CA2124580C - Method for selecting recombinant microorganisms containing at their surface at least one molecule having enzymatic activity - Google Patents
Method for selecting recombinant microorganisms containing at their surface at least one molecule having enzymatic activity Download PDFInfo
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Abstract
A method for selecting recombinant micro-organisms of which the sur-face comprises at least one molecule having enzymatic activity, wherein the rec-ombinant micro-organisms are immobilized on a solid support by a covalent inhibitor of the active site of said molecule, which is connected to the solid sup-port,by an arm; the recombinant micro-organisms are recovered and all or part of their genome is isolated and cloned.
Description
F I L E . ~-f-~l~f~l 1 1 f , ~ ' ~ ° ' I : l ! l t...?
2 ~j[' T~3A[Q;;~.. ~.°, ~'_: i 1 PCT/BE92/00052 METHOD FOR SELECTING RECOMBTNANT MICROORGANTSMS
CONTAINING AT THEIR SURFACE AT LEAST ONE MOLECULE
HAVTNG ENZYMATTC ACTIVITY
Subiect of the invention The invention relates to a method for selecting recombinant microorganisms containing at their surface at least one molecule having enzymatic activity.
The invention also' extends to the means for implementing this method and to methods for obtaining p them: .
Prior state of -the art' and technolo ical back round formin t~xe lc~asi.s of the invention The discovery of enzymes having new properties (catalytic activity, new enhanced stability, altered 15 specificity, shifted pH profile and the Like) is of great interest for the improvement of biochemical processes.
Although very substantial progress has been achieved in theunderstanding of'the structure-activity relationship fog proteins (Atkins et al : , Current opinion 20 in structural biology (199Ia, 1, p. 611 to 623, the prediction of the type of modification to be introduced into a protein in order to alter its properties in a desired dir~ation remains difficult: , Consequently', a' strategy is a ually adopted in 25 which an existing enzyme, whose gene is mutated, is used as starting material. This mutagenesis i.s carried out i:n a random manner, thus creating a -asst population of mutated enzymes: Among these, it is hoped that some of them will,have the desired new properties (Cunningham et 3p al:, Protein Engineering (1987j, 1, p. 319 to 325 and Lehtovaara et al. Protein Engineering (1988, 2, p. 263 to 268 y..
xn order to increase the chances of success, it is necessary to test a very large number of mutants and to have available a'teohnique which makes it possible to.
select., within the largest possible population, the .
l. enzymes having the:appropriate properties.
l proteins haring new enzymatic properties can also be produced by resorting to the monoclonal antibody.
_ 2 -technique. In this case, the selection of the antibodies-enzymes (also called abzymes). having the appropriate v properties also necessitates an effective screening technique.
A technique for,deteotion of the mutated enzymes rahich is frequently used is based on the development of a sensitive test for the enzymatic activity. A search is then directly carried'out on a population of mutants plated on a Petri dish for those which produce the desired enzyme. 8owever; this-technique is laborious and pexmits 'only a limited number of clones, to be tested (Pcallack et ale, J. Am. Chem. Soc., 111, p. 5961-5962 (1989) and Huse et al., Science, 246. p~ 1275-1281 (1989))0 An advantageous alternative arises when the desired catalytic activity rnay be useful for the survival or for the development of the microorganism expressing the'corresponding gene.: It is sufficient, in this case, to exert a selection pressure on the medium for culturing the population of mutants in order to select the strains which produce the desired enzyme (Hermes et al., PNAS, USA; 87; p. 696-700 (1990) and Evnin et al., PNAS, USA, 87, p. 6659-6663 (1990))~
This technique has the disadvantage of not being applicable to all the molecules having enzyma~.ic act3.vity .
It is also known (Smith, Science (1985), 228,'p.
1315-1317, Parmley and Smith, Gene (1985), 76, 305-318, de la, Cruz et al.,- J. Hiol. Chem. (1988), 263, p. 4318-4322, Bass et al., Proteins (1990), 8, p. 309 to 314;
Cwirla et al., Proc: Nath. Acad. Sci: USA (1990), 87, p.
6378-6382, Devlin et al:, Science (1990), 249, 404-406, McCafferty et al., Nature (1990); 348, p. 552-554, Scott and Smith, Science (1930), 249, p. 386-390, Clackson et al:, Nature (191), 352, p. 624-628, Zowman et al., Biochemistry, (1991), 30, p: 10832-10838, J. McCafferty et a1.1 Prot. Engng (1991) 955-961, Kang et al., (1991) Proc. Natl: Acad. Sci. USA, vol:' 88, p. 4363-4366; Barbas et al. (1991), Proc: Natl. Acad. Sci. USA, vol. 88, p. ?978-7982; Roberts et al., Proc. Natl. Aced. Sci. USA
(lgg2j, 89, p. 2429-2433j to be able to expose certain foreign peptide sequences, in particular proteins, at the surface of "filamentous" phages and of phagemids.
However, the in vitro selection of phages which correctly expose active polypeptide sequences at their surface is not resolved either Various affinity chromatography techniques have been de~aribed: Among the affinity supports developed, several use immobilized streptavidin, or avidin, the latter being advantageously in monomeric form (EP-A-0,423,~38j. Phages with peptide sequences having an affinity for streptavidin have thus begn selected with the aid of agarose carrying this protein (Devlin et al., Science (1990jr 249e 404=406j.
However,; these techniques have the disadvantage that the selection, during affinity chromatography, of these phages is carried out on the basis of their ability to form a complex with peptide receptors or specific antibodies which indiscriminately bind active ox non-active molecules:
With' the aim of detecting specific microorganisms, various other' labeling techniques have been developed. Far ekample, certain microorganisms have been detected with the aid of bacteri:ophages which are specific ' for these hosts and ' labeled (WO-A-8, 804, 326 j .
Transducing particles of known host specificity and containing genes which alter the phenotype can he used for lab~Iing bacteria (WO-A-9,004,041j.
, The sensitivity to certain toxic substances which is associated ~ai.th the detection by bioluminescence encoded by a plasmid has also' been used to detect specific microorganisms.
However, none of these techniques permits the selection of microorganisms carrying catalytic peptides.
Moreover, covalent inhibitors, used as substrate by Certain enzymes, are also known for their capacity to inhibit all enzymatic reactions by binding covalently to the active site of the enzyme.
.., _~_ Some covalent inhibitors are also called "suicide" inhibitors (ATOR et al., The Enzyme, vol. 19, p. 214-282, edit. Sigman Bayer 3rd edition (1990)) because they bind irreversibly to the active site of the enzyme using the catalytic mechanism of the latter and thus render any reuse of the enzyme impossible.
It is also aCnown that it is possible to immobilize a substrate by first binding it to biotin and by placing the product obta3.ned in the presence of avidin bound to a water-insoluble support (JP-A-3,080,098). This immobilized substrate permits the easy determination of certain enzymatic activities, but cannot serve as chrome-tographic support to select microorganisms carrying catalytic peptides, Aims of the invention The invention: aims to develop a method for selecting'recombinant microorganisms containing at their surface at leasf. one molecule having enzymatic activity and which does not carry the disadvantages of the prior state of the art.
Tie invention aims i~ particular to develop a method which permits the detection and selection of recombinanfi microorganisms exposing at their surface molecules having enzymatic activity, which are performed on tl~e basis of the catalytic activity of these molecules rather than on'the basis of their complexing activity.
Chara~ter~ati.c features of the invention The invention gelates to a method for selecting recombinant microorganisms Containing at their surface at lest one molecule having enzymatic activity, in which the recombinant nni:craorganisms, preferably viruses such as filamentous pages ~r .1 phages or phagemids, are immobilized on a solid support by a covalent inhibitor of the active site' of the molecule having enzymatic acti.va.ty, connected to the solid support by an arm; the recombinant anicraorganisms or all or part of their genome are then recovered and all 'or part of their genome is isolated., The term "recombinant microorganism" is , , n ~,~.~~':~~~3 understaod to mean any macromolecular structure which satisfies the English definition of '°Genetic Display Package" (GDP), that is to say any macromolecular struc ture incorporating genetic material and containing a peptide sequence encoded by this genetic material.
This definition includes especially yeasts, bacteria, viruses, phages such as filamentous phages or phagemids.
The term "solid support" is understood to mean any solid structure on which the microorganisms can be temporarily immobilized.
Such a solid support can be made by a person skilled in the art according to the techniques known in the prior state of the art.
Thie support may consist of a chromatographic support, paramagnetic parta.cles, a polystyrene support or any other support on which complexing proteins such as avidin ox streptavidiri or-an antibody capable of binding the microorganism are immobilized:
Preferably, the molecules having enzymatic activity are chosen from the group consisting of enzymes, abzymes, catalytic pegtides or a mixture thereof.
~3ccording to a preferred embodiment of the method of the invention, prior to their'immobilization on the solid support, the recombinant microorganiazns are labe~.ed with the covalent inhibitor connected by an arm to a ligand which can be immobilized on the solid support.
According to another preferred embpdiment of the method of the invention, the microorganisms or alb. or part of ,heir genome are recovered by destruction of the microorga~aism or by detachment of the rnicroarganism from '-its solid support.
Th~.s detachment can be performed ~ according to 'several methodss'either by detaching the ligand from the solid support w~;th an eluting salu~ion, preferably with a solution at an acidic pH or by cleaving the arm connecting he covalent inhibitor to the solid support;
or' by cleaving the bond between he covalent inhibitor and the molecule having enzymatic activity or by ~~'~~,~::~~~
- _ 6 _ cleaving a peptide linkage between the molecule with enzymatic activity and the microorganism.
The cleavage of the peptide linkage between the molecule having enzymatic activity is preferably performed with a specific protease such as factor 1UA or collagenase; ayanogen bromide which cleaves a peptide linkage containing a methionine or hydroxylamine according [sic] to a peptide linkage containing the peptide ~sparagine--glycine have also been used.
1p Such oleavage makes it possible to advantageously increase the selectivity of the method according to the in~rention .
Indeed, the suicide inhibitor in the method according to the invention is capable of also binding to the microorganism at sites other than the active site of the molecule having enzymatic activity.
Hy cleaving the specific peptide linkage existing between the molecule having enzymatic activity and the microorganism,.only the m.icroorganis~ containing'at its surface an active enzymatic molecule will be recovered during the: elution.:
Furthermore, this method o selection can be applied for the detection and selection of microorganisms which expose at their surface, on a native or recombinant protein of the microorganism, one or more specific peptides cagoble of inducing a cellular or hura~oral immune response.
Indeed, it was observed that a mere antibody-antigen recognition of such peptides did not p~rrinit 'selection of the peptides most 'capable of eliciting. a cellular or humoral immune response (6nletzel ( 1991' , Prot.
Eng., vol 4, p: 371-37:4).
Consequently, a cleavage carried out according to the invention at-the level of the peptide linkage existing between the said peptide and the native molecule of the miaroar~gan.ism also makes it possible to select in a specific manner the ;peptide(s) must capable of eliciting a cellular ax humoral reaction.
In addition, all or .past of the genome of the f L
microorganisms can also be recovered by destroying the ni.croorganism or by recovering all or part of its genome.
This genome is then isolated, amplified (far example by PCR) and cloned.
The invention also relates to a marker consisting of a covalent, preferably irreversible, inhibitor of the active site of a molecule having enzymatic activity, connected by an arm to a ligand which can be immobilized on a solid support. .
1(~ Preferably, the ligand is a biotin or an imino-biotin.
Advantageously, the arm contains a cleavable chemical functional group, preferably a disulfide func-tional group.
According to a preferred embodiment of the marker according to the invention, the covalent inhibitor is an analog of the specific transition state of the enzymatic reaction performed by he molecule having enzymatic activity Another aspect of the invention relates to a method for preparing the marker according to the inven ion,.in which a covalent inhibitor of the active site of a molecule having en2ymati.c activity is coupled by an arm to a ligand which can be immobilized an a solid s~~pc~rt.
~5 The invention also relates to a solid support~in which a covale~xt inhibitor of the active site of a molecule; with enzymatic activity is connected by an arm to a solid support.
Advantageous3.y, the covalent inhibitor connected 30; to the solid support consists of the marker according to the invention.
According to a preferred embodiment of the inven 'tion, tJhe arm is connected to the solid support by an ester linkage or contains a vicinal diol.
35 A final aspect of the invention relates to the method for preparing the solid support according to the in~rention in which:a covalent inhibitor of the active site of a molecule having enzymatic activity is connected by an'arm to a solid support.
Hrief descri~ation of the firnares Figure 1 .represents the nucleotide sequence of the ~i-laatamase--encoding gene of the plasmid r pH~322.
Figure 2 schematically represents the steps of the method for inserting the gene encoding the mature (3-lactamase into the phage fdDOGl.
0 Figure 3 ~'ep~esents the nucleotide sequences of the primers comple-mentary to the gene encoding (~-lactamase:
Figures Q to 9' schematically represent the 15 method of synthesis of the markers 'according to the ~;nvention Eigur~ 10 schematically represents the binding of a microorganisan onto a 20 ~~lid support, by the marker according to the invention, as well as the possible cleavage sites which snake it possible to recover the said microorganism.
25 Descri ti.on of a referred -eaebadiment of the inventa.o~x Figure 10 schematically represents a recombinant ..
microorganism ( 1 ) containing at its surface a molecule having enzymatic activity ( 2 ) , immobilized on a solid support (3)- containing a complexing'protein (9) such as 30; avidin or streptavidin, by a marker consisting of a covalent inhibitor,(5) of the active site of the molecule having enzymatic activity (2),,an a~ (S) and a ligand ( 7 j which can be ~:mmobili~ed by the complexing protein (r~) on the solid support (3).
35 The microorganisms or all or part of their genome can be recovered:
by detaching the ligand (7) from the solid support with ~n eluting solutian, such as a so7.ution at a pH
of the order of 4 (~);
.- by cleaving the arm (6) of the marker (B);
- by cleaving the bond between the inhibitor (5) and the molecule having enzy~aatic activity (2) (C);
- by cleaving the peptide linkage between the microorganism (1) and the molecule having enzymatic activity (2) (D)~
- by destroying the microorganism (1) and by recovering all or part of its genome (E).
The method will be described in greater detail with ref~zence t,o the acc~ampanying figures, in the following examples.
Example Is Construction and selection of phages carrying active (3-lactamase (fd-bla) The viruses having at their surface molecules with enzymatl.c activity are constructed from "f~.lamentous' phages.
The filamentous phages such as M13 or fd are bacterial viruses iahich, in tlae free form, contain a 6. ~1 kb single-~trarrded circular DN~1 endapsulated in a 2~ - protein en~relope era the foam of a very long cylinder consisting of five proteins (Heck'& 2ink, Gene (1981), 16 ~ p,: 35; to 5g; Giant et al . , J. Hiol. Chem. ( 1981 ) , 25:6, p: 539 to 596; Isopez & Webster, Virology (1983), 127, p. 177 to 193.
Among these, the product of the gene III (pIII) is a complex protein, present in an amount of 3 to 5 copies, which closes ore of the ends of the virion and is composed of three parts: a'signal peptide which is used for exporting: the protein to the ~er~:plasm of the 30, bacterium and 'is cleared during excretion; a globular part necessary for infection which recognizes the F-pilus of the E. coli target cell; a hydrophobic, transmembrane part during the virus morphogenesis phase which subsequently ensures anchorage of pIII in the particle ' (J. Armstrong ~t al., FENS T,etters (1981), 135, p. 167-172; C:W~ Gray et al., J. Mol. Biol. (1981) 146 p. 621 to 627; G. Glasez-Wuttke et al., BBA (1989) 985, p. 239 to 2~7).
Following their work on the role and function of /~ i~ t-.A ~t ,~ ~ a the protein px~I in the physiology of the phage fd, Smith and Crissman (Virology (1984), 132, p. 445 to 455) showed that it was possible to introduce, by recombinant DtdA
methods, a foreign peptide g~~~E~~~e inside the sequence ofi protein pTTfi ( Scienoe ( 19 ~~ ~ ~ p . 13'15 ts~ i31? y or between the signal popfi : . and the sequence of the mature protein (Gene (1988), 73, p. 305 to 328). .'his insertion dogs not prevent the viability of the phdge and the introduced sequence is accessible.
1U The phage°enzyn~;: fd-tet-~°lactamase ( fd-bla is constructed from the pt~age fdDOGl (Clackson et al, Nature, 3~2, p. 62'4 to 628 (1991) in which the sequence encoding the mutated ~-lactamase has been introduced.
This phage is derived from the phage fd-tet (Heck et al : , Nucl. Acids. Res. , 5, p. 4495 to 4503 (1978 ) ) , in which a polylinker is inserted between the sequence enco~3ing the signal peptide of protein III (pTII) and the sequence encoding the mature pIII; in this manner, the ~i-~actamase will farm, avith pITI, a Fusion protein. The phage fd-tet is itself derived from the phage fd (Zacher et alo, Gene, _9 p. 127 to 140 (1980)) by insertion of the tetracycline resistance gene of the transposon Tn 10, The phage fdDOGI comprises two restriction sites (Notl and-Apal.l) constructed by mutations in the region '.25 encoding the protein pIII, between the signal peptide and the globular part.
The ph~ge fdDO~l also Comprises the tetracycline gene inserted in, the region of he origin of replication o~:rthe ~hage and permitting a propagation of the phage as 3U plasmid.
isolation of the~nucleotide se ence encodin -lactamase The ~°lactamase gene of the plasmid pBR322 of H: coli (whoa~ sequence is,.givey by Sutcliffe, PNAS USA, 7~5, p. 3737 to 3741 (1987) and in figure 1) was isolated 35 from the phagemi.d ~Hluescript SIKt~.
Given that the cloning into fdDOGl will use the ApaZ1 site and that the sequence of the ~i°lactamase Contains such ~ site, the latter should first be removed b~ site°directed mutagenesis. This mutation conserves the protein sequence.
The ph~gemid pBluescript S/K+~ (from the company Stratag~ne~, sold by Ozyme~ j , is mutated according to the method -of Eckstein (Nucl. Acids Res., 14, p. 9679 to 9698 (1986] with the aid of a kit marketed by the company Amersham'~ .
After in vitro mutagenesis, the double strand of the mutated phagemid pHluescript S/K+~ (pBluescript-Mj is used to transform the strain TG1 of E. coli (Gibson, Studi.e~ on the Epstein-Harr virus genome, PhD Thesis, Camb~~:dge University England (1984).
The transformed cells are plated on LB-Amp mediuan (Sambrook et al., Molecular Cloning, A laboratory manual, second edition, Cold Spring Harbor Laboratory Press (1989) Appendice A).
Colonies are recovered and cultured in LB-Amp medium. The single--stranded form of pBluescript-M is produced by addition of the helper phage 8408 to the medium (Russet et al., 4~5, p. 333 to 338 (1986jj~
The strand gHluescript-M is then sequenced acdosding to the Sanger method (PNAS USA, 74, p. 5463 to 5467 (19?7)) with the aid of the primer 5'-TTTA~AAGTGGCCATCATTGGA centered at the position encoding S~r 68 in order to verify that, in the chosen claneg, the mutation is indeed introduced.
Clot~iz~ of --lactamase into the ha a gdDOGl The soqcaence of the gene encod.i.ng the mature p-lactamase is ~.nserted into the phage fdDOG~, between the sequence encoding -the signal peptide of pIII and that encoding:the mature pIII. The Gloving sites ApaL1 and Notl are used according to the scheme given in figure 2.
To this effect, the ~-lactamase gene is recovered from the phagemid pBluoscript-M with introduction of the aPProPriate restriction sites by PCR.
The grimers used, shown in Figure 3, contain the foTlawing characteristics:
The primer in 5' is a 30mer complementary over 18 bases of the mature ~-lactamase gene starting from the second colon (encoding Pro 2j. The 5' extension (12 .8.. tJ
bases) introduces the ApaLl site and causes a mutation which results in the replacement of His 1 by Gln 1. This mutation is introduced in order to avoid the phages carrying a ~,-lactamase having the terminal amino sequence His-Pro-Gln capable of exhibiting an affinity for avidin or streptavidin (Devlin et al. Science, 249, p. 404 to 406 (1990)) subsequently used for their selection.
The primer in 3' is a 47mer complementary over 2I
bases from 'the end of the ~-lactamase gene. The e~ctension replaces the stop codon by a codon encoding a glycine, introduces four colons between the gene encoding the ~3-lactamase and the gent encoding the protein pIII, as well as the Notl restriction site for the cloning. These faur additional amino acids constitute a~ potential cleavage site between the two proteins for factor Xa. The protein pIII provides, indeed, the capacity for infection of the phage~ (Goldsmith et al., Eiochemistry, 16, p.
2686-2694 (1977). While the presence of a protein as voluminous as (~-lactama.se affects the capacity for infection, an incubation with factor Xa makes it possible to restore the latter:
7Cn addition, thia site constitutes an additional cleavage point for recovering the miaroo~ganisms.
The phage fdDOGl is digested for 18 hours with ApaLl and Notl anti purified on agarose gel. Moreover, the sequence of the phag~mid pBluesc~ipt-M is amplified with the primer9 described above using 34 PCR cycles (1 min.
at gSoCr 1 min. at 60°C and 2:5 min. at 72°C) in the presence of Taqpolymerasea (Saiki et al., Science, 239, p, 487 (,1988). ,After gel purification, the product is digested with ApaLl and Notl for 18h and repurified; then 1.5 ~g of digested fdDOGl and 250 ng of the ~i-lactamase gene are mixed in 50 girl with ~ units of T4 DNA ligase (Gibco-HRL~) and maintained avernight at 16°C. The purified ligation mixture is used to transform the strain TG1 of E. Coli.
The cells are plated on LB-tet and LB-Amp medium (Sambrook et al.) where it is observed that the presence of the phage provides the resistance to the antibiotic.
'~I
Colonies are then cultured in 2xYT-Tet medium or in 2xYT-Amp medium in order to prepare the phage fd-bla.
Instead of constructing phages-enzymes by mani pulation of the protein pIII, it is also possible to construct a fusion protein with the protein pVIII (1) which is a small protein of 5.2 kd constituting the cylinder body o~ the ghage fd and which is normally present in 2700 copies (Newman et al., J. Mol. Biol 0.977), 116, p: 593 to 606). If the size of the DNA
~p molecule is modified, the number of copies of the protein pVIII can be adjusted in order to include the said DNA
molecule.
This variant is achieved with a phagemid in order to b~ able to control the number of copies of the fusion protein Kang et al:, (1991) Proc. Natl. Acad. Sci. USA, vol. 88, p. 4363-~1~66.
Characterization of the phade carrYinct the active ~--lactamase ~(fci-blal The phage thus constructed is characterized in 'the fdlloraing manner: The fact that the inf~:ction by the phage confers on the cells a resistance to ampicillin is first demonstrated.;Then it is shown. directly that the phage itself exhibits an actauity for hydrolyzing a y-lactam, nitrocefine. The fact that the enzymatic aativi.ty . is not pxesent in the ultrafiltrate after filtration on a membrane which alle~ws the (3-lactamase to pass through already indicates that the enzyme is bound to the phac~e. Finally, the phage proteins are analyzed by gel electrophoresis and Western Hlotting and it is shown that the protein gIII is present in the form of a fusion protein with the ~3~-lactamase. This confirms that the construction is indeed correct and that, during the marphog~nesis of the virus, there is no proteolytic cleavage ~rhich would result in the detachment of the (i-lactamase from the phage:
Hy infection of cells of E. Coli TG1 with this phage and plating ors Z~-Amp medium, ampicillin-resistant c~lonies are obtained. The p=lac~amase activity is dgrectly detected in the Petri dish by a nitrocefine test '~,~'~ ~ ~~~
(Pliickthun et al., J. Biol. Chem., 262, p. 3951 to 3957 (19~3'~)). Likewise, the phage isolated from the culture medium after centrifugation of the cells, precipitated with 4 ~ PEG, 0.5M NaCI (final) and resuspended in 50 mM
tris buffer, 150 mM I~1'aCi at pH 7.5, at a concentration of the order of l0bz transducing units (tu) per ml, hydrolyzes the nitrocefine.
The specific activity of the enzyme carried by the phages was identical to that of the ~3-lactamase RTEM
ZO in solution (Boehringer). The measurement of the activity was also performed:af~ter ultrafiltration'of the phages on Centri/por membrane with an MW cut-off of 100,000 which allows commercial p-lactamase RTEM to pass through. bees than 5 ~ of the ~i-lactamase activity was detected in the ultrafiltrate, which indicates that the enzyme remains attached to the phage.
Viruses fd=tet arid fd-bla are prepared and cancentrated (1Q'a tu/ml). 20 ~1 of each solu~tian are loaded onto a x.2:5 ~ SDS-polyacrylamide gel and analyzed 20~ (miniprotean~ Il from Hiorad~). By S~Testern Blotting, the proteins are transferred onto nitrocellulose (minitraaashlot~ from Hiorad~) and the revealing is perf~ra~ed with an anti-protein III rabbit ant~.body, followed by a goat antibody against a rabbit antibody coupled to IiltP ( horseradish peroxidase ) . The chromophoric substrate o~ FiRP reveals bands o~ apparent M~1 of 70 kd , and 105 kd for fd-tet and fd-°bl,a respectively. The rev~eala.aag taas also performed with an anti-~°-lactamase antibody; a band was fpund only for fd-bla at the position.e~cpected for the fusion protein.
Example II: Construction and characterization of phages carrying an inactive ~-lactamase ( fd-bla-) In order to test the efficiency of the selection method according to the invention, phages carrying an inactive ~i-lactamase (fd-bla') are constructed and characterized.
The phagemid pBluescript-M is subjected to a mutagenesis in which the colon encoding the essential ~~.~~:.~~~
serine 68 is muted to alanine in order to inactivate the enZyIIle a The mutated gene is recovered according to the same technique as that used for the active ~i-lactamase (PCR amplification with introduction of restriction sites (cf. figure 2)). The phage carrying inactive p-lactamase (fd-bla-) is characterized by restriction fragment analysis, camplete sequencing of the Hla gene, detention of enzymatic ac~.ivi.ty and analysis of the proteins.
1p The S68A mutation is introduced according to the same procedure as the mutation of the Apal~l site. The transformed cells are dated on X-Gal medium (Horwitz et al., J. Med. Chem., 7, p. 574 (1964)) without ampicillin.
The blue colonies are cultured in 2xYT medium and the single-stranded form is produced by addition of the helper phage 8408. The mutation is sequenced by the S~.nger method with the aid of the primer 5' -TA~TGTATGCGGCGACC cor~glementary to the region encoding G90-Y95.
After amplification of the gene by PCR, cloning, transfc~rnbation and platine~ on tet mediuan as performed for the active ~i-lactaanase, colonies are recovered and cultured in 2xYT~-tet u~edium. The double-stranded DNA of the phage is isolated anti digested with the restriction nucleases HamHl and He~R1. The- digestion products are separated on agarosethe transforr~ants show 2 fragments of 9.7 kbp and 5.5 khp, in agreement with what is expected. The complete sequencing Qf the mutated Hla gene is performed with the primers 5'-TAGTGTATGCGGCGACC, 5'-Ajlacuna]GCGA~TTACjlacuna]G, 5'-CCAGCCAGCCGGAAG and 5'-TGCTAAACAACTTTC located respsetive3y in the regions encoding G90-Y65, H156-L160, 2223-W227 of the ~-lactamase and E5-A9 of the mature protein pIIT). It thus is verified that the PCR amplification did not introduce mutations other than that aimed at inactivating the active site.
Tha presence of a ~-lactamase activity is then detected by performing, on the supernatant of a culture of phage fd-bla-, a ni.trocefine test as described above.
~~2~~.~ ~~~
16 _ In agreement with what is expected, no activity is observed.
Finally, analyses of the phage coat proteins by denaturing gel electrophoresis, Western Blotting and immunological detection is performed as for the phage fd-bla. The fusion protein PIIT-p-LACTAMASE is correctly identified.
Example III: Synthesis of the active ~i-lactamase marker provided with a small-sized arm between auioide inhibitor and ligand 'rhe phagee whose DNA contains the active (3-lactam~se gene and which carry the corresponding enzyme attached to the soot protein;pTII will be selected by a reaction with the markex according to the invention. This marker consists of a bifunctional molecule which contains, at one end, a covalent inhibitor of the ~-lac amp a and, at the other, a lig~nd, in this case biotin, which can be easily immobilized on a solid support:
The sdicide inhibitor module consists of a penam sulfone (see Fisher et al., Biochemistry (1981), 20, 2?26~-2731:; Metes et al., FENS I,et. (1982), 143, 265-267, Cl.ark~ et al. ; ( 1,983 ) , 748, 389-3g7, Dmitrienko et al . , Hioorg. Chem. (1985), 13; 34-4'6) fun~tionalized'so as.to allow its attachment Day an arm to a ligan~l ar a support.
Inei.no-biotl.n can also be advantageously used because its affinity for streptavidln can be considerably reduced by decreasing, the pH of the solution. This will give:us an additional possibility to isolate the phages 30, fxom the supp~rt after immobilization.
These tan entities are connected by an arm of sufficient length to allow the interaction of the ligand carried by the marker with the immobi.Iized protein.
The scheme for synthesis of the bifunctional molecule is given in figures 4 to:6. Most of the steps of this synthesis are described in he chemical literature ox~ involve coventional procedures. The molecules are characterized by TR spectroscopy and 1H-NMR.
Sodium p-nitrobenzyloxycarbonyl-2-aminoethane-suTfonate (3) is prepared by addition of two equivalents of p-nitrobenzyl chloroformate to dissolved tsarina (2) one equivalent [sic] of 1N NaOH at 0°C, continuously neutralizing the medium by addition of NaOH, under the Schotten-Haumann conditions. After extraction of the p-nitrobenzyl alcohol with ethyl acetate, the product is freeze-dried. The yield was 66 ~.
The sulfonate {3) was converted to sulfonyl chloride {~j with phosphorous oxychloride according to the method of Fujita (Synthesis, 423-424 (1982)). Hy washing the oil obtained with dry ether, the product was obtained in the form of a white powder with a yield of 97 ~:
In _parallel, 6-aminopenicillanic acid (5) was converted to its methoxymethyl ester (6) according to the method of Manhas et al: (Synthesis, 549-552 (1983)) with the aid of m~thox~rm~thyl chloride after temporary protection with ethyl acetoacetate.
The acid chloride (~) is coupled with the 6-aminoperaicilla.nic acid ester ( 5 ) in dichloromethane in the presence ~f tri~thylamine. The product (7) purified on a silica column {eluantc ethyl acetate-dichloromethane 80 ~ 20 ) , was obtainsc~ with a yield of S0 ~ .
This product was oxidized into a sulfone (8) 2.5 according to the method of Johnson et al > ( J. Org. Cher~i. , 28, 1927-28 ( 1963 ) ) ; 'the product obtained was washed with ether to give a solid white [sic] which was purified on silica (eluants ethyl acetate-dichloromethane 50:50j. The Meld was 75 ~.
On deprotection of the amine functional group by hydrogenation on palladium/carbon according to a conven-tiona3. method, the product (9) was obtained which was coupled directly with sulfosuccinimidyl 2-(biotinamido)-ethyl-1,3--dithioprapionate (10) (marketed by PTERCE~), to 3,5 gave the marker precursor (1-11) which was purified by chromatography on silica {eluant: ethyl acetate-dichloromethane SOt50)a The yield was 25 The protection of the carboxylic functional group in position 3 of the penam ring in (11) was removed with ' y~'.~:~.3ii t .1.
the aid of magnesium bromide in dichloromethane according to the method of Rim et al. (Tetrahedron Letters, 32, 3099- °3100(1991)). The crude product recovered (12) after evaporation of the solvent was used directly to inhibit the enzyme and, to label the phages carrying active enzyme .
Exame,le IV: Immobil.lzation of the phages carrying active (3-lactamase ( fd-bla ) The use of a covalent marker irreversibly binding the active site of the enzyme and making impossible any reuse of the enzyme, dyes not constitute a disadvantage in the method for selecting the enzyme. Tndeed, in this method the desire is only to select and recover the genetic sequence encoding the act~.ve molecule.
Ths optimum conditions for inhibition, by the hifunctional marker, of the phages carrying the active p~-lactaan~se arm first determined. To this end, the rate of disappearance of the ~i-1'actamas~ activity of the phages is measured, by the nitrocefine~test, as a func-Lion of time after addition of the inhibitor. phages are then incubated i,n the presence of the marker.
Then the reaction product is passed through an .sgarose-coupled streptavidin column. The phages are then eluted by cleaving the disulfide functional group of the 25' marker. They are used to'reinfeat an E. coli. culture.
E'xom this culture, the gene encoding the active p-lac~amase can be easily recovered.
A culture of bacteria infected with the fd-bla phag~s is pregared in a 2xYT medium containing 15 ~g per ml of tetracycline and incubated, with stirring, at 37°C
for 16 hours. After centrifugation of the culture for 10 minutes at 10000 rpm .(revolutions per minutej, the supernatant is recovered. The phages are precipitated by additibn of 1/5 , volr~me of 20 ~ polyethylene glycol solution, at 2.5M NaCI. After incubation for 1 hour at ~°C end centrifugation, the phages are resuspended in a 50 mM acetate buffer at pH 5:0.
The marker ( 1-2 ) in solution i.n ~M~O, at a concentration oø l mg/25 ~1, was added at a final ' v;~<~cR~
concentration bf 0.25 mNt to the solution containing the phages (approximately 10" tu/mlj and 1 $ BSA. The inhibi-tion is continued for 30 minutes.
Moreover, a suspension of 250 gel of agarose e~rc~p~avidin (PIEP.CE~j is washed with 20 ml of PHS buffer containing 1 ~ HSA (Bovine Serum Albumin, Sigmaj. 0.3 ml of the solution, containing 1.25 x 109 phages, was loaded onto the column, washed with 6 ml of PBS/1 ~ BSA and 6 ml of PHS. The phages axe then eluted with 1 ml of a 500 mM
dithiothreitol solution in PHS buffer. One aliquot of recovered ghage~ was incubated, after change of buffer, with 10 ml of E, cpli TG1 cells in exponential growth at 37pC. The recovery of the phages was demonstrated by plating various dilutions on I,B-tet medium and counting the colonies. 1.0 x 10° phages carrying active ~i-lactamase, equivalent to 14.4 ppm, were recovered in this manner:
Excmple Vs Tmmobilization oø phages carrying inactive p-lactamase (fd-bla'j ~, . 05 x 10' phagss ( df-bla' j contained in A . 3 mi of acetate buffer at pH 5 were plcced in the presence of the marker (12j according to the invention and the reaction product was passed through an agarc~se-coupled strep-tcvidin column and eluted according to a procedure identical to that described in example 4. 2.0 x 10' phages carrying inactive ~-lactamase, equivalent to 1.9 PPm. wire recovered'in ,this case. .
Comparison of the results of examples IV and V
shows that the active phages are retained better than the 30, inaative.Pha9es by ~ factor of 8.
Example VIc Enrichment, caith active phages, of a mixture containing active (fd-blaj and inactive' ( fd-bla' ) phages a elution with dithiothreitol An experiment of the same type was carried out with a mixture of active phages and inac~ti.ve phages to determine directly the active phage enrichment factor.
A mixture containing F . 4 x 109 ,active phages and 1.4 x 10'° inactivd phages in 0.3 m1 of acdtic buffer at s' a v j .a~ .'; '' pH 5 in the presence of 1 $ BSA was planed in the presence of the marker (12) at.a final concentration of 0.25 mM. Excess marker was removed by ultrafiltration on a unit Ultrafree 30, 000 from Millipore . The solution was Loaded onto streptavidin-agarose. After washing the column with 5 ml of PBS/1 ~ BSA, the phages were eluted with 500 mM dithiothreital. An aliquot of eluted phages was plated at various dilutions on LH-tet dishes; the co?~onies were thus counted: A total of 1.3 x 106 phages were recovered in this manner. The number of phages carrying active ~i-lactamase was determined by directly measuring the ~i-lactamase activity of the colonies by the nitrocefine plate test as described above. This number was also verified by plating an aliquot of the eluate on I5 LB-Amp dishes. The number of eluted active phages was 1.1 x .106. The enrichment factor obtained was therefore 5.5.
Exa3a_p1e V'rI: Enrichment, with active phages, of a mixture containing active (fd-bla) and inactive (fd-bla') phages: elution with factor Xa ~,n experiment of the same type was carried out with a mixture of active phages and inactive phages to dixectly determine the enrichment factor; the elution was 25 this time carried out with factor Xa cleaving a peptide linkage between the (i-lactamase and the protein ILT.
mixture containing 2.E x 10'° active phages and 2.2 x 1Q'° inactive phagds in 1.5 ml of acetic buffer 2 ~
BSA was placed in the presence of the marker (12) at a 30 final concentration of 0.75 mM, passed over streptavidin agar~se. After washing the column with 80 ml of PBS, 2 ml of 0.1 M TR1CS buffer at pH 7 containing 0.'02 ~ I~aN3 and 2 ml caf Tris buffer at pH 7; 1 ml of a solution containing 10 ~g of factor Xa (Boeringher) was added to 35 the suspension and it was allowed to stand overnight at room temperature, with gentle stirring. After centrifug~ti.on, the supernatant was analyzed by plating at various dilutions on LH-tet dishes; the colonies were thus counted. A total of 6 x 105 phages were recovered in f ~.~~-'_3~~
this manner. The number of eluted active phages was 5.7 x 105. The enrichment factor obtained was therefore 15.
Example 'IYII: Enrichment, with active phages, of a mixture containing active (fd-blaj and inactive ( fd-bla' j phages :
preimmobilization of the marker Another experiment was carried out with a mixture of active ph~ges and inactive phages, but with prior immobilization of the marker on the streptavidin-agarose column.
250 Nl of streptavidin-agarose suspension are incubated for one hour in an acetic buffer at pB 5 in the presence of l ~ HSA~ after washing wl.tta 2.5 ml of acetic buffer without BSA, 2 ~1 of a 50 mM solution of marker ( 12 ) in DM~O axe added: After 1 minute and 5 cycles of washing and centrifugation with, each time, 1 ml of acetic buffer, 0.8 ml of phages in an acetic buffer 1 ~ BSA containing 9 ~ 6 x 101° active phages and 5 x 10'0 inactive phages were added to the suspension with gentle stixr~.ng, ov~rraight at 4°C. After, washing with 20 ml. of PBS/1 ~ BSA and 50 ml of solution] 0.1 ~ Tween 20 in PBS, the ph~ges ~ ase eluted with factor Xa. and counted as in example VT. 1.15 x lOs active phases and 1.85 x 105 inactive phages were recovered, in this manner. the enrichment factor obtained was therefore 3.2.
Expose gX: Enrichment, with active phages, of a mixtuxe containing active (fd-blaj and inactive ( fd-bla- ) phages : elution by hydrolysis of the aryl-enzyme linkage An experiment similar to that of example VIII was carried out with a mixture of active phages and inactive phages to verify the possibility'of recovering phages by cleaving the linkage between the ~-lactamase and the suicide inhibitor, namely the ester linkage of 'the aryl enzyme. This cleavage was carried out by hydrolysis.
A mixture containing 2.9 x 10'° active phages and 1.05 x 10'° inactive phages wire [sic] loaded onto a column where the marker had been immobilized. After extensive washing as in example ~T11I, the suspension was allowed to stand overnight at room temperature and the supernatant from a centrifugation was analyzed. 1.4 x 105 active phages and 2.5 x 105 inactive phages were recovered in this manner. The enrichment factor obtained was therefore 2.1.
In the examples mentioned, the percentage of immobilized phages is low because the size of the arm between the immobilizable ligand and the inhibitor is quite small. In order to avoid this disadvantage, another marker having a larger-sized arm was prepared.
Example X: Synthesis of a marker of active (i-ldctamase provided with a large-sized arm between suicide inhibitor and ligand Carbobenzyloxy-2-(2-ami.noethoxyjethanol~ (15) ( figure 7 j was prepared by addition of 1 equivalent of benzyl chloraformate (13j to 2-(2~-aminoethoxy)ethanol (1~4) in water saturated by bicarbonate. The product was extracted' in organic phase and purified on a silica column ( el:xant : ethyl acetate-dichloromethane 50 : 50 j . The yield was 5g ~:
Ethyl vinyl-sulfQnate (17), has prepared'from 2-chloroethane-sulfonyl chloride (16j according to the , method of Whitmore and handau (J. Am. Chem. Soc., 68, p.
1'i97-1798 (1946j). It eaas purified bY horizontal distil-lation at 8g°C under 0.6 mm of mercury to give a colorless o~.l (Yield 45 ~j. One equivalent of the protected amino alcohol (1~5j was' added onto (17) in dry a~etonaarile to which finely ground potassium bicarbonate was added After heating, for 3 days at 80°C, filtration and evaporation of the solvent, the product (18j was purified on silica (eluant: ethyl acetate-dichlorometh~ne 50:50). ThE yield was 60 ~: The sul.fone functional group was deprotected according to the method of Tipson et al, (J. Org. Chern., 12, 133-137 (1947)) to give, after 24 hours o~ reaction, ( 1-9 ) in the form of a white powder after washing with hexane (yield 86 ~). This product was converted to an acid chloride (20) according to the method of Fujita (Synthesis, 423-424 (i1982j).
The acid chloride (20) (figure 7) was coupled directly with the ester of of [sic] 6-aminopenicillanic acid (6) in dichloromethane in the presence of triethylamine -for one hour. The product obtained (21), pur.ifi.ed on silica (eluant: ethyl acetate-dichloromethane 50:50) was obtained with a yield of 34 $. It was oxidized into a sulfone ( 22 ) according to the method of Jotmson et al. (J. Org. Ohem., 28, 1927-28 (1963)), the product obtained was washed with ether to give a white solid which was purified on silica (eluant: ethyl acetate-dichloromethane 50:50). The yield was 72 ~.
pn deprotection of the amine functional group by hydrogenation on palladiumlcarbon in ethanol according to the conventional anethod, the product (23) (figure 8) was obtained which was coupled directly with sulfosuccinimidyl 2-(biotinamido)ethyl-1,3 dithiopropionate (10) (marketed by PIERCE~) in order to obta~:n the marker precursor ( 24 ) which was purified by chr~matography on silica (eluant: ethyl acetate dichloromethan~ 50:50). The yield was 13 ~.
The protection of the carboxylic functional group in positian-3 of the penam ring in (2~,4) was removed with the aid of magnesium bromide in dichloromethane according to the'method of lKim et al. (Tetrahedron Letters, 32, 3099-310U (1991)). , .
Exa~ngle xL: Enrichment, with active phages, of a gn~.xture containing active ( fd-bla ) and inactive (fd-bla') phages: labeling with the inhibitor (24), elution with factor Xa An enrichment experiment was carried out with labeling with the inhibitor (24) of a mixture of active phages and inactive phages in order to determine the-effect o.f the size of the arm connecting the biotin 2igand with the suicide inhibitor module.
A mixture containing l.2 x 10'° active phages and 3.3 x 10'° inactive phages in 1.5 ml of acetic buffer 1~
BSA was placed in the presence of the marker at a final concentration of 0.75 mM, passed over streptavidinagarose. After washing the column, elution as described in example VTT and analysis, 6.8 x 106 activo phages and 1.9 x 10~ inactive phages were found. The.
enrichment factor obtained was therefore 90.
EXA~ipLE KIT : synthesis of a second active ~9-lactaxnase marker provided with a large-sized arm between suicide inhibitor az~d ligand In order to have a general method for the synthesis of a marker provided with a large-sized arm between the ligand and the inhibiting head, a precursor carrying an activated ester functional group and there-fQre capable of being coupled to numerous inhibitors and containing itself a large-si.zed arm waa prepared. It was coupled to the suicide inhibitor of the (i-lactamase.
1,5 The activated ester of the N-hydroxysucci.nimide with biotin-N-e-aminocaproic (26j was prepared as described (Wilchek and Bayer, Methods Enz. (1990), .~04, 123-160) , It was dissolved in DMF and added to a solution of 3 equivalents of cystamine (26) [sic] in DMF (figure 9j.
The crude product, isolated by precipitation with ether (10 times the volume ~f the so2ution) was purified on ~ Sepharose ~ (Pharmacia~ j coluann equilibrated with 1'ythl.um chloride, using as eluant 25 ~ ethanol. The addition product ( 20 j caws eluted with 50 mM LriCl . The solution was freeze-dried: The yield was 79 ~.
Three equivalents of glutaric anhydride (29j, dissolved in acetonitrile, were added in portions to the purified prodr~et (containing 7Gi:CI) dissolved at a 30' concentration o~ the arde~ of 0.01 M in buffered aqueous medium { K~iCO,-KzCO" 5 0 / 5 0 , 10 mM at pI3 9 . 6 j containing 10 ~k ethar~ol. The acid obtained (30.j was precipitated by acidification with 1N HC1 up to pH 2. The product was filtered and dried, the yield was 43 ~.
The acid (3a) was converted .into an activated ester under standard conditions by coupling with Id-hydroxysuccinimide' (31j with the aid of c~icyclohexylcarbodii.mide (DCCj in the DMF. After 72 hours, the reaction.medium was precipitated with dry - 2~ -ether. The precipitate was dried in a desiccator (yield:
B? ~D, this product (32) was used as sucks for the coupling reaction with the deprotected ester of 6-aminopenicillanic acid (6) in dry ethanol.
E_xampl~ XI~~e Enrichment, with active phages, of a mixture containing active (fd-bia) and inactive ( fd-'bla' ) phages : immobilization on paramagnetic particles.
.An experiment similar to experiment VI was I0 carried out, the only di~fesenae being that the streptavidin is carried by paramagnetic particles. The washes are carried out by resuspension in fresh buffer.
Tlxe elutioh of the ~.mmobilized phages is carried out with 50 mM dithiothrei.tol; The enrichment factor is determined as in example V'I.
Ea~ample XIVa Enrichment, with acti~re phages, of a mixture aontaini-~g active (fd-bla) and inactive ('fd-bla'j phages: immobilization by "panni.ng't ire a polystyrene tube 2~ An' experiment similar to experiment VII is carried out, the only difference berg that the streptavidin is immobilized by "panning" at the surface of a polystyrene .tube . following a technique conventionally used for EL~ISA tests. The washes are 25 carried out by resuspensic~n in-fresh buffer. The elution of the iman~bilized phages is carried out with factor Xa.
The enrichment f aotor i.s detern2ined a~ in example III I .
Given that, in'examples XIII and XIV, the washes can be carried out more rapidly than in the examples 30 using the chromatographic technique and the phages less easily entrapped here than in a gel, these variants of the enrichment experianents are more suitable for the observation of high enrichmea~t factors Example ' XIIL: ( i.c ] Sel.ec'tiora of phages carrying an 3~ abzyme In this case, the covalent inhibitor will consist of a suicide a.mhibitor characteristic of the reaction to be catalyzed by the abzyme or by an analog of the specific ~rarasition state.
~%~'~ ~~~
~- Zs -The detection and selection of these phages are then carried out in .a rraanner identical to the detection and selection of the phages which expose active enzymes at their surface.
CONTAINING AT THEIR SURFACE AT LEAST ONE MOLECULE
HAVTNG ENZYMATTC ACTIVITY
Subiect of the invention The invention relates to a method for selecting recombinant microorganisms containing at their surface at least one molecule having enzymatic activity.
The invention also' extends to the means for implementing this method and to methods for obtaining p them: .
Prior state of -the art' and technolo ical back round formin t~xe lc~asi.s of the invention The discovery of enzymes having new properties (catalytic activity, new enhanced stability, altered 15 specificity, shifted pH profile and the Like) is of great interest for the improvement of biochemical processes.
Although very substantial progress has been achieved in theunderstanding of'the structure-activity relationship fog proteins (Atkins et al : , Current opinion 20 in structural biology (199Ia, 1, p. 611 to 623, the prediction of the type of modification to be introduced into a protein in order to alter its properties in a desired dir~ation remains difficult: , Consequently', a' strategy is a ually adopted in 25 which an existing enzyme, whose gene is mutated, is used as starting material. This mutagenesis i.s carried out i:n a random manner, thus creating a -asst population of mutated enzymes: Among these, it is hoped that some of them will,have the desired new properties (Cunningham et 3p al:, Protein Engineering (1987j, 1, p. 319 to 325 and Lehtovaara et al. Protein Engineering (1988, 2, p. 263 to 268 y..
xn order to increase the chances of success, it is necessary to test a very large number of mutants and to have available a'teohnique which makes it possible to.
select., within the largest possible population, the .
l. enzymes having the:appropriate properties.
l proteins haring new enzymatic properties can also be produced by resorting to the monoclonal antibody.
_ 2 -technique. In this case, the selection of the antibodies-enzymes (also called abzymes). having the appropriate v properties also necessitates an effective screening technique.
A technique for,deteotion of the mutated enzymes rahich is frequently used is based on the development of a sensitive test for the enzymatic activity. A search is then directly carried'out on a population of mutants plated on a Petri dish for those which produce the desired enzyme. 8owever; this-technique is laborious and pexmits 'only a limited number of clones, to be tested (Pcallack et ale, J. Am. Chem. Soc., 111, p. 5961-5962 (1989) and Huse et al., Science, 246. p~ 1275-1281 (1989))0 An advantageous alternative arises when the desired catalytic activity rnay be useful for the survival or for the development of the microorganism expressing the'corresponding gene.: It is sufficient, in this case, to exert a selection pressure on the medium for culturing the population of mutants in order to select the strains which produce the desired enzyme (Hermes et al., PNAS, USA; 87; p. 696-700 (1990) and Evnin et al., PNAS, USA, 87, p. 6659-6663 (1990))~
This technique has the disadvantage of not being applicable to all the molecules having enzyma~.ic act3.vity .
It is also known (Smith, Science (1985), 228,'p.
1315-1317, Parmley and Smith, Gene (1985), 76, 305-318, de la, Cruz et al.,- J. Hiol. Chem. (1988), 263, p. 4318-4322, Bass et al., Proteins (1990), 8, p. 309 to 314;
Cwirla et al., Proc: Nath. Acad. Sci: USA (1990), 87, p.
6378-6382, Devlin et al:, Science (1990), 249, 404-406, McCafferty et al., Nature (1990); 348, p. 552-554, Scott and Smith, Science (1930), 249, p. 386-390, Clackson et al:, Nature (191), 352, p. 624-628, Zowman et al., Biochemistry, (1991), 30, p: 10832-10838, J. McCafferty et a1.1 Prot. Engng (1991) 955-961, Kang et al., (1991) Proc. Natl: Acad. Sci. USA, vol:' 88, p. 4363-4366; Barbas et al. (1991), Proc: Natl. Acad. Sci. USA, vol. 88, p. ?978-7982; Roberts et al., Proc. Natl. Aced. Sci. USA
(lgg2j, 89, p. 2429-2433j to be able to expose certain foreign peptide sequences, in particular proteins, at the surface of "filamentous" phages and of phagemids.
However, the in vitro selection of phages which correctly expose active polypeptide sequences at their surface is not resolved either Various affinity chromatography techniques have been de~aribed: Among the affinity supports developed, several use immobilized streptavidin, or avidin, the latter being advantageously in monomeric form (EP-A-0,423,~38j. Phages with peptide sequences having an affinity for streptavidin have thus begn selected with the aid of agarose carrying this protein (Devlin et al., Science (1990jr 249e 404=406j.
However,; these techniques have the disadvantage that the selection, during affinity chromatography, of these phages is carried out on the basis of their ability to form a complex with peptide receptors or specific antibodies which indiscriminately bind active ox non-active molecules:
With' the aim of detecting specific microorganisms, various other' labeling techniques have been developed. Far ekample, certain microorganisms have been detected with the aid of bacteri:ophages which are specific ' for these hosts and ' labeled (WO-A-8, 804, 326 j .
Transducing particles of known host specificity and containing genes which alter the phenotype can he used for lab~Iing bacteria (WO-A-9,004,041j.
, The sensitivity to certain toxic substances which is associated ~ai.th the detection by bioluminescence encoded by a plasmid has also' been used to detect specific microorganisms.
However, none of these techniques permits the selection of microorganisms carrying catalytic peptides.
Moreover, covalent inhibitors, used as substrate by Certain enzymes, are also known for their capacity to inhibit all enzymatic reactions by binding covalently to the active site of the enzyme.
.., _~_ Some covalent inhibitors are also called "suicide" inhibitors (ATOR et al., The Enzyme, vol. 19, p. 214-282, edit. Sigman Bayer 3rd edition (1990)) because they bind irreversibly to the active site of the enzyme using the catalytic mechanism of the latter and thus render any reuse of the enzyme impossible.
It is also aCnown that it is possible to immobilize a substrate by first binding it to biotin and by placing the product obta3.ned in the presence of avidin bound to a water-insoluble support (JP-A-3,080,098). This immobilized substrate permits the easy determination of certain enzymatic activities, but cannot serve as chrome-tographic support to select microorganisms carrying catalytic peptides, Aims of the invention The invention: aims to develop a method for selecting'recombinant microorganisms containing at their surface at leasf. one molecule having enzymatic activity and which does not carry the disadvantages of the prior state of the art.
Tie invention aims i~ particular to develop a method which permits the detection and selection of recombinanfi microorganisms exposing at their surface molecules having enzymatic activity, which are performed on tl~e basis of the catalytic activity of these molecules rather than on'the basis of their complexing activity.
Chara~ter~ati.c features of the invention The invention gelates to a method for selecting recombinant microorganisms Containing at their surface at lest one molecule having enzymatic activity, in which the recombinant nni:craorganisms, preferably viruses such as filamentous pages ~r .1 phages or phagemids, are immobilized on a solid support by a covalent inhibitor of the active site' of the molecule having enzymatic acti.va.ty, connected to the solid support by an arm; the recombinant anicraorganisms or all or part of their genome are then recovered and all 'or part of their genome is isolated., The term "recombinant microorganism" is , , n ~,~.~~':~~~3 understaod to mean any macromolecular structure which satisfies the English definition of '°Genetic Display Package" (GDP), that is to say any macromolecular struc ture incorporating genetic material and containing a peptide sequence encoded by this genetic material.
This definition includes especially yeasts, bacteria, viruses, phages such as filamentous phages or phagemids.
The term "solid support" is understood to mean any solid structure on which the microorganisms can be temporarily immobilized.
Such a solid support can be made by a person skilled in the art according to the techniques known in the prior state of the art.
Thie support may consist of a chromatographic support, paramagnetic parta.cles, a polystyrene support or any other support on which complexing proteins such as avidin ox streptavidiri or-an antibody capable of binding the microorganism are immobilized:
Preferably, the molecules having enzymatic activity are chosen from the group consisting of enzymes, abzymes, catalytic pegtides or a mixture thereof.
~3ccording to a preferred embodiment of the method of the invention, prior to their'immobilization on the solid support, the recombinant microorganiazns are labe~.ed with the covalent inhibitor connected by an arm to a ligand which can be immobilized on the solid support.
According to another preferred embpdiment of the method of the invention, the microorganisms or alb. or part of ,heir genome are recovered by destruction of the microorga~aism or by detachment of the rnicroarganism from '-its solid support.
Th~.s detachment can be performed ~ according to 'several methodss'either by detaching the ligand from the solid support w~;th an eluting salu~ion, preferably with a solution at an acidic pH or by cleaving the arm connecting he covalent inhibitor to the solid support;
or' by cleaving the bond between he covalent inhibitor and the molecule having enzymatic activity or by ~~'~~,~::~~~
- _ 6 _ cleaving a peptide linkage between the molecule with enzymatic activity and the microorganism.
The cleavage of the peptide linkage between the molecule having enzymatic activity is preferably performed with a specific protease such as factor 1UA or collagenase; ayanogen bromide which cleaves a peptide linkage containing a methionine or hydroxylamine according [sic] to a peptide linkage containing the peptide ~sparagine--glycine have also been used.
1p Such oleavage makes it possible to advantageously increase the selectivity of the method according to the in~rention .
Indeed, the suicide inhibitor in the method according to the invention is capable of also binding to the microorganism at sites other than the active site of the molecule having enzymatic activity.
Hy cleaving the specific peptide linkage existing between the molecule having enzymatic activity and the microorganism,.only the m.icroorganis~ containing'at its surface an active enzymatic molecule will be recovered during the: elution.:
Furthermore, this method o selection can be applied for the detection and selection of microorganisms which expose at their surface, on a native or recombinant protein of the microorganism, one or more specific peptides cagoble of inducing a cellular or hura~oral immune response.
Indeed, it was observed that a mere antibody-antigen recognition of such peptides did not p~rrinit 'selection of the peptides most 'capable of eliciting. a cellular or humoral immune response (6nletzel ( 1991' , Prot.
Eng., vol 4, p: 371-37:4).
Consequently, a cleavage carried out according to the invention at-the level of the peptide linkage existing between the said peptide and the native molecule of the miaroar~gan.ism also makes it possible to select in a specific manner the ;peptide(s) must capable of eliciting a cellular ax humoral reaction.
In addition, all or .past of the genome of the f L
microorganisms can also be recovered by destroying the ni.croorganism or by recovering all or part of its genome.
This genome is then isolated, amplified (far example by PCR) and cloned.
The invention also relates to a marker consisting of a covalent, preferably irreversible, inhibitor of the active site of a molecule having enzymatic activity, connected by an arm to a ligand which can be immobilized on a solid support. .
1(~ Preferably, the ligand is a biotin or an imino-biotin.
Advantageously, the arm contains a cleavable chemical functional group, preferably a disulfide func-tional group.
According to a preferred embodiment of the marker according to the invention, the covalent inhibitor is an analog of the specific transition state of the enzymatic reaction performed by he molecule having enzymatic activity Another aspect of the invention relates to a method for preparing the marker according to the inven ion,.in which a covalent inhibitor of the active site of a molecule having en2ymati.c activity is coupled by an arm to a ligand which can be immobilized an a solid s~~pc~rt.
~5 The invention also relates to a solid support~in which a covale~xt inhibitor of the active site of a molecule; with enzymatic activity is connected by an arm to a solid support.
Advantageous3.y, the covalent inhibitor connected 30; to the solid support consists of the marker according to the invention.
According to a preferred embodiment of the inven 'tion, tJhe arm is connected to the solid support by an ester linkage or contains a vicinal diol.
35 A final aspect of the invention relates to the method for preparing the solid support according to the in~rention in which:a covalent inhibitor of the active site of a molecule having enzymatic activity is connected by an'arm to a solid support.
Hrief descri~ation of the firnares Figure 1 .represents the nucleotide sequence of the ~i-laatamase--encoding gene of the plasmid r pH~322.
Figure 2 schematically represents the steps of the method for inserting the gene encoding the mature (3-lactamase into the phage fdDOGl.
0 Figure 3 ~'ep~esents the nucleotide sequences of the primers comple-mentary to the gene encoding (~-lactamase:
Figures Q to 9' schematically represent the 15 method of synthesis of the markers 'according to the ~;nvention Eigur~ 10 schematically represents the binding of a microorganisan onto a 20 ~~lid support, by the marker according to the invention, as well as the possible cleavage sites which snake it possible to recover the said microorganism.
25 Descri ti.on of a referred -eaebadiment of the inventa.o~x Figure 10 schematically represents a recombinant ..
microorganism ( 1 ) containing at its surface a molecule having enzymatic activity ( 2 ) , immobilized on a solid support (3)- containing a complexing'protein (9) such as 30; avidin or streptavidin, by a marker consisting of a covalent inhibitor,(5) of the active site of the molecule having enzymatic activity (2),,an a~ (S) and a ligand ( 7 j which can be ~:mmobili~ed by the complexing protein (r~) on the solid support (3).
35 The microorganisms or all or part of their genome can be recovered:
by detaching the ligand (7) from the solid support with ~n eluting solutian, such as a so7.ution at a pH
of the order of 4 (~);
.- by cleaving the arm (6) of the marker (B);
- by cleaving the bond between the inhibitor (5) and the molecule having enzy~aatic activity (2) (C);
- by cleaving the peptide linkage between the microorganism (1) and the molecule having enzymatic activity (2) (D)~
- by destroying the microorganism (1) and by recovering all or part of its genome (E).
The method will be described in greater detail with ref~zence t,o the acc~ampanying figures, in the following examples.
Example Is Construction and selection of phages carrying active (3-lactamase (fd-bla) The viruses having at their surface molecules with enzymatl.c activity are constructed from "f~.lamentous' phages.
The filamentous phages such as M13 or fd are bacterial viruses iahich, in tlae free form, contain a 6. ~1 kb single-~trarrded circular DN~1 endapsulated in a 2~ - protein en~relope era the foam of a very long cylinder consisting of five proteins (Heck'& 2ink, Gene (1981), 16 ~ p,: 35; to 5g; Giant et al . , J. Hiol. Chem. ( 1981 ) , 25:6, p: 539 to 596; Isopez & Webster, Virology (1983), 127, p. 177 to 193.
Among these, the product of the gene III (pIII) is a complex protein, present in an amount of 3 to 5 copies, which closes ore of the ends of the virion and is composed of three parts: a'signal peptide which is used for exporting: the protein to the ~er~:plasm of the 30, bacterium and 'is cleared during excretion; a globular part necessary for infection which recognizes the F-pilus of the E. coli target cell; a hydrophobic, transmembrane part during the virus morphogenesis phase which subsequently ensures anchorage of pIII in the particle ' (J. Armstrong ~t al., FENS T,etters (1981), 135, p. 167-172; C:W~ Gray et al., J. Mol. Biol. (1981) 146 p. 621 to 627; G. Glasez-Wuttke et al., BBA (1989) 985, p. 239 to 2~7).
Following their work on the role and function of /~ i~ t-.A ~t ,~ ~ a the protein px~I in the physiology of the phage fd, Smith and Crissman (Virology (1984), 132, p. 445 to 455) showed that it was possible to introduce, by recombinant DtdA
methods, a foreign peptide g~~~E~~~e inside the sequence ofi protein pTTfi ( Scienoe ( 19 ~~ ~ ~ p . 13'15 ts~ i31? y or between the signal popfi : . and the sequence of the mature protein (Gene (1988), 73, p. 305 to 328). .'his insertion dogs not prevent the viability of the phdge and the introduced sequence is accessible.
1U The phage°enzyn~;: fd-tet-~°lactamase ( fd-bla is constructed from the pt~age fdDOGl (Clackson et al, Nature, 3~2, p. 62'4 to 628 (1991) in which the sequence encoding the mutated ~-lactamase has been introduced.
This phage is derived from the phage fd-tet (Heck et al : , Nucl. Acids. Res. , 5, p. 4495 to 4503 (1978 ) ) , in which a polylinker is inserted between the sequence enco~3ing the signal peptide of protein III (pTII) and the sequence encoding the mature pIII; in this manner, the ~i-~actamase will farm, avith pITI, a Fusion protein. The phage fd-tet is itself derived from the phage fd (Zacher et alo, Gene, _9 p. 127 to 140 (1980)) by insertion of the tetracycline resistance gene of the transposon Tn 10, The phage fdDOGI comprises two restriction sites (Notl and-Apal.l) constructed by mutations in the region '.25 encoding the protein pIII, between the signal peptide and the globular part.
The ph~ge fdDO~l also Comprises the tetracycline gene inserted in, the region of he origin of replication o~:rthe ~hage and permitting a propagation of the phage as 3U plasmid.
isolation of the~nucleotide se ence encodin -lactamase The ~°lactamase gene of the plasmid pBR322 of H: coli (whoa~ sequence is,.givey by Sutcliffe, PNAS USA, 7~5, p. 3737 to 3741 (1987) and in figure 1) was isolated 35 from the phagemi.d ~Hluescript SIKt~.
Given that the cloning into fdDOGl will use the ApaZ1 site and that the sequence of the ~i°lactamase Contains such ~ site, the latter should first be removed b~ site°directed mutagenesis. This mutation conserves the protein sequence.
The ph~gemid pBluescript S/K+~ (from the company Stratag~ne~, sold by Ozyme~ j , is mutated according to the method -of Eckstein (Nucl. Acids Res., 14, p. 9679 to 9698 (1986] with the aid of a kit marketed by the company Amersham'~ .
After in vitro mutagenesis, the double strand of the mutated phagemid pHluescript S/K+~ (pBluescript-Mj is used to transform the strain TG1 of E. coli (Gibson, Studi.e~ on the Epstein-Harr virus genome, PhD Thesis, Camb~~:dge University England (1984).
The transformed cells are plated on LB-Amp mediuan (Sambrook et al., Molecular Cloning, A laboratory manual, second edition, Cold Spring Harbor Laboratory Press (1989) Appendice A).
Colonies are recovered and cultured in LB-Amp medium. The single--stranded form of pBluescript-M is produced by addition of the helper phage 8408 to the medium (Russet et al., 4~5, p. 333 to 338 (1986jj~
The strand gHluescript-M is then sequenced acdosding to the Sanger method (PNAS USA, 74, p. 5463 to 5467 (19?7)) with the aid of the primer 5'-TTTA~AAGTGGCCATCATTGGA centered at the position encoding S~r 68 in order to verify that, in the chosen claneg, the mutation is indeed introduced.
Clot~iz~ of --lactamase into the ha a gdDOGl The soqcaence of the gene encod.i.ng the mature p-lactamase is ~.nserted into the phage fdDOG~, between the sequence encoding -the signal peptide of pIII and that encoding:the mature pIII. The Gloving sites ApaL1 and Notl are used according to the scheme given in figure 2.
To this effect, the ~-lactamase gene is recovered from the phagemid pBluoscript-M with introduction of the aPProPriate restriction sites by PCR.
The grimers used, shown in Figure 3, contain the foTlawing characteristics:
The primer in 5' is a 30mer complementary over 18 bases of the mature ~-lactamase gene starting from the second colon (encoding Pro 2j. The 5' extension (12 .8.. tJ
bases) introduces the ApaLl site and causes a mutation which results in the replacement of His 1 by Gln 1. This mutation is introduced in order to avoid the phages carrying a ~,-lactamase having the terminal amino sequence His-Pro-Gln capable of exhibiting an affinity for avidin or streptavidin (Devlin et al. Science, 249, p. 404 to 406 (1990)) subsequently used for their selection.
The primer in 3' is a 47mer complementary over 2I
bases from 'the end of the ~-lactamase gene. The e~ctension replaces the stop codon by a codon encoding a glycine, introduces four colons between the gene encoding the ~3-lactamase and the gent encoding the protein pIII, as well as the Notl restriction site for the cloning. These faur additional amino acids constitute a~ potential cleavage site between the two proteins for factor Xa. The protein pIII provides, indeed, the capacity for infection of the phage~ (Goldsmith et al., Eiochemistry, 16, p.
2686-2694 (1977). While the presence of a protein as voluminous as (~-lactama.se affects the capacity for infection, an incubation with factor Xa makes it possible to restore the latter:
7Cn addition, thia site constitutes an additional cleavage point for recovering the miaroo~ganisms.
The phage fdDOGl is digested for 18 hours with ApaLl and Notl anti purified on agarose gel. Moreover, the sequence of the phag~mid pBluesc~ipt-M is amplified with the primer9 described above using 34 PCR cycles (1 min.
at gSoCr 1 min. at 60°C and 2:5 min. at 72°C) in the presence of Taqpolymerasea (Saiki et al., Science, 239, p, 487 (,1988). ,After gel purification, the product is digested with ApaLl and Notl for 18h and repurified; then 1.5 ~g of digested fdDOGl and 250 ng of the ~i-lactamase gene are mixed in 50 girl with ~ units of T4 DNA ligase (Gibco-HRL~) and maintained avernight at 16°C. The purified ligation mixture is used to transform the strain TG1 of E. Coli.
The cells are plated on LB-tet and LB-Amp medium (Sambrook et al.) where it is observed that the presence of the phage provides the resistance to the antibiotic.
'~I
Colonies are then cultured in 2xYT-Tet medium or in 2xYT-Amp medium in order to prepare the phage fd-bla.
Instead of constructing phages-enzymes by mani pulation of the protein pIII, it is also possible to construct a fusion protein with the protein pVIII (1) which is a small protein of 5.2 kd constituting the cylinder body o~ the ghage fd and which is normally present in 2700 copies (Newman et al., J. Mol. Biol 0.977), 116, p: 593 to 606). If the size of the DNA
~p molecule is modified, the number of copies of the protein pVIII can be adjusted in order to include the said DNA
molecule.
This variant is achieved with a phagemid in order to b~ able to control the number of copies of the fusion protein Kang et al:, (1991) Proc. Natl. Acad. Sci. USA, vol. 88, p. 4363-~1~66.
Characterization of the phade carrYinct the active ~--lactamase ~(fci-blal The phage thus constructed is characterized in 'the fdlloraing manner: The fact that the inf~:ction by the phage confers on the cells a resistance to ampicillin is first demonstrated.;Then it is shown. directly that the phage itself exhibits an actauity for hydrolyzing a y-lactam, nitrocefine. The fact that the enzymatic aativi.ty . is not pxesent in the ultrafiltrate after filtration on a membrane which alle~ws the (3-lactamase to pass through already indicates that the enzyme is bound to the phac~e. Finally, the phage proteins are analyzed by gel electrophoresis and Western Hlotting and it is shown that the protein gIII is present in the form of a fusion protein with the ~3~-lactamase. This confirms that the construction is indeed correct and that, during the marphog~nesis of the virus, there is no proteolytic cleavage ~rhich would result in the detachment of the (i-lactamase from the phage:
Hy infection of cells of E. Coli TG1 with this phage and plating ors Z~-Amp medium, ampicillin-resistant c~lonies are obtained. The p=lac~amase activity is dgrectly detected in the Petri dish by a nitrocefine test '~,~'~ ~ ~~~
(Pliickthun et al., J. Biol. Chem., 262, p. 3951 to 3957 (19~3'~)). Likewise, the phage isolated from the culture medium after centrifugation of the cells, precipitated with 4 ~ PEG, 0.5M NaCI (final) and resuspended in 50 mM
tris buffer, 150 mM I~1'aCi at pH 7.5, at a concentration of the order of l0bz transducing units (tu) per ml, hydrolyzes the nitrocefine.
The specific activity of the enzyme carried by the phages was identical to that of the ~3-lactamase RTEM
ZO in solution (Boehringer). The measurement of the activity was also performed:af~ter ultrafiltration'of the phages on Centri/por membrane with an MW cut-off of 100,000 which allows commercial p-lactamase RTEM to pass through. bees than 5 ~ of the ~i-lactamase activity was detected in the ultrafiltrate, which indicates that the enzyme remains attached to the phage.
Viruses fd=tet arid fd-bla are prepared and cancentrated (1Q'a tu/ml). 20 ~1 of each solu~tian are loaded onto a x.2:5 ~ SDS-polyacrylamide gel and analyzed 20~ (miniprotean~ Il from Hiorad~). By S~Testern Blotting, the proteins are transferred onto nitrocellulose (minitraaashlot~ from Hiorad~) and the revealing is perf~ra~ed with an anti-protein III rabbit ant~.body, followed by a goat antibody against a rabbit antibody coupled to IiltP ( horseradish peroxidase ) . The chromophoric substrate o~ FiRP reveals bands o~ apparent M~1 of 70 kd , and 105 kd for fd-tet and fd-°bl,a respectively. The rev~eala.aag taas also performed with an anti-~°-lactamase antibody; a band was fpund only for fd-bla at the position.e~cpected for the fusion protein.
Example II: Construction and characterization of phages carrying an inactive ~-lactamase ( fd-bla-) In order to test the efficiency of the selection method according to the invention, phages carrying an inactive ~i-lactamase (fd-bla') are constructed and characterized.
The phagemid pBluescript-M is subjected to a mutagenesis in which the colon encoding the essential ~~.~~:.~~~
serine 68 is muted to alanine in order to inactivate the enZyIIle a The mutated gene is recovered according to the same technique as that used for the active ~i-lactamase (PCR amplification with introduction of restriction sites (cf. figure 2)). The phage carrying inactive p-lactamase (fd-bla-) is characterized by restriction fragment analysis, camplete sequencing of the Hla gene, detention of enzymatic ac~.ivi.ty and analysis of the proteins.
1p The S68A mutation is introduced according to the same procedure as the mutation of the Apal~l site. The transformed cells are dated on X-Gal medium (Horwitz et al., J. Med. Chem., 7, p. 574 (1964)) without ampicillin.
The blue colonies are cultured in 2xYT medium and the single-stranded form is produced by addition of the helper phage 8408. The mutation is sequenced by the S~.nger method with the aid of the primer 5' -TA~TGTATGCGGCGACC cor~glementary to the region encoding G90-Y95.
After amplification of the gene by PCR, cloning, transfc~rnbation and platine~ on tet mediuan as performed for the active ~i-lactaanase, colonies are recovered and cultured in 2xYT~-tet u~edium. The double-stranded DNA of the phage is isolated anti digested with the restriction nucleases HamHl and He~R1. The- digestion products are separated on agarosethe transforr~ants show 2 fragments of 9.7 kbp and 5.5 khp, in agreement with what is expected. The complete sequencing Qf the mutated Hla gene is performed with the primers 5'-TAGTGTATGCGGCGACC, 5'-Ajlacuna]GCGA~TTACjlacuna]G, 5'-CCAGCCAGCCGGAAG and 5'-TGCTAAACAACTTTC located respsetive3y in the regions encoding G90-Y65, H156-L160, 2223-W227 of the ~-lactamase and E5-A9 of the mature protein pIIT). It thus is verified that the PCR amplification did not introduce mutations other than that aimed at inactivating the active site.
Tha presence of a ~-lactamase activity is then detected by performing, on the supernatant of a culture of phage fd-bla-, a ni.trocefine test as described above.
~~2~~.~ ~~~
16 _ In agreement with what is expected, no activity is observed.
Finally, analyses of the phage coat proteins by denaturing gel electrophoresis, Western Blotting and immunological detection is performed as for the phage fd-bla. The fusion protein PIIT-p-LACTAMASE is correctly identified.
Example III: Synthesis of the active ~i-lactamase marker provided with a small-sized arm between auioide inhibitor and ligand 'rhe phagee whose DNA contains the active (3-lactam~se gene and which carry the corresponding enzyme attached to the soot protein;pTII will be selected by a reaction with the markex according to the invention. This marker consists of a bifunctional molecule which contains, at one end, a covalent inhibitor of the ~-lac amp a and, at the other, a lig~nd, in this case biotin, which can be easily immobilized on a solid support:
The sdicide inhibitor module consists of a penam sulfone (see Fisher et al., Biochemistry (1981), 20, 2?26~-2731:; Metes et al., FENS I,et. (1982), 143, 265-267, Cl.ark~ et al. ; ( 1,983 ) , 748, 389-3g7, Dmitrienko et al . , Hioorg. Chem. (1985), 13; 34-4'6) fun~tionalized'so as.to allow its attachment Day an arm to a ligan~l ar a support.
Inei.no-biotl.n can also be advantageously used because its affinity for streptavidln can be considerably reduced by decreasing, the pH of the solution. This will give:us an additional possibility to isolate the phages 30, fxom the supp~rt after immobilization.
These tan entities are connected by an arm of sufficