .
There are other documents ~hich descrlbe fu~ion proteins en~bllng the de6ired ~ec~eted or exportod product to be obtained. There may be mentioned e~peclally ~he paper in the n~me of T . P - HOPP et al -, publlshed ln Biotechnol., 1988, 6, 1204-1210, which d~scrlbes ~ short N-torminal fu~ion ~equence u6ed a~ a m~rker for the ldentlfLc~tLon ~nd purification of recombin~nt prote~n~;
the remov~l of ~ald marker has the ~dvantage of not 0 nece~sit~ting drastlc treatment~ of the ~usion prot~in, It should be noted that, Ln thi~ paper, T.P. HOPP et al.
state that the production of fusion proteins h~B 2 numb~r of drawbacks at the present time; in e~fect, Lt i~ not an obv~ou~ m~tter to obtain fus~on psotelns which po~es~
quAtsrn~xy ~tructure whlch corre6ponds to th~t of the n~tive protein, which ~re stablo ~nt whlch ret~in the blologlcal ~ctivlty of the l~tter; there may al~o be mentLoned the p~per published ln BlochemLc~l cnd B~ - !
physlc~l Research, 19~7, 149, 2, 607-614, which doscribes a hybrid protei~ which co~prises ~ucc~ssively proin~ulin or a fragment o~ the latter and alk~l~ phospha~ and i~ obtained from a DNA ccmprl~ing successively the proinsulin ~ an~ the alkallne phoRphata6e gene (bindlng vLa the 5' end of t~
~ene coding for mature pho~
~owever, the ~scription~ o fu~ad ~nd/or ex-ported proteins described in tho litor~ture ~pply only to ~pccl~l c~ses and c~nnot b~ c~p~blo of genor~liz~tion. I
The Applican~ h~ accordlngLy ~.~m~d to provlde ~ j unil~of hybrld prote~ dire~t1y ~xport~d in the pe~ipl~c ~pace or ~rated cut of ~he oe11.in ~heir hybrl.d forms,~ co~istlng of ~ fr~t cf a pro~in Pl, a pr~te~l P2 ~xl an enz~ icl hy~rid proteln~ retainlng the propcrtie~ of th~ prot~i~ P2 ~nd of the en~yme ~nd being c-~p~ble of b~lng uaed ~
re~gent for dlagnosi~ and/or detection in the contex~ of ~ssAys of the RA (receptor a8B~y) or EIA (enzyme immuno-ass~y) type, especially of the ELISA type, and for the ~electlon of recombinant c10nes or for the 6cre~ning of a DN~ 1ibrary; such hybrid proteins m~et pr~ctlcal needs better than t~e hybrld proteins of the prior ~rt, in I - 20~65 particular in ~h~t said Qxported or ~e~reted hybrld protein~ are ~rectly u~able a~ a diagno~tic reagent without a purif ication ~tep, ~nd ~re st~ble.
In ~ddltion, ~uch hybrid proteins m~ke po~sible, in p~rt~cular, the a5say of 8m811 molecule5, whLCh u~ually, even if they c~n be coupled chemlc~lly to ~n enzyme, are u~able to find appllc~tlQn ~ a re~gent, ~nd which h~ve hitherto been essentlally a~s~yed by ~IA.
lt is ~lso an ob~ect of the invention to provlde the tools for the production of s~id hybrld prote,ns.
The sub~ect of the present invention i5 a nucleic acld sequence, which consist~ of ~ hybrld ~e~uence compri~ing ~ucce~s~vely the leader sequence of the ~tructur~l gene for a suitable pro~ein Pl exported 4r secreted by a mlcroorganL~m, in partlcu1ar ~hosen f~om the group whlch comprlse~ bacteria and yea6t8, a nucleic acid ~ Ke ~x~ or a NH2 ~ r~al fra~t of ~aid mature proteln P1, A n~1eic acid 6eq~x~ ooding ~or a p~tcln r?. nnd t~n fragment coding for at least one functlona~ fragment of

2~ the mature ~x~ e of a sult~b1e extrac ~ la~mic enzyme, the asse~ly o~ the~ lr~gments belng in a 6ingle readlng fr~me and codin~ for a hybrld proteln posseH~lng ~mult~neously the propert~e~ of the enzyme ~nt some properties of ~ld protei~ P~ in par~icul~r that of interacting speclfl-c~lly with ~n ~nt~body, ~n antigen or a receptor, In the 6en~ of th~ pr~en~ lnvention~ nuclolc ~cld i~ under~tood to mean oLth~r a ~in~la-~tranded or double~str~nde~ nucl~lc ~cld 6equcnce, the nuclelc ~cld being eithe~ ~ DNA or ~n RNA.
Accordlng to another ~dv~nt~geou~ em~od~ment of ~ho ~eq~cncc according t:o th~ invention, tho enzyme i~
cho~en rom the group which compri3e~, ln partlcul~r, ~l~allne phosph~ta~e, ~cid phosph~t~e, acld glucos~
pho~ph~t~se, cyclic phosphodle~terase and ~-lact~e.
~ccord~ng to another advant~geous embod~ment of the sequence accordlng to ~he lnvent~on, the ~r~gmont o~
sequenoe codlng for the protein P2 1~ ~ in p~rticul~r, cho~en fro~ the group which compri~es the sequence~
coding for pep~ide hormone~, the ~equenc~s coding for toxln and ~he ~quences codin~ for a Blngl~-chaln fr~g-mQ~t analogous to the vari~ble domains of Lmm~no-qlobulins ~recomblnant Lmmunoglobulin~).
These 12tter fr~gments are, ~n p~rticular, S de~crl~ed in Proc- Natl. Acad. Sci. USA, 1988, 85, 5879-5883.
Accord~ng to an arrangement of ~hi~ em~o~ment, the tox~n is adv~nt2geou~1y a neurotoxin.
Accord~ng t~ the invent~on~ the pro~ei~ Pl is ld~nticdl to or di~ferent f-om the enz~me defined above.
Accordlng to another advantageous ambodLment of the sequencQ accor~ing to the invention, s~d nucleic acid se~uence con5~st& of a hybri~ sequence compris~ng succe6sively the leader fr~gment of the ~tructur~l gene of an enzy~ exported or 6ecreted by a microorgani~m, in particu~r cho~en fro~ the qroup which COmpri6eB b~cteri~
~n~ yeas~, a nucleic acid ~ oe coding for a ~2~n~nal fragm2nt of said mat ~ ~yme, a nucleic acid 6eq~e ~X~ng ~or n ~ro.~n r2 and then a ~ragment coding for at least one functional fragment o~ the mature sequenco of the enzym~, the &ssembly of these fragments being in ~ ~ingle readin~
fr~me and coding for A hybrid p~otein pos~es~ing ~im~l-taneously the propertieR of the en~yme ~nd BOm~ proper-ti~s o~ the proteln, in parti~ular that of ~nteractln~
sp~ci~ic~lly with ~n antlbody, an antigcn or ~ receptor.
Accordlng to sn advsntag~ou~ arrAng~ment of thls embodimen~, the hybrld ~equ~nce comprl~es succea~i~ely the l~de~ ~equenc~ of the ~tructural gen~ for alkalin~
phosph~t~se, ~ ~ragm~n~ codlng for, a~ mo~t, ~he ~ N-term~n~l ~mlno acid~ of m~ture alkal~n~a pho3phatA~e, DNA ~eq~enco codlnq for n protoln P2 or a frayment of the l~tter, ~nd ~hen fl fragmHnt codlng for, ~t least, the 422 C-termln~l r~m~lnin~ ~ino acld~ of ~lkallne phosph~t~e, the a~emb~y o~ the~e fr~ment~ ~eln~ in a ~lngle re~dlnq ~S fr~me and formlnq a hybrid DNA sequenc~ codlng fo~
hybrld pro~ein possessing 6imultaneously the propcrties of alXalln~ phosphalase, especially its enzym~tlc actl-vity, ~nd some of tha prop~rtics of the prote~n P2~ in p~rtic~l~r that of lnter~cting Rpocif~c~lly with ~n antigen, an antibody or a recepto:c.
According to an advant~geous v~ri~nt o~ thi~
arr~ngement, the hybrid DNA sequen~o com~ es succe~-s~vely tl~ n~leic acld sequ~l~ce eoding for tbe al~li~ ~sp~a~ase lea~e~ ~e~1~, th~ s~u~e c3~lng for t~ ~8 ~ ninal a~nlne ac1~ls of aLlczlir~ p~phatase, the s~e codLng .fo~~.~ a zbu-toxln a (ea) ar~i the s~ce c~ng for ~ 422 C-æ~al arn~r~ acid~
o, aL'caline p~sphatasP.
Accordin5 to this ~-arl ant, th~ sequence cod~ nq for erabutoxLn 2 compr~ se~ 192 ba~a pai-s and .wo addi-tional Sau3AI rastrlct' on si~e~ .
~he hybrid sequence comprises the ~ormula I
below; ~ T p I~r fraç~t o~ the str~c~r~l g~3 f~r p~- CGG ACA CCA
E M P V 1, E h R A A Q G
GAA ~G CC~ GT~ CTG GAA AAC CGG GC~ GCT CJ~C GGC G~ ATT .~C~
A P G G A R ~ ~ T G ~ O P R ~ C
GCA ~CC GGC OG~ GCT CGC. CGT `r~A ACG GCT C~ CCC AC~ A ~CT
r N B ~ ~ ~ Q P Q ~ ~ X ~ C S
m ~AC c~ c~c ~ ~cc C~A CCG C~ ~CO AC~ ~ ~C ~ ~CT rc~
P C ~ S S C Y ~ X Q ) S D r ?~
cc~ ~o a~c ~CC 5cr ~ A~ ~C aAa C~ cc C~ ~C Car S ~ ~ ~ C G C ~ ~ V J;
G~A AC~ ~TA aT~ G~A ACo CCA ~G~ GC CCC ~ C~C ~¢ ~CC
G ~ C C ~ 8 1~ V C )7 N ~, D
t;aT ~ A C~C ~GT ~ CC ~ 5~ C.AC C~C ~;C ~C ~r G~
CAG ~CT~fr~gment coding ~or ~t lo~st the 422 C-~ermin~l rem~l~lng amino acld5 of a~ ~ phc~pha~ ~ ~nd is designa~ e~/phoA28.
The irst arrow corre~ponds to the beqlnnlng of the ln~rt containlng er~butoxln a~ thu ~cond arrow corre~pond~ to tho end of ~d in~er~, The complete saquence o~ th~ ~tr~c~ural g~na ~or al~allno phosphsta~a (p~ gene) 1G ~escr~ ~ G.~$, 1~86, 44, 121-125.
According tO nnother ~dvantageou~ arr~ngement of thls embodlment, th~ hybrid nucleic ~cld s~uence ~ccord-lng to the lnvention comprl~e~ ~ucce~lvely the ler~der ~squence of the gtructural gena for al~aline pho~phc ~6e, a fragment coding for the 6 N-ter~inal ~mino ~cid~ of mature al~line phosphatase, a nucleic acld ~oquancc coding for a proteln P2 or Q fr~ment of the l~tter, ~d t~en a fragment coding for the 444 c-termln~l rem~ln~ng -7- 20196~
~mlno acids of alkaline pho~phat~e, wh~ch ~equence compri~es, in addltion, in particular in order to place ~he ~s3emb1y of the~e .ragment6 in ~ 5ingle re~d~ng fr~me, downstream ~nd/or upstream from the f~~gment codlng for the protein P2, a ~uit~ble nuclslc ~c ~ d Crag~ent, whlch hybrid ~eouence, ~n a single re~dl~q fr~me, cod~ for 8 hybrid protein poss~6~ng s~mult~n~-ously the propert~ eR Oc alkaline phosphatasQ and some of the p~oper~ies of the protain different ~rom th~ enzyme, in par~~cular that of interacting 6~ecifica!1y with ~n antlgen, an antl~ody or a receptor.
A hybrid nucleLc acid ~e~uenc~ accordinq to the lnvent~on, ~n which the ~equence coding for the p~ote~n P2 18 inser~ed after the trlpl~t codlng for the 6th ~mino ~cid of mature alkallne phosphat~e, 1B, in partlcul~r, prepared by me~n~ of the lnsertlon of At leA8t one unl~u~ I
re~trlctlon slte in the sequence codinq ~or nlk~lLne phosphat~se, ~hifting the reading fr~me of the phoA geno out of phAse ~nd preventing expression of the alk~llnQ
phosph~t~s~, where~s in8ertlon of the protcin P2 ~t the s~te ensble~, under the condl~ion~ of the inventlon, the alXallne pho~ph~ta8e gene to bQ ~hlfted back into ph~a nnd the en~yme ~o be expre~sed.
Accordlng to sn advant~geous vAri~nt of thl~ :
arr~ngemen~, the hybrld DNA 6equence compr1ses suCces-sLv~ly the lcader sequence o~ tho ~tructur~l gene f~
alkallne phosphst~se, the sequenc~ coding for ~he 6 N-te~mln~' ~mino ~cids of al~.~line pho6ph~t~c~, ~h~ nucleio ~cid sequence ~oding ~or ~nglot~ns~n I, a fragm~n~
compriaing ~he ~equenc~ AGG G and whlch en~b~ea thc ~lkallne pho~p~at~ qene to be sh~f~ed b~ck into phase, ~nd then ~ ~r~gment codlng for, ~ le~6t, ~ho 444 C-term~ l remAinlng ~mlno ~c~ds of a1k~11ne pho~ph~ta~e.
Th~ hybrld sequence co~pri~e~ the formul~ II
be10w~
~2~
G.G A~A CAA ~GC ACT r~TT GC~ CTG GCA CTC ~ C'`~ CTG
~et lys gln s~r ~h~ al~ 1e~J als ~eU !eu P~0 leu 19u --e-- 201966~ j -1 +1 +6 ,.T ,~C~, C'~'^ G,~, ACA ~A~ GCC CGu ~C~ Crh, ~`~ A-G c_- G~
ph~ tr~; p~o val t,~r ` ys ala arg ~hr pro g~u me~ pro ~sp ~ 7 C~ G~C T.~. A~l CAC C,G ~.~ CA~: CTl Ag~ g~l C~G GAA AA~, a~c val ~ ~ ile his 5~0 t)he hls ~eu arg val leu ~'u as~l ~ . _ . . .
~G~ T ~ G G~,C G~ C'" GCA C_C GGI_ Gvl G~ - C~C
ar~ a` ~ ala s!r s!y asp ' le thr ~la pr~ gly gLy ala a g CG TT~ G ...
a~ ~ 1 eu ~hr . . .
;
t450 .,, ~T G A~A TA~
... ~au lys sCop This ~equence of formul~ I; i8 desig~ated An~io/
phoA6.
In thls formula II, the ne~at~e nu~ber~n~
corresponds to the amino ~cids of the alk~llne pho~ph~-t~se ~lgnAl peptide and the po~iti~e numb~rlng corres-ponds to the ~mlno ac~ds of mature alkaline phosph~tase~
The underlined ~equence i6 th~t of angiotensln I. The arginlne preceding the ~aline + 7 has been lntrodu~d on account of the need~ of cloning and ~hift~g of th3 phoA
1~ geno bac~ lnto ph~se . The nuc~eotides correspondlng to the uni~ue re~trlction s1te defined above lntroduced into the phosphata6e gene are 8hown in lower-case latter~1 these corre~pond, ln the p~esent C~8G ~ tO nn Sma~ ~ite (ccc..... ggg). ~h$~ mdke~ Lt po~ibl~ to demonst,r~te ~he lS 5hlftlng of th~ pho~ gone out of ph~e, cau~ed by the pre~e~c~ of ~nld s~te, snd tho ~hlftin~ back Lnto phs~e brou~ht about by ~he Lntroduction of th~ ~ngioten~in 6equence. ~he ~ortion of phosph~tase 3e~uence not ~hown ~dotted) co~respon~s to th~t publlshod by C~NG 0t ~1, (Ct~ G, C.N., W.-J. ~UANG, and E.Y. CHEN, 1986, Gone, 44~121-12~).
Accordlnq to another ~dvant~g~ous ~rlant of thl~
~rrsnqement, the hyb_id DNA se~uence comprlse6 8UCco~-sively the léader sequence of the ~tructur~l gene for alk~line pho6ph~ts6e, the sequence codLng for thq 6 N-terminal ~mlno ac~d~ of alkaline phosphata~l a frag~ent comprising the sequence &AT CCC, the nucleic acid se~uence coding for erabutoxin a, a f~agment co~pri8ing 9 201966~
th~ sequence GAT C, which fr~gment e~bl~3s the alk~line pho~phac~e gen~ to be ~h~ ftç~d back lnto pha6e, ~nd then ~ fr~glTent coding for, at le~t, the 44~ C-tennlnal rem~nLng amino aclds of ~lkaline p}lo~ph~t~se.
S Ths hybrid sequence comp~se the fon~ul~ III
~elo~:

~ . G AA~ C~ AGC AC~ ATT GCA C~-G GC~ CTC TTA CCG T T~ CTG
Y.et lys ~ln ser ~hr ile ala ' eu ala leu leu pro !eu leu T~. ACC CCT GTG AC.~ P~ GCC CGG ACA CCA GAA A-G cc~ GAT
phe th. pro val ~hr lys ala arg th~ pro ~lu mQt pro asp CCC AGG ATA TGT . T . P~AC C~T C,~G TCA TCG CA~ CCG CAA ACC
pro arg ile cyq ~he asn hls_~ln ser 6~r qln pro qln thr AC~ AAA ACT ~Gr TC~ CC . GGG GAG ~GC ~CT TGC TAT ~AC AA~
thr l ys thr cY~ ser pro qly c~ù ~or çor CY8 tY. ~sn lyc C~A TG~ AGC GAT TTC CGS GGA ACT AT~ ~TT C~ AGG GGA ~G T
gln ~rp 3er asP Phe arq qLy thr lle llo qlu_~rq qly CY8 GGT T~C CCC ACA GTG AAG CCC GGT A'rT AAA C~C AGT TG~ TG;C
qlY cy3 p o thr val ly~ D~O ~1~ lle lys leu ~e cv~ cy~

GAA T~ GAG CTC SGC A~C AAT GAT Cg~ gTT CT~ GAA AAC CG~
~lu g~r glu ~l cYS asn asn a~p ~ val leu glu 13n arg GCT ~CT C~G GGC GAT ~TT AC~ GC~ CCC GGC GG~ GCT CGC CG~
al~ al~ ~ln gly ~8p il~ thr ala pro gly sly ala ~rg ~_g ACG GGT GAT C~ AC~ GCC ...
l~u thr gly ~sp gln the a!.a ...

~SO
~ G AAA ~AA
... l~u ly~ s~op ~III) Thi~ ~equence of formula III i~ de~iqnated e~iphoA6.
In thi~ formula III, the negAtive numberlng correspond~ to the ~mlno acid~ of the alksl~ne phc~ph~-t~e ~igna~ peptlde and the posit~e numb~r~ng corresponds to the ~mlno ~c~d~ of m~ture ~lk~lLne pho~--lO- ~0196~
ph~tA~e. The underllned ~equence i8 that of er~butoxln ~.
The æmino acids A~p-Pro and ABP_Arg on elther sid~ o~ the -. er~butoxin sequ~nce have been introduced on ~count of the ~ of clo~ng and shiftlng of the phoA gene b~ck into phese . The nucleotides corr~spondlng to the Sm~I res-trict~on site lntro~uced into the pho~phat~se gene are shown ln lewer-case letters (coc.... ggg). This makes it pos~ble to demonstrate the shlfting of the phoA gene out of pha~s, caused by the presence of the slt~, snd the shiftLng back into phase brought about by the introduc-t1on o~ the erabutoxin sequence. The portlon of pho~pha-ta8e sequence rot shown (dotted) corresponda to that published by CHANG et ~1. (CHANG, C.~., W.-J. XUANG, and E.Y. C~EN, 1986, Gene 44:121-125)..
15The sub~ect of the present invention Ls ~l~o 2 protein, whLch conslsts of a hy~rid ~equence compri~l~g 8ucces8~vely a fr~gment of ~ prot~ln Pl exported or 6e~reted by a mlcroorganlsm, in particular chosen fro~
the group which comprlse~ bactex~a and yna6tn, ~ sult~ble 20protein P2 or ~ fragment o~ the latter ~nd ~t le~t one function~l ~r~g~ent of a suitable enzyme, the hybrld protein pos3es~1ng 81multaneously the propertle~ of the enzymo and ~o~e properties of the protein P2, in p~rtl-cular of interacting specific~lly w~th ~n antlbody, ~n 25~nti~on or ~ roceptor.
~ccordln~ to ~n ~dvant~geou~ ombodiment of the hybrid prot~in, the prote~n Pl 1~ dl~ferent from the on~ymQ, AccordLnq to ~nother ~dvant~geou~ embodLmont of ~0the hybrld protein, tho proteln Pl i3 Idontlc~l to the en~yme, ~ccording to ~n ~dv~nt~geou~ ~rr~n~ement o~ thl~
embodimen~, the the hybrld proteln compr~sea ~n en~ym~
exported or ~ecreted by a microorqanicm, in p~rtlcul~r 35chosen fror~ the group which comprises bacterla ~nd ye~ts, into whlch a suita~le proteln P2 LB lnserted.
According to another advantageou~ arrangement o f em~od~ment, the hybrid protein comprise~
succes~ively a fraqment of an en2yme exported or secreted -11- 2~6~g~
by a r,ic~oorganism, in partlcular cho~en from ~he gr~up which comp-ise~ b~ct2r~ and yeast~, a ~u~abl~ pzotein P2 ~nd ~hen the complete mature sequenc~ o~ e enzyme, According .o ~nother advantageous embodiment of s the hybr~d proteln accordlng to the lnventlon, it c~n-tains ~ protein P2, ln p~rt~cular oho~en from the group which comprlses peptide ho~mone~, 8 ~ngle-cha~n rra~ment~
an~logous to the va~lable fragments o lmmunoglobulin~
~nd toxln~, inserted i~to an enzy~e chosen rom the s~oup wh~ch comprises, in part~cul~r, ~lk~line pho3phat~e ac~d pho~ph2ta8e, acla ~lucose phosphatase, cycllc phosphodiesterasc and ~-lactam~,e, According to an ar,angement of th~ 8 em~odiment, ~aid hybrid protein advantageously.compriRes 8 neurotoxin ln8srted into a~ka~ine phosphntase.
According to an advant~gèous variant of thl~
~rranyement, the neurotoxin ~s erabutoxln ~ (ea), which hybr~d protain possesse8 the sequenco deduced according to the fo~mula I or III above.
2~ According to anothe~ nrrangQment of thl~ e~bodi-ment, the hybrid prote~n advantn~eously compri~e~
angioten~in I ~n8erted into alkallne phosphata6e, snd po5Be8se8 the 8equence deduced ac~ording to the formul~
II a~o~e, 25 Tho pre~ent lnvention h~, in p~rt~cul~r, th~
advantage of expresslng, uncxpectedly, both .~n onzymo and a fu~ction~l protein P2, whethor lt iu inserted into ~h~
enzyme or sAndwlchod betwoen a prot~ain Pl nnd the enzy~o, and of po880~81ng grent ~t~bllity. It ~l~o ha~
the ~dvAn~ge 0~ ~xpressing ~ 8m~11 func~lon~L prot~ln ~ni~h, hltherto, even if lt could be coupled chc~lcally with ~n enZymQ~ could no~ lnd ~ppllcat~on ~a ~ re~gont ~n~, accordin~1y, could not b~ u~Qd in a~ys af EI~ o~
ELISA type, but only ln a~ys of the ~IA type, wh~ch t~re much mara ~wXw~rd to c~rry out.
The sub~ect of ~he pre~ent inv~antion 18 al~o ~mily of vectors fo~ t~e expression and/or clonln~ of a hybr~d proteln ~ccordlng to the ~nvention~ whereln each vector inc1~des ~n expression syst~m comprl~lngl -12- ~019~6~
- a ~uitable p~omo~er, - ~ ribo~o~e-bindlng ~i~e, - ~ nuclelc ecid ~equence cont~in~ng~ leacer sX~X~e of ~he s~tu~al gene for a p~o~in P1, a ~ ~ ~x~ng for a N~2-~ ~ ~al fr~n~ ~f ~ t~ ~c4~1n rl, a cO~,~ ~lng for a protein P2 'nserted at a n~t~ral -eot-lction slte ~r.d the m~ture sequence codlng fo. an enzy~e or a fr~g-ment of the latter, which nuclelc ~cid sequsnce cor-responds to ~ hybrld seque~ce ~ccordlng to the lnvention, lo and - ~ transc_iption termlnato~, whlch expression sy~tem i8 in~erted into a 8U~ table genetic ~tructure, in particular cho~en from the group which comprlse~ plasmid~, phag~s, cosmLds or sultable chromo~omes, Accordlng to an advantag~ous e~bodiment of the vector, the senetic struct~re ~ 6 a plas~ld and the p.rotein P1 Ls identlcal to the en2yme .
According to an advantageous arrangement of thL~
vector, when P2 i~ advantageously er~bu~oxLn a, ~ pl~mid 8 obtained po~sesslng the following propertie~
- it compri~e~ 6.1 kb;
- it is obta~ned by l~gation of plasmLd pJC24~1 carry~ng the structural qene for alkaline phosphat~se ~nd the gene for resl8~nc~ to ~mp~cillln (Ap~) - the pl~mid belng llne~ri~ed at the site Bcl~, ~ t codon 28 of ~he 0tructur~1 gena Ior ~lk~llne phosph~t~s~ - with the 192-b~e p~L~ S~u3AI-Ssu3~I
fr~gment cecling for exa~utoxin c~.
This pla~mid h~ boen deslqn~tod pEP1726 by tho invontor~.
Pla~mld pJC2411 ls de~crlb~d ln the p~p~r by J.C.
LAZZARONI et ~L., publi~hed ln J. a~cterio~ 985, 1$4, l376-l3a~ .
The sub~ect of the present inventlon 18 alsO
~nother famlly of ~ec~or~ for the axpression ~nd/or clonlns of a hy~rld protein according to th~ in~ention, wherein e~ch vector lnclude~ an expresslon oy~te~
compr~ing~

201966~

- ~ suit~Dle promoter, - a r~bo~ome-binding ~ite, - a nuclelc acld ~quenc~ cont~ining the lead~r ~x~Y~ce of ~ s~.~al 9~ for a ?~in P1~ a ~x~ ccdln~ , 5 for a ~i2-~,in~l frac~t of tne mature ~ro~e~.n P1, the ~es~ ~x~ng for an extrac~ la~c ~y~e or a _rag-ment of the latter and one or more uniqus restrlctlon site3, cap~ble of ~eceLving a sequence codlng for ~
p_oteln P2, s~tuated at the ~unction between the fragment of ~equence cod'ng for the prote~n Pl and the seouence co~ing for the cnzyme, and ' - a tran~cript~on termin~to~, which expr~ssion system i~ insc~ted into a 3ult~ble genetic structure, ln particular chosen from the group which comprises plas~ids, ph~ges, cos~ids or suit~ble chromosomes.
Accordlng to an adv~ntageous embod~ment of the the voctor, ~t least one of the uni~ue re~trictlon ~i~e~
introducad causea A shifting of the gene for the enzym~
out of pha~e, the shifting o~ the gene b~c~ into ph~se boinq e~fected by the introductlon o~ the sequence f~r the proteln P2.
Accord1n~ to an advantaqeous arrangement of thi~
embodim~nt, a pl~smid 1~ obt~ined possess1ng the follo~-lng proportloss 1t comprlse~ approxim~tely 5.9 kb, ~nd - it 1~ obt~ined by d~rocted mut~gone~ o~ the phoA gon~ c~rrLod by pl~smid pJC243l, ~ to 1ntsoducc ~ unlquc SmaI r~strlction gite correspondi~$ to position ~6 o~ the m~ture pro~eln~
Such ~ pla~mld h~ b~n de~iqn~ted pI,IPl by tho in~ntor~ ~nd lg s~id to b~ unladen.
When ~he ~equence codlng for the proteln P2 l~
ln~erted at ~ re3~rictlon site natural!y pre~ent ln the structural gene for pho~, or when the 3equ~nce cod~nq fo~
the prote~n P2 18 inserted at ~ unique restrlctlon ~ite lntroduced beforeh~nd 'nto the ~tructur~l gene ~or phoA, such vectors contaln hybrld ~equence~ accordlnq to t~e in~ention ~nd directly express the hybrid prote~n when 20~9~5 thoy ~re pre~ent ~n a suit~ble microorg~nlsm; ~uch ~ectors are said to be loaded, whereas the ~ec~or deslg-nat~d pLIP1, capable of receiving ~ proteln P2, 1~, for lts part, 3~id ~o be unlade~, The sub~ec~ of t~e present ~nvention ~s ~lso a m~croorganlsm obtained by gQnetic tran~form~tion, whlch - 18 obtalned by suitabls modificatlon of a su~tab1e ~tr~in of E. coli with a vector according to th~ lnventLon, and in part~cul~r by transformat~on when the ~ctor i~ a pla~mld.
According to an embodlment of tha lnvention, ~id microorganism is advantageously ~n ~. co1~ strain CC118 tran~formed by plasmid pEP1726.
Such a str~ tran~formed by R~ld pla~mld w~
depo~ited on the d~te 2 June 1~89 under number I-a62 with ., the Collection N~tlonale de3 Culture~ de ~icroorg~ni~es ~N~tlona1 Collection of ~ic~oorgAnlsm Culture~] held by the Pa~teur Instltu~, Such ~traln i~ design~ted SEP 1726 by the inven-tor~, Accordlng to ~nother e~bod~ment of the lnventlon, s~ld microorg~ni~m i~ advantageou~ly ~n ~ st~in CC118 tr~nsformed by p1as~1d pLIPl.
;~j Such ~ Rtr~in tr~ns~ormed by the pl~mld wn~
depo~ited on the date 7 june 1990 undex num~er ~-9S4 with the Collec~lon Na~lonalo de~ Culture9 de ~icro-o~g~nlsme~ [N~tLon~l Col1ection o Mlcroorg~nlsm Cul-turea~ held by the Pastour In~t~tute.
Such ~n unl~den vec~or has ~ha ~d~nt~ge o~
en~bling ~ny sequence codin~ for ~ protein to be lnoert~d ~t thls unl~ue restrlction slt~, ~hls modlication of the stxuctur~l gene for llne phosph~ta3e introduce~, ln t~dditlon, a fr~me-shlft in the nucleotide se~uence sltu~ted downotrenm, which sequence 18 ch~racteristlc of the functional portiorl of phoA.
~his unlsden vector, whlch corresponds to pl~mld pLIP1 defined above, hence does not Qxpress ~lk~llne pho~h~t~e activity.

20196~5 ~ he formu~a IY b~low pres~ntc an alignm~nt of ~he nu~l20tide and protein sequences corr~pondlng ~o the natlve phoA gene (~), the modified pho~ gene carri~d ~y the unladen vector pLIPl (B) and the hybr~d phoA gene 5 carried by the l~aded vector (C).
A. Native phosphatase~
CGC. ACP. CCA GAA A~G CG. GST CTG G~ AAC CG~ GC~ G-T C~
a-g t~r pro glu ~.et pro val leu g~u asn arg ala ala ~'n tl 16 t7 ..... CTG AAA TAA
..... leu ly9 ~t~op ~45~
B. Introduct~on of the Sm~I site ~unladen vector pLIPl):
~CG ~CA CCA ~AA A~G ccc ~gg ~C T~G AAA AcC CG~ C~G C,C
~r~ thr pro glu ~e~ pro gly p~e trp lys thr gly leu l~u ~I t6 .,.,. TTT 'rGC TGA
..... ph~ cy~ ~top ~49 C. In~ertl~n and rhLftlng ba~k lnto pha~e en~bllng ~ I
pho~ph~tase h~brid to be obtained (loaded Vector)~ j ~n6~rt CGG ACA CCh ~A ASG ccc 3n t 1 ~g gTT CTG GAA ~AC CGG GC~
arg thr pro glu met p~o xxx, x Xx val leu ~!u ~sn ~r~ al~
+1 ~6 t7 GC~ ... CTG ~A TAA
~1~ ... luu lys stop ~50 (IV) ~he nuclootlde~ o~lyinatlng from thu n~tlve pho~ I
gen~ a~ ~hown ln capi~al let~er~. Thc numberlng co~-responds to the aMino ~cids o mature al~aline phoJph~-tn~o. Tho dot~ed lln~ corro~pond to the portion~ of sequence~ not shown. The nucleotldeff corrospondlng to the SmsI rc~trlction site introduced lnto ~he pho~ g~ne are ~hown ln luwer-case letters (cccggg) (B). The f~me~hlft ~hereby introduced leads to the uynehesls of an ~berr~nt protein o~ 49 ~mino acid~ he introduction ~t the SmaI site of a sequence of 3n+1 base pa~rs (~n~ert) not containlng the termination codon en~bles the phoA gene ~o be sh~ted b~ck lnto phase and a hybrid proteln to b~
I

-16- 2~1956~
obta~ned (C). Furth~rmore, if the 3n+1 ~equence in its opposlte ori~antation contair~s a t~rmination cod~n, only the introduction of thl~ 5equsncH in the de~ired orlent~-tlon w~ll en~ble a pro~eln po6sessing pho8ph~ta~ actl-vlty to be o~t,~ned.
According ~o ~he invention, the promoter 18chosen from the group which compri~Qs the pho~ gene promoter ,qnd ~ny ~tronger promoter than the phoA gen~
promoter.
The phoA gene promoter, ~n p,rt~cul~r, i~s an inducible promoter wh~ch is ~ctiv~ted when the pho~ph~te concentr~tlon in the culture medium of the bacterlQ
becomes very low.

Such a promotex ha~, in part~cul~r~ the ~dv~nt~ge 1~ of en~bling hybrid proteins to be produced $n phosph~te-poor culturo medium. The depletlon of phocsphat~
in the medlum durl~g the culture of the b~cterl~ perml~s an lnduction o~ the synthes~ls of the hybrid protelns after the b~cterl~l m~sss hss lncre~sed. The adv~ntage of ~Such an induclble Syste~ is to reduce tho rlsks ~f proteoly~sL3 during productlon, ln~smuch ,~s the protelns extr,~cted ~re synthes~sized ,~t the end of culturing. ~he homogenelty of the hybrld i~ thereby Sncre~sed, The 8ub~ect of the pres-~sent inv~ntion 1B al~o a proce~q ~or the ~xpre~Sion of a hybrld proteln ~ccord~ ng to the lnve~t~on~ whlch ~mploys ~n Rxprec,sslon vector ~ I
de~ined ~bove ln a ~sicroor~nlesm accordlng to th~ lnvon- I
tlon, 'l~o prop@r~ies of ~he hybrld prot~m must be enp~sizcds ( l) the extreme s3~mplieity of it~s productiosn and ~he ,.~bsen~ of los~g ~nd dif f lcult purif ~ cat~on st~p~ 1 :
~2) it~ st~bility during its p~ductlon by bacteria~ duxing its 3torage i~ fcrent cor~ltla~s ~ ~uring ~ tlo t~st~ ~in~
t~ enzv~t.l-: activitv rx~.xsl~ts ~or ~n~ t.h~n 24 l~urs ln th~ pre6enco of the ~ubstrat~3. 1 The ~ub~ect of the presont inventlon ls ~180 a diagno~tlc re~gent, which con8ists of a hybr~d protein accord~ n~ to the invent~ on.
Such a reagent finds, ln partlcular~ application ln lmmunoen~ymologlcal ass~ys ~ ~ n the detect~on of -17- 2~19~5 receptor~ cr ~s histochamical labels.
Th~ sub~ect of the pre~ent invention is ~lso a method for the ~mmunoenzymological assay of proteins, ~hlch consists in detecting proteins pre8ent in a blo-loqical f!uid by brlnging Ithe biologic~l f!uld ~n~contact ~ith the dlagno~tic reaqent accordlng to the invantion ~nd wherein the presence of the reagent ln the form of ~ complex or in free fo~m i~ visuali~ed b~
~ui~ble c~lorimetrlc reaction.
Accordinq to ~n advant~eou~ embod~'~ent of t:~e method, the prot~ins are 2nt~en~.
Accordlng to another advant~geouR embodiment sf t~e ~ethod, the proteins are ant~odie~.
The sub~ect of the pre3ent~inventlon i8~ ~rther-more, a ready-to-u8e kit for c~rrying out the Lmmuno-enzymological as~ay, whlch compri~es, ~art ~rom the ~pproprlat~ quantitie~ of buffe~ and reagents u~eful for car~ying out the a85ay, cultable quantlt~ 98 of the reagent accordin~ to the invent~n, The reagent accordlng to the lnvention h~ ~h~
ad~antage of being dlrectly usable, whore~ usually, in the context of an ~lA o~ ELISA as~ay, the antlg~n or antibody is bound coval~ntly to an enz~me, the coupling be~ng c~rried out chomlcally. To be effectlve, ~uch a 2S coupllng mu~t be carried out ~l~ing two highly purifLed components .
The sub~ect of ~he pr0~en~ invontion L~ o a method for 8creenlng llbr~ri~ o cDN~ or of qenom~c nuclelc acld, or for the ~eloction o~ rocombin~nt clone~, whereln tho po~lble pro~encH of an enzymo exportod or sec~eted rom clon~ of b~ctesi~ or o eU~Aryotic cell~
whLch h~e, where ~pproprl~t~, in~egr~ted ~ pL~mid, ph~ge, a cosmld or a recombin~nt chromo~ome under i~re~-tiqation contalnlng a hybrid nucleic ~cld sequence 3S ~ccordlng to the lnventlon i~ vi6uali~ed by a color~-metric ~eaction or a selectlon on a 8uit~ble culture modium.
Accordlng to an advantageous em~od~ment of the ~thod, w~en t~e en~y.T~3 ls alkall~ ~sphat~Eie, ~ pre~s o~

201966~

exported or secretad enzy~e i~ u~lized u~i~g ~ ~uit-abl~ ~ubstrate ~or the e~zyme.
According to another advant~geous em~od~ment ~f the method, when the enzyme i8 ~-lac~m~e, the presenc~
of .he ~xported or sec_eted enzyme 18 V~ su~lize~ u~l~g medlum containing amplcillin.
In effect, ~n expre~s~on ~y3tem con~alnln~, downstre~m from the in~e_tad 3eqU~nc~ for the protein P2, the gene specifylng ~-lac'~mase en~le~ open -e~d~ng fr~mes to be cloned effectively ~nd exclusivel~. Tha~e construc~ions lead to the synthe~i~ of hybrid protelna contàining the de5ired polypeptide~ followed by func-tlon~ ctamase. ~he bacterla expre~slng such prot~in~
~re then 8elected by theLr re~l~tance to ~mpiclllin.
In the accom~anying drawings:
~igu~e 1 shows pla~ld pJC2431 c~rrylng the ~tructur~l gene for ~lkaline phosphat~se und~r the cont-ol of its own promo~er Figure 2 ~ llustrates an a~ay of erabutoxln ~ by an ~ISA test usLng the hybrid ea/phoA6.
Fl~ure 3 ~llu3tr~te~ ~n ~ y of anqloten~ln I by an ELIS~ te~t u~lng the hybrLd Angio/~hoA6.

~EPl72~.
Prep~r~tlon o the Sau3AI~S~u3AI fr~gment codlng for er~utoxLn al ~he 1~2-ba~e palr Sau3AI-S~u3AI r~-t:cictlon r~gmen~ cod~nq fo~ e~butox~n wl~hout ~ ~ignnl se~uence was prepared from the repllcati~e form of th~
phage M13mp19 (-2 G~T, -1 CCC, ~63 GAT)ea cont~lning ~he Ea gane c~rrying mut~tLon~ ln respect of 3 codon~, th~e modi~cations leading to the ~orm~t~on of two new Sau3AI
restriction ~lte~ Ln the genel -2 TAC (Tyr) become~ GA~
(~sp); -1 ACc (Thr) become~ CCC (Pro) t l 63 TAG (A~) becomes G~T (Asp) (se~ formul~ I), ~chl~ frag~ent ~5 put ln t~
pre~ence of the vec~or pJC2431 opened by psr Lal clesv~ge with BclI and DNA l~gase. ~he lig~t~ons WQre performed ln the buffer 50 mM TrLs-HCl pH 7.4, 10 m~ ~gCl2, 10 m~ DTT, O.5 m~ ATP ~nd lOO ~g/ml sSA, ~ith 1 U of ph~ge T4 DNA

20~966~

llgase and a vector/lnsert conc~ntr~tior~ r~tio of 1:10.
Dc pno~phoryletion of the vector was ca~ried out ~ccordlng to M~.ni~ti~ et al.
~igu-e 1 shows plzsmid pJC2 4 31 c~r~yLng the S ~r~lctu.~l gene fo~ alkaline pho~phat~se und~r the cont~ol of lts own ?romoter (L~zzar~ni et ~1., 1985).
"`his pla~imld posEeSSes three BclI restrictlon ~it~; Bcl sltu~ted upstre~m from the phoA p_omoterl and BclI2 a~
BclI~ sltudted, -espectively, at codon~ +28 ~nd ~363 of the phoA gene; the ~nsertion of ea Lnto the site ~clIl ena~las pla~mld pEP1726, of lnt9rest for the production of a hybrld protein according to th~ invention, to be obtalned~ .
Since the enzyme BclI c~nnot cut DNA ~t re~-lc-tlon ~Ltes mothyl~t~d by ~am-methyl~se, a d~m- ~t~in G~2163 i8 ~ransformed with pJC2431 in order to pr~p~re unmcthylated pl~ntld. A partlal digestlon of pJC2431 w~th Bcll ~nd ~ dephospho~yl~tion are then performed, The DN~
corresponding to the opened plasmid (~ single cut by BclI
out of the three pos6ible cut~) i8 then i301~tcd on low-melting point agarose gel accordin~ to the protocol of M~ni~tl~ et al. (1982).
Exam~le 2. ~x~re~ion o~ th~ h~rid ~ptel~
ea/~hoA2~ (in~ertion ~t ~a28 o~ alkal~ne phos~hat~se) A
~h~ ~train CC118 (~phoA20) w8~ tr~nsform~d wl~h the lig~tion mlx~ure de~crib~d ~n Ex~mpl~ 1. Th~ Ap~
(~mpi~illin-re~i~tant) tran~form~nt~ obt~lnod were purl~iad on alk~lLn~ phos~h~t.l~e-l~duclng medium cont~ln-ing XP ~nd .ampiclllin.
The lnducing madlu~ ES (~olution of(N~)2S0~
2 g/l, FeS0~.7H~0 0.5 ~g/l, ~Cl 7S mg/l ~nd trl~thanol-amine 7~5 g~l, ad~u~ted to pH ~.5). The additlon of ~he chromogonic substr~te 5-b~omo-4-chloro-3-Lndol~l pho~-pha~e (XP C.4 g~ o thi~ medium ~nabl~s the dl~
~5 phosphatase actlvity to be ~isuali~ed on P~t_l dLs~es, the XP ~mpartlng .~ blue coloratlon to the colonle~
which hydroly~e it.
Three t~es of tran~form~n~s h~ve been dlstingu1shad according to their cap~city t~ hydrolyze 20~ 966~

XP: colo~less clon~s, blue clones and f inally very deep blu~ clones. The latter two c~tegori~s hence po~e~ ~n alkallne pho~phataRe ~ctivity due to th~ pl~smid which they contain~
Analy8is of the re6tr~ction fraqments o~ th~
plasmid DNA of these different t-an3formants h~ enablçd the slte and orientation of th~ ln~ertion of the ea gene to be deter~ined in each c~se.
The deep blue clones corre~pond to insertlons of one or two Sau3AI-Sau3AI fragments at th~ ~lte BclIA~ th~t i~ to ~ay up~trea~ from the phoA genQ and it5 promoter.
The al~llne phosphata~e actlvlty of th~e clone~ 1 identical to th~t of the ~train CCl18 ~pJC2431).
The les~ pronounced blue clonns correspond to an in-fr~me insertion of a copy of the ea ~ene at the ~ite BclI2 (pE~1726~, The colorless clones origln4te from insertion o~
the e~ gene ~t the site BclI~, but in the opposite orien-tation to thst of the phoA geno.
~0 The protocol used for extraction of the periplas-mic proteln8 by o~otic shock i8 th~t described by Dvorak et al. (1~67).
Example 3~ Method for obt~lning plasmid pLIPl ~unladen expres~ion vector) and pl~mld PLlpl/p2 ~loadod 2~ expres~ion vecto~), l. Introduct~on of a cloning sLte lnto tho pho~
ger~q, Th~ phoA gen~ coli al~alln~ phospha~R) c~rrl~d by pl~mld pJC2431 was mod~flQd by directod mu~Agenesi~ in o~der to introduce a un1~ue SmaI rc~trl~-tion 3~te, after the codon correspondlng to tho ~lxth amlno acid o~ th~ mature protein, ~nd wa0 cho6~n for two re~on~ l - the N-terminal region of the phooph~tase c~n be modlflcd wlthout m~or Lmpalrment of Lts enzym~tic activity, - the presence of the first N-terminal Amlno acids $8 nece~ssr~ ln order to permit cleavage of the ~lgn21 peptide.

-~21- 201966~
Thi~ site w~s introduced ~o a~ to bring ~bout frams$hift in the phoA gene. In consequene~, this modi-fled gene i3 inc~pable of expre~ng a function~l pro- ~
tein. I
2. Introduction of exogenoug DNA 8equence~ ~nto the phoA gene.
The DNA fragment5 intend~d for introduction into the phoA gene at th~ S~aI 81te are obt~ned by nu~leot~de 6ynthesis or originate ~rom cloned gsne ~equences. They ~re prepared ~o aB to meet the followin~ requirements:
(1) their 5' snd 3' end6 mu~t be blunt, the Sm~I
~ite generat~ ng blunt ends;
~2) the~ introduction in the desi~ed orientat~on must enable ~ phoA gene~equence down~tre~m from the lnser-tLon slte to be 8hifted back into pha~e and mu8t perml~
the synthesls of a hybrid protein posse~sLn~ phosph~tase nctl~lty; ~nd

(3) the~r 6equence ~n th~ oriontation opposlte to ~at desired mu~t posseY~ a termln~tion codon in ph~e with the reAdLng ~r~me. This mu~t bring about an ab~ence of function of the hybr~d qene obta~ned Ln the case of ln~ertion Ln this orlent~ti~n.
The gone conatructions are obtalnQd by tho cla~lcal techniques of genetic en~ineerLng.
Only vecsors which h~vo in~ertod ~ exoq¢no~s noe~ in th3 oor~t d~r~ctlon wlll h~ve nn open ~eadlng frame p~rmlttlng tho expr~aslon o a hybrld prote1n con~i~tln~ of ~lle pol~peptide encoded by th~ in~c~ted s~ce .~d ~ lln~3 phoAph~t~@, 3. S~l~ctlon o tho hybrld genCG.
P~fter lig~tlon of the do~i~ed DNA fragment ln tho vector pLIPl, the recom~inant pl~mid~ are u ~ to tr,~-~or~ the bacterl~L str~in CCl18, sn E, col~ mut4nt not expres~ing alkAline phosphata~e. Only the bacSerl~l clone~ cont~ining the expected gene con~truction wlll ~e c~pable of recoverin~ a pho~phnts~e activlty. Thia activlty m~y be ~i6ualized by culturing the bacterla on n sultnble selection medium contalning a chromogen~c ~u~strate for the pho~phatase. ~he po~ltive clone~ appear ~01~

blue, Thu~, the appearance of t~e blue color~tlon, ~ndic8tlng that the bncteriu~ ~xpre~ a proteln po~-se~slng pho6phatase activ~ty, me~n~:
- that the exogenous DNA fragment ha8 been ~n8~rted correotlyr in the appropriate orientation, enabling thç
pho~phatase coding sequ~nce to ~e shlfted ~ack 1nto pha8e, - that the hybrid protain, ~ product of the recom-bLnant gene, L~ correctly exparted lnto the peripl~om of the bacterium, - that the hybrld protein produce~ dimers ~n order to form the homodimer corrc~ponding to the actlvo form o~
the pho~phatas~, - and hQnce that th~ inRertion o~ th~ exogonou~
protein sequence ~nto the phosphntao~ do~a not grently ~mpnlr it~ enzymntic actlvity.
Example 4~ ExPres8ion Of tho hybr~d ~roteln~
ea/p hoA6 ~nd Angio~p~oA6l zo ~. Introduction of a protein P2 lnto nlkallne pho~phata~e~
~ hlS i~ carried out b4~woen ~mlno ao1d~ 6 ~nd 7 . of the m~tur~ phosph~ta8e. S~ld hyb~ld protein~ consl~t .,. o~
- th~ 21 amino acldB o~ tho pho~ph~ta~e ~lgna1 pepclde~ This p~ptide enable~ the prote~n to be cxported to the perlpla~m~c 8paCe of the b~ct~rlum. Thl~ ~oc~ c-tlon 1~ ~8~entl~1 for the acqul81tlon of pho~ph~t~ae acti~lty and for a c~n~enient ext~actlon o tho hybrl~
prote~n) - the 6 N-termin~l a~lno acld~ o~ the ~ho~phat~o, ~heir pre~enCe per~its clea~ge of tho ~ignal peptid0 durlng the p~sa~e into the perlplasm;
- the amino acid ~equence of the exogonous protein ln~erted $nto tho phosphata~e. Tho dem~nd8 of clonlng can, where appropriate, introduce ono or more additlon~l ~mi~o acids at the end~ of thi~ ~equence~ ~nd - the last 444 amino acids o~ the phosphat~so.

-23- 2~19~i65 2. Production of the hy~rid prot~ln~:
The ~acterla are cultured ~n ~ pho~ph~te-poor llquid medlum. When the phosph~te i6 exh~u~ted, the phosphatase promoter i~ sct~vated and le~ds to tr~n~rlp-tlon of the hybrid gene. The a~v~ntage of this lnduc~ionph~nomenon i~ to permit a rapld synthesl6 of the hyb~ d proteins at the end of culturing (4 to 5 hours of cul- -ture), and hence to limit th~ rl~ks of proteolyt~c degradation. The proteins of the bactQri~l perlpl~m are extracted by o~motic shock. The UBe of the proteln hyb~lds (a9 reagent~, fo~ example) doQ~ not nece~s1tate further purlfication.
3. Tox~n/pho6phatase hybrid (e~/~hoA6).
- Gene construction~
15A 19 5-base p~i~ fragment co~prl~lng th~ er~b~-toxLn ~ codi~g sequence, fl~nked ~t the 5' and 3~ en~ by a Sau3AI ~ite as ~pecifled ~bo~e in Bxample 1, w~
rep~ired with Rlenow poly~er~o ~nd Lntroduced ~t tho SmaI site of plaamid p~ he pres~nce of a term~n~tion c~don in pha~e, in the fr~g~ont $n ~he oppo~te or$ent~-tion, permitted a Yl~u~l selectlon of tha ~#~hed re~ Ant clone~ acco~ding to the princlple de~crlbed ~bove in Ex~mple 2. The pl~m~d theroby obta~n-d w~ d-~1gnate~
pLIPl/ea. ~he expected polypoptido ch~ln (ea/phoA6) as compri~e~ ~ucce4~i~0ly~ tho pho~pha~ase ~gnal peptido, the ~irst 6 ~mino ~clds of the phoAphat~se~ two ~ddi-tional amino ~cld~ lntroduced on account of thc need~ of cloning (Asp, Pro), the 62 ~mino acidn o~ ~r~butoxln ~, two ~dtlti~n~l amlno ~cld~ lntroduced on ~ccount of th~
need~ of clonlng (A~p, ~rg) ~nd thn l~t 444 a~lno ~cld~
of thc pho~phstas0. The si~n~l peptldo 1~ cl~Aved durlng the p~sage ~nto the perlpla~m.
- Ch~racter~s~ics of the hybrld protoln e~/phoA6~
The hybrld protein e~phoA6 w~s produc~d ~ccord-ing to the procedure de~cribed ~bo~e, Three characteristlc6 of tho hybrid were studledT
~l) lts en~ym~tic ~ctl~ity, (2) the presence of ant~gonic determin~nt~9 pecific to erabutoxin, ~d -24- 201~665~
(~) the po~si~Llity of u~in~ tha hybrid for perfor~ins ~n a~ay of free toxin.
These characteri~tlc6 were comp~r~d with tho~ of the hybrid ea/phoA28 (Example 2), a product of pl~id pE~1726, obtaln~d by the introduction e~ er~butoxln lnto the phosph~ase after position 28 w~hout prlor ~odlflca-tion of the phoA qene, u3ing a pre-exl~ting restriction slte.
a~ The enzymatic acti~lty of the hybrid~ w~
evaluated by compar~ son wLth that of natlve alkall~e pho~phat~e obta~n~d under thQ ~ame condit~ons of produc-tion. ~he specLfic acti~ity of bAct~rl~l perlpla~mi~
extract~, obt~ined by 08motic ~hoc~, cont~lnln~ ~h~
dlffercnt proteins was determlned. Th~ proteln concentr~-tion of the ext~ct i~ d~termined from lt~ absorb~nc~ ~t465 ~m and ~rom a ~tandard cur~e produced by prote~n ~8Ay by ~radford's m~thod (~radford, 1976). ~he enzy-m~t~c ~ctlvity ~ mea~ured on 20 ~1 of extrsct, Afte~
~ncubation for 15 min at 37~C ln th~ preY~nce of 25 mM p-nitrophenyl phosphate (pNPP), tha reactlon 18 stopped ~y~ddln~ 1 ml of 1 M NaOH. The ab~orbance o~ the ~mpl~ i~
re~d ~t 410 nm (yellow color~tlon duè to the form~tLon of pNPO~). The ~pecl~c en~y~e ~ctivity ~S~), ln nmol o~
p~PP hydroly~ed per minu~e per mg Qf prot~ln, 1~ en by the formul~
A410 x 5.5$
S ~
(mg proto~n/ml)x2xlO'2 The measure of th~ activlty of tho hybrid~
exprc~sed a~ ~ percent~ge of ~peclfLc ~ctl~lty corre~pon-ding to ~h~ n~tiv~ pho~phat~e t Na~LvQ phosph~t~Ao~ 100~ (SA) e~ oA6~ 50 eA/Ljho2at 30 b. The pre~ence of ~ntlg~nlc deter~Ln~nt~
~peci~ic to erabutoxin carrl~d by the hybrld prot2in~
ea/bhoA6 ~nd e~/~hoA28 w~8 studied by ~n ~ A te~t. The~e prote~ arecapable of blndlng to the ~onoclona1 antlboqy M~2-3, ~pecLfic for an ant~genic determinant carri~d by 201966~

the n~tive toxin. Furthermore, there i8 ~ competlt~on for b~x~ng be~n ea/phoA or ea/phoA2~ and th~ na~ve toxin. In c~der to e~aluate the level of ~tructurLng of the toxln ln the hybridg, we comp~red by an ELISA test it~ recognltlon by ~ serum directed toward~ the n~tivo toxin ~nd by ~ ~erum dlrected towards the den~tured toxin. To the extent of 6 0~ of the hyhrld eaiphoA6, the toxin is correctly ~tructured, ~herea8 this proportlon ~ 8 only 32~ for th~
hybrid e~hoA~8.
1~ ..
C. Fi~ure 2 Lllustxates an a3~y of or~butoxin ~ by ~n B~IS~ test u~ing the hybr~d en~phoA~.
The competltion for binding betw~en ea/phoA6 ~n~
the free toxln to the ~onoclonal ~ntibody ~2_3 ~
~tudled by ~n ELISA test. A~te~ ad~orptlon of the ~nti-body on an ELISA pl~te, ea/pho~6 (o~motlc ~ho~k d~lutod 10-fold) 1B added ~n the pre~ence of d~ffer~nt quantltle~
of toxln.
quantity o~ hybrld bound in tho presence B of toxin The rn~io ~
Bo qu~ntity of hybrid ~ound in the ~b~ence of toxln i~ exprR~ed ln term~ of th9 qu~ntity of frae toxln. The senJltivity o~ this te~t, e~luated by the qu~ntlty of toxln for whLch the ratio B~Bo~0.5, i8 2.5 pmol, ~qul~lent to 17 n~. This ~ ltivlty i~ equlv~l~nt to th~ o~ ~n ~IA test u~lng th~ trltiatod toxln,

4. Angiot~n~in I/phosph~t~o hybrld (An~lo~hoA6).
~ne construc~lon~
~ 31-b~ p~lr fragment c~p~ble of codlng for ~n~ioten~ln ~ w~ obt~lned by hybridls~lon o~ two compl~menta~y synthe~lc ollgonucleotide~ Thl~ fr~gment w~ lntroduced At the 6maI ~lte of pl~id pLIPl. The ~equence of this fragment waq determined ~o ~ to lntro-~uco ~ termln~tlon codon ln ph~e with the readlng fr~m~
~5 ~019~65 of the phosphata~e when lts integr~tlon t~kes place in the orienta~lon opposlte to that de~lred. Thu~, after the ~equencing of sever~l clones ~elected for the expres~on of a pho~ph~t~e sctlvity ~blue clone~), they ~ll corre~-ponded to the des~red ~onstruction. The pl~mid thereby obta~ned waB designated pLIPl/Ang~o. The ~xpect~d poly-peptlde chain (Anglo/~ol~6) comprisQ~ succo~s~vely~ the pho3phat~s~ sLgnal peptide, the ~ir~t 6 amlno scids ~
the pho~phata~e, the 10 amino ac d~ cf ~ngioten~ln I, an additional ~mino acid introduced on account of th~ n~eds of cloninq (Arg) snd the last 44~ amino acid~ of the phosphat~e. The 8ignal peptide i8 cle~ved during the pasB~ge into thQ peripl~sm.
- Ch~racteri~tic6 of th~ hybrid p~otein Anqlo/
lS phoA61 The hybrid protein AngioJphoA6 w~ produced accordin~ to the procedure ~e~cribed ~bove.
Three characterlstics of the hybr~d w~re studle (l) 1ts ~nzy~m~tic activlty, (2) itu reco~nitlon by A ~erum dlrected tow~rda Angiote~Bin 1 ' ~nd ~ 3) the pos~lblllty of us1nq tho hybrid for performlng a hormone ~s~y.
a . The en~matLc ~ctlvity of the hybrld Ang10~
25 PhoA6 wa~ ev~lu~t~d ~ccording to th~ proced~re de~cribed ~bove for the hybrid ea/~hOA6. Thi0 m~ur~ 1~ expressed a8 ~ porcentage of ~peclflc actlvLty corr~pondln~ to the nAtiVe pho~ph~ta~e:
NA~1~re pho8phatA~e: 10096 (Sl~) .
Anglo/pho~6: 93~
b. The recognltion o~ th~ hybrid pro~oin Angio/phoA~ by ~ ~erum dlr~cted townrd~ Angloten~1n I w~
~tud$cd ~y an ELISA te8t . A plg ~erum d~ rect~d toward3 rabbit lm~unogLobullns w~s ~dsor~ed on an ~LISA pl~te .
r~bbit serum dlrected tow~rds ~Lngloten~ln I coupled to bo~ne ~erum ~lbu~in was then ~ound to the pig ~erum.
Pln~lly~ the hybrld protein Angio/phoA6, produced by o~mot~c ~ho~k, W~8 added to the sy~tsm. ~t- b$nding to the ~ntlbodlQs directed tow~rd~ angiotensin WC18 -27- 2~19 6 ~5 visu~llzed through it5 phosphat~e ac~L~lty by adding a chromogenlc substrate for the enzyme~ Th~ speciflcity o~
thia reoogn~tion i~ demonstrated by th~ ~xi~tence ~f a competltion for binding betwe~n the hy~r~ d and ~ro~
ho~on~.
c,Flgur~ 3 illuBtr~te~ ~n a~y of ~ngloten~n I by ~n EBISA teBt uslng the hybrid Angio/ohoA6.
The compet~tion for blnd~ng bet~en Anglo/phoA6 0 ~nd free angiotensln I to a r~bb~t ~erum dlrect~d tow~rd~
th~s hor~one W~8 studled by an E~ISA ta~t. Th- rabb~t ~erum ~8 bound beforehand to ~ plg antl-r~b~it lmmuno_ globul~n ~erum ~dso~bod on an EL~SA platQ~ Tha hybrld Anglo/phoA6 (o~otic ~hock d~luted 20-foid) 1~ add~d ln 1~ ~he presence of dlfferent quantitles of anglotun~ln I.
quantity of hybrid bound ln the pre~ence ~ of hormone The ratio - ' -Bo quantity of hybrld bound in the ab~nce 0~ hor~one i~ expre~ed ln terms of the qu~ntlty of free hormone.
~he s&nsltlvi~y of thl~ te~t, evaluated by the qu~ntity of hormona for which ~he r~tio B/Bo-~.5, 1~ 0.09 pmol, e~uiv~lent to O.12 ng. ThLs sen~iti~lty i~ equiv~lent to th~t o~ RIA test~ u~ed in com~on di~gnoRtlc pr~ctice tXlt m~rket~d by CO~SPAGNI~ ORIS INDUS~RIE FR~NCE~ .
~x~mple 5 ~ St~billty o th~ pho~ph~t~e hybrid prot~ins.
1. Inductlon of the synthn~l~ of the pho~ph~t~e hybrld~
In the expre~ion vector produced, p~IPl, thc ph~ gene promoter ha~ been re~lned in order ~o ~n~ble the hy~ld genea to be oxpress0d. Thi~ promoter LB ~n ~nducible promoter which L8 ~cti~ted when the pho~ph~t~
concentr~tion ln the culture modlum o~ th~ b~cter~

2~19665 --2~3--become6 very low.
Under th0 conditions chosen (~ee Ex~mpl~ 4.~), the syr.thesl~ of the hybrid p~oteinB ~ me~ur~d by the appearance of phosphatase activity, beglns ~fter 3 hour~
30 m~nute6 of culture. Extract~on i8 carrled out when the maxlmum ~ccumulation of the hybrid~ l~ reached, n~mely after 4 hour8 30 minutes to 5 hour~.
2. St~bility o~ the hybrld proteins;
The ~tabllity of th~ hybrld proteln~ W8~ evalu-~ted in three ~ituations:
(l) stablllty durlng their production, (2) ~tabillty during thelr use, (3) stability during storage oper~tion~.
~. The stabillty of the hybrLds and of the n~tl~
pho8phatase was studled by v~su~ lng their lnteqrl~y afte~ labeLlng wlth ['sS~meth$onlne, Lmmunoprecipit~tLo~
Rnd polyacrylamlde gel electrophore~i9. The bacteri~ ~e culture~ under the usual cond~tions o~ productlon~
L~bellng of the hybr~ds w$th ~S)me~hionlne i~ carrle~
out ~t the ti~e of induct~on of their ~ynthe~is for 30 minutes~ The proteLn~ immunoprocip~tated w~th ~ mono-clonal antibody speciflc for al~aline pho~ph~ta~o appe~
in tho form of a slngle mol~cular ~pecleo. ~he ~ol~cul~r masses observod a~e very clo~e to thr expected theoretl-cal Yalue8~ app~oxi~ately 46,000 dalton~ fo~ the pho~ph~-t~se, approxlmntely 5~,000 dal~on~ for e~phoA6 ~nd ~pproxim~toly 4~,000 d~lton~ for ~ng~o/~hoA6. By prolonq-lng the bact~rlnl culturo by 4 hour~ or l~belln~
tchn~), no degr~datlon of tho l~beled prot01n~
observed. These result~ ~how ~hat the pho~ph~t~o hybr~d protctn~ do not underqo ~ny proteolytlc degrad~tLon durlng their productlon. Thls 1~ by no mo~nn th~ c~e ln respect o~ the hybrid protoln~ obtalned by ~u~lan of 3t~phylococcus protein A or of ~, coll ~-g~lacto~ldase.
In th~e systems, the ma~orlty of the expected hyhrld proteln undergoe~ a proteolytic degr~d~tlon.
b. After ~4 hours' incubatlon under the ~l~u~
z~tien conditions of the ELISA te~t, no slgnlfLc~nt f~ll ln enzym~tlc ~ctivity of the hybrlds 18 ~b~e~ved. ~hLs 20~9~6~
.--29--re~ult demonotrate~ t.he ~tsb~lLty of th~ reagent durln~
U5R . In cons~qu~nce~ thc vl~uAliza~io~ ~ime of an ELISA
test may be prolonged ln order to incre~e Lts ~en~l-tlYi~, c. After lQ cycleo of freezLng at -180'C followed by tha~ing at 37 C, that i6 to Bay ~nd~r dra~t~c ~ondi-tiono, 9~ of the phosphat2~e activi~y ~f th~ hybrid~ i~
preserved. Prelimin~y otudies have yielded s~mil~r results ln reopect of lyophlliz~tlon. It h~nce appea~
that the pho~phatase hybrid6 can remaln p~rf ectly otab~
under norm 1 condit~ons of packaging.
Example 6~ ~ethod or o~reenin~ cDNA or ~eno~lc llbrarl~, sccordlng to the ~n~ent~on, ~he comblned cloning and exp~ossLon sy~tem~
curren~ly on the market are integr~ted ln bacteriophage~
or pla~mids~ Their e~8ential common characterlotics aro a~ ~ollows t they po~es~ a po~tion of the lacZ geno o r E. coli (~-gal~ctosid~se), mod~fied ln ~uch a way tha~
it contalns one or, more often, a serio~ of unique restrictlon 8ite8. The inse~tion of DNA fragmenta ~f thL8 slte ge~erally leado to lo~ of ~he ~-g~lacto~idase ~ctiv~ty. Thl8 property m~ke~ Lt po~olble to recognL~e the recombinRnt clone~, wh~ch form colorle~
colon~e~ or lyt~ c pl~que~, whereAs the clone~ without ~n in~ert appe~r blu~ in the prQs~nce of X-G~ Soreo~er, the in~ertod DNA ~equr3ncos m~y be ~xpre-~ed ln the fo~m o~ fusion pro~ein~ wi~h a fragment of tho ~-gal~¢~o~i-da~e. Their d~sctlon is then pos~ible ualng ~ntibodles ~peclfic ox ~h~ desired proteln~. ~he vlsu~ at~on ~
3~ par~ormed by d~fferent technl~ue~t l~b~llng with lodlne~25 cr the u~e of ~n~Lbody-enzymQ con~ugate~ (Mel0cul~r Cloni~g, 1989, 12~1~). Substltutlon in the~o nyntem~ of tho modlLed l~c~ gene by the modif led pho~ g~ne, in part~cul~r a~ de~crlbed for plasmld pLIPl~ le~d~ to the appe~rance of blue And colorless clones ln the pre~ence of XP. In thls ca~e, the ~ituatlon 1~ the oppo~lte o~
th~t occuxrin~ in the ~ystems using ~-g~lactoaldase, the clone~ cap~ble of convertin~ the colorimetrlc aub~tr~te, XP ln thls in~t~nce, b~lng reco~b~n~nt clonos.

_30_ 2~1 9~6~
Fu~th~rmore, the ~Lmple color~t~on of A clone ls Indlc~-tlv~ o~ ~e~aral properties. It means th~t: 1) a DNA
fr~gment has been inserted into the phoA ~ene, ~L) thl~
fragment code~ for an open re~d~ng frame, lii) the in3ertion ha8 enabled the phoA gene t~ b~ ~hlfted ~ack lntc pha~ and to be expres~ed in the form of ~ hybrld prote~n, iv~ thi~ hybr~d prot6in i8 exported ~nt~ the peripl~sm and v) the recomb~nant alkalln8 phosphat~
dlmeri2es to for~ an actl~e en~yme whlch hydrolyzs its ~ub8trate~, and in particular th~ colorimetric sub-~tr~te~. Thus, the informat~on re~led by the colorl_ metrLc test i~ ~uch more exten8ive than in the case of ~-gal~cto~ldase. The u8e of the latter en2ym~ p~r~it~
distinction between the clones wIth ~n in~ert And the clones without an in~ert without ~nvoking the exi~tenc~
or the properties of a recombinant protoln, ~hereas tho u~e o alk~l~ne phosphata~e en~bl~ the clone8 capabl~ ~
Rynthesi2lng an exporta~le hybrid protein posJe~sing a ~tructu~ed ~nd enzymatic~lly activ~ phoA domain to be ~dentlfled dlrectly. Moreover, the c~paclty of the hybrl~
protein3 to be exported to the p~iplasm 18 not without Qn effect on ~he ~tructurlng of the polypeptlde ln~arted in~o phoA. In the ca~e o~ erabutoxin a, lts export~tlon i8 ~ccompanled by the form~tion of it~ di~ulflde brldgo~, It thu~ ~dopt~ a ~onformation ena~llng lt to be recogni~ed by ~ntlbod~es specific for the ~tructurad form of the toxln. ~o~t antlb~diou havo thl s type ~
~onf~rm~tLon~dependen~ ~pociflclty, ~nd recognL2e only cor~ctly 3tructured prote~n~. Fln~lly, l~ l~ po~iblo to turn to good ~c~ount the onzym3tlc ~ctlvity o~ the hybrld proteln8 s~nthesl~ed in order to d~monntr~te very ~Lmply ~n inter~c~ion wlth ~ntlbodies sp~clfic for the lnserted prot~n. A aimpl~ blottlng of tho colonlea ~ynthe~i~lng the recomhln~nt proteins onto ~ nltrocellulo~e fllte~
Ratur~ted beorehand with ~ solutlon of specl~c ~nt~bodle~, ~ollowed ~y a visualization wlth a coloF~
metr~c re~gen~ fo~ the ph~sphat~se, wlll enable th~
cLone8 se~ret~ ng a hybrLd proteln cap~ble of re~ctlng ~it~ the ~ntibodies to be v~su~lized.

-31- 2019~
Thl~ method can al 80 prove ext~emely ~3f f lc~c ~ ou~
for identifylng the epitope6 of a defined proteln. DNA
fr~gmen~s coding for thi~ prot~ln may be obt~lned by synthesis, by the action of re~trict~on enzyme3 on ~he ; whole gene or by any othe~ method ~uch ~ the U8~ e~ PCR
(polymarase chain reactlon) techniques, f~r ~x~mple, ~nd integrated ln the phoA gene. A study of the lnter~ction of the recombin~nt proteln~ result~ng from these c~n-gtruction~ with 8er~ or monoclon~l antlbodie~ dlrected toward~ the whole proteLn then enables the region~ of the prote~n bearing the epltope~ to be ~dentified, Ex~mple 7: Method for the selection of rw om-.
blnant clone~, ~ccording to the inventlon.
, The us~ of the bl~ gene which code~ for ~-lac-tam~se, ~n en~yme whlch confer~ ~m~lcillin re~i~tanc~ on the bacterium which expres~es it, ~ke~ av~llable a ~creen fo~ selectlon of the recom~lnant clone~ wh~ch alone c~n grow ~n the pre~ence o the antibiotic. ~hla type of ~election enables very r~re coding sequences to be lnvestlgated.

Claims (45)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A nucleic acid sequence, which consists of a hybrid sequence comprising successively the leader sequence of the structural gene for a suitable protein Pl exported or secreted by a microorganism, in particular chosen from the group which comprises bacteria and yeasts, a nucleic acid sequence coding for a NH2 terminal fragment of said mature protein P1, a nucleic acid sequence coding for a protein P2 and then a fragment coding for at least one functional fragment of the mature sequence of a suitable extracyto-plasmic enzyme,the assembly of these fragments being in a single reading frame and coding for a hybrid protein possessing simultaneously the properties of the enzyme and some properties of said protein P2, in particular that of interacting specifically with an antibody, an antigen or a receptor.

2. The hybrid sequence as claimed in claim 1, in which the enzyme is chosen from the group which com-prises, in particular, alkaline phosphatase, acid phos-phatase, acid glucose phosphatase, cyclic phosphodi-esterase and .beta.-lactamase,

3. The hybrid sequence as claimed in claim 1 in which the fragment of sequence coding for the protein P2 is, in particular, chosen from the group which comprises the sequences coding for peptide hormones, the sequences coding for a toxin and the sequences coding for a single-chain fragment analogous to the variable do-mains of immunoglobulins (recombinant immunoglobulins).

4. The hybrid sequence as claimed in claim 3, in which the toxin is advantageously a neurotoxin.

5. The hybrid sequence as claimed in claim 1 , in which the protoln Pl is identical the enzyme,

6. The hybrid sequence as claimed in claim 1 in which the protein Pl is different from the enzyme.

7. The hybrid sequence according to claim 1 which consists of a hybrid sequence comprising successively the leader fragment of the structural gene of an enzyme exported or secreted by a microorganism, in particular chosen from the group which comprises bacteria and yeasts, a nucleic acid sequence coding for a NH2-terminal fragment of said mature enzyme, a nucleic acid sequence coding for a protein P2 and then a fragment coding for at least one functional fragment of the mature sequence of said enzyme, the assembly of these fragments being in a single reading frame and coding for a hybrid protein possessing simultaneously the properties of the enzyme and some properties of said protein, in particular that of inter-acting specifically with an antibody, an antigen or a receptor.

8. The hybrid sequence according to claims 1 which comprises successively the leader sequence of the structural gene for alkaline phosphates, a fragment coding for, at most, the 28 N-terminal amino acids of mature alkaline phospha-tase, a DNA sequence coding for a protein P2 or a frag-ment of the latter, and then a fragment coding for, at least, the 422 C-terminal remaining amino acids of alkaline phosphatase, the assembly of these fragments being in a single reading frame and forming a hybrid DNA
sequence coding for a hybrid protein possessing simul-taneously the properties of alkaline phosphatase, espe-cially its enzymatic activity, and some of the properties of the protein P2, in particular that of interacting specifically with an antigen, an antibody or a receptor,

9. The hybrid sequence as claimed in claimed 8, which comprises successively the nucleic acid sequence coding for the alkaline phos-phatase leader peptide, the sequence coding for the 28 N-terminal amino acids of alkaline phosphatase, the sequence coding for mature erabutoxin a (ea) and the sequence coding for the 422 C-terminal amino acids of alkaline phosphatase.

10, The hybrid sequence as claimed in claim 8 in which the sequence coding for erabutoxin a comprises 192 base pairs and two additional Sau3AI
restriction sites.

11. The hybrid sequence as claimed in claim 10, which comprises the formula I below:

Leader fragment of the structural gene for -fragment coding for at least the 422 C-terminal remaining amino acids of alkaline phosphatase.

12. The hybrid sequence as claimed in claim 1 which comprises successively the leader sequence of the structural gene for alkaline phosphatase, a fragment coding for the 6 N-terminal acid acids of mature alkaline phosphatase, a nucleic acid sequence coding for a protein P2 or a fragment of the latter, and then a fragment coding for the 444 C-terminal remaining amino acids of alkaline phosphatase, which sequence comprises, in addition, in particular in order to place the assembly of these fragments in a single reading frame, downstream and/or upsteam from the fragment coding for the protein P2, a suitable nucleic acid fragment which hybrid sequence, in a single reading frame, codes for a hybrid protein possessing simultaneously the properties of alkylene phosphatase and some of the properties of the protein different from the enzyme, in particular that of inter-acting specifically with an antigen, an antibody or receptor.

13. The hybrid sequence as claimed in claim 12, which comprises successively the leader sequence of the struc-tural gene for alkaline phosphatase, the sequence coding for the 6 N-terminal amino acids of alkaline phosphatase, the nucleic acid sequence coding for angiotensin I, fragment comprising the sequence AGG G and which enables the alkaline phosphatase gene to be shifted back into phase, and then a fragment coding for, at least, the 444 C-terminal remaining amino acids of alkaline phosphatase.

14. The hybrid sequence as claimed in claim 13, which comprises the formula II below:

15. The hybrid sequence as claimed in claim 12, which comprises successively the leader sequence of the stric-tural gene for alkaline phosphatase, the sequence coding for the 6 N-terminal amino acids of alkaline phosphatase, a fragment comprising the sequence GAT CCC, the nucleic acid sequence coding for erabutixoin a, a fragment com-prising the sequence GAT C, which fragment enables the alkaline phosphatase gene to be shifted back into phase, and then a fragment coding for, at least the 444 C-terminal remaining amino acids of alkaline phosphatase,

16. The sequence as claimed in claimed 15, which comprises the formula III below:

17. A. protein, which consists of a hybrid sequence comprising successively a fragment of a protein P1 exported or secreted by a microorganism, in particular chosen from the group which comprises bacteria and yeasts, a suitable protein P2 or a fragment of the latter and at least one functional fragment of a suitable enzyme, said hybrid protein possessing simultaneously the properties of the enzyme and some properties of the protein P2, in particular of interacting specifically with an antibody, an antigen or a receptor.

18. The hybrid protein as claimed in claim 17, in which the protein P1 is different from the enzyme.

19. The hybrid protein as claimed in claim 17, in which the protein P1 is identical to the enzyme.

20. The hybrid protein as claimed in claim 17 which comprises an enzyme exported or secreted by a microorganism, in particular chosen from the group which comprises bacteria and yeasts, into which a suit-able protein P2 is inserted.

21. The hybrid protein as claimed in claim 17 which comprises successively a fragment of an enzyme exported or secreted by a microorganism, in particular chosen from the group which comprises bacteria and yeasts, a suitable protein P2 and then the complete mature sequence of said enzyme.

22. The hybrid protein as claimed in claim 17 which contains a protein P2, in parti-cular chosen from the group which comprises peptide hormones, single-chain fragments analogous to the vari-able domains of immunoglobulins and toxins, inserted into an enzyme chosen from the group which comprised, in particular, alkaline phosphatase, acid phosphatase, cyclic phospodiesterase and .beta.-lactamase.

23. The hybrid protein as claimed in claim 22, which advantageously comprises a neurotoxin inserted into alkaline phosphatase.

24. The hybrid protein as claimed in claim 22 or claim 23, in which the neurotoxin is erabutoxin a (ea), which hybrid protein possesses the sequence deduced according to the formula I or III as claimed in claim 12 or claim 16.

25. The hybrid protein as claimed in claim 22, which advantageously comprises angiotensin I inserted into alkaline phosphatase, and possesses the sequence deduce according to the formula II as claimed in claim 14.

26. A family of vectors for the expression and/or cloning of a hybrid protein as claimed in claim 17 wherein each vector includes an expres-sion system comprising:
- a suitable promoter, - a ribosome-binding site, - a nucleic acid sequence containing the leader sequence of the structural gene for a protein P1, a nucleic acid sequence coding for a NH2-terminal fragment of said mature protein P1, a nucleic acid sequence coding for a protein P2 inserted at a natural restriction site and the sequence coding for a mature extra-cytoplasmic enzyme or a fragment of the latter, which nucleic acid sequence corresponds to a hybrid sequence according to the invention, and, - a transcription terminator, which expression system is inserted into a suitable genetic structure, in particular chosen from the group which comprises plasmids, phages, cosmids or auitable chromosomes.

27. The vector as claimed in claim 26, in which the genetic structure is a plasmid and the protein P1 is identical to the enzyme.

28. The vector as claimed in claim 26 wherein, when P2 is advantageously erabutoxin a, plasmid designated pEP1726 is obtained, possessing the following properties:
- it comprises 6.1 kb;
- it is obtained by ligation of PLASMID pJC2431 carrying the structural gene for alkaline phosphatase and the gene for resistance to ampicillin (Apr) - said plasmid being linearized at the site Bc1L
at codon 28 of said structural gene for alkaline phosphatase - with the 192-base pair Sau3AI-Sau3AI
fragment coding for erabutoxin a,

29. A family of vectors for the expression and/or cloning of n hybrid protein as claimed in claim 17 wherein each vector includes an expression system comprising:
- a suitable promoter, - a ribosome-binding site, - a nucleic acid sequence containing the leader sequence of the structural gene for a protein P1, a nucleic acid sequence coding for a NH2-terminal fragment of said mature protein P1, the sequence coding for a mature extracytoplasmic enzyme or a frag-ment of the latter and one or more unique restriction sites, capable of receiving a sequence coding for a protein P2, situated at of the junction between the fragment of sequence coding for the protein P1 and the sequence coding for the enzyme, and - a transcription terminator, which expression system is inserted into a suitable genetic structure, in particular chosen from the group which comprises plasmids, phages, cosmids or suitable chromosomes.

35. The vector as claimed in claim 29, in which at least one of the unique restriction sites introduced causes a shifting of the gene for the enzyme out of phase, the shifting of said gene back into phase being effected by the introduction of the sequence for the protein P2.

31. The vector as claimed in claim 29 or claim 30, wherein a plasmid designated pLIP1 is obtained, possess-ing the following properties:
- it comprises approximately 5.9 kb, and - it is obtained by directed mutagenesis of the phoA gene carried by plasmid pJC2431, so as to introduce a unique SmaI restriction site corresponding to position +6 of the mature protein.

32. A microorganism obtained by genetic transforma-tion, which is obtained by suitable modification of a suitable strain of E. coli with a vector as claimed in claim 26

33. The microorganism as claimed in claim 32, which is advantageously an E. coli strain CC118 transformed by the vector pEP1726.

34. The microorganism as claimed in claim 33, desig-nated SEP 1726, which was deposited on the date 2 June 1989 under number I-862 with the Collection Nationale des Cultures de Microorganisme. [National Collection of Microorganism Cultures] held by the Pasteur Institute.

35. The microorganism as claimed in claim 32, which is advantageously an E. coli strain CC118 transformed by plasmid pLIP1.

36. The microorganism as claimed in claim 35, which was deposited on the date 7 june 1990 under number I-954 with the Collection Nationale des Cultures de Micro-organismes [National Collection of Microorganism Cul-tures] held by the Pasteur Institute.

37. A process for the expression of a hybrid protein as claimed in claim 17 , which employs an expression vector as claimed in claim 26 in a microorganism as claimed in claim

38. A diagnostic reagent, which consists of a hybrid protein as claimed in any one of claim 17

39. A method for the immunoeonzymological assay of proteins, which consists in detecting proteins present in a biological fluid by bringing said biological fluid into contact with the diagnostic reagent as claimed in claim 38 and wherein the presence of said reagent in the form of a complex or in free form is visualized by a suitable colorimetric reaction.

40. The assay method as claimed in claim 39, in which said proteins are antigens.

41. The assay method as claimed in claim 39, in which said proteins are antibodies.

42. A ready-to-use kit for carrying out an immuno-enzymological assay as claimed in claim 39 which comprises, apart from the appropriate quanti-ties of buffers and reagents useful for carrying out said assay, suitable quantities of the reagent as claimed in claim 38.

43. A method for screening lilbraries of cDNA or of genomic nucleic acid, or for the selection of recombinant clones, wherein the possible presence of an enzyme exported or secreted from cones of bacteria or of eukaryotic cells which have, where appropriate, inte-grated a plasmid, a phage, a cosmid or a recombinant chromosome under investigation containing a hybrid nucleic acid sequence as claimed in claim 1 is visualized by a colorimetric reaction or a selection on a suitable culture medium.

44. The method as claimed in claim 43, wherein when the enzyme is alkaline phosphatase, the presence of exported or secreted enzyme is visualized using a suitable substrate fox said enzyme.

45. The method as claimed in claim 43, wherein when the enzyme is .beta.-lactamase, the presence of the exported or secreted enzyme is visualized using A medium contain-ing ampicillin.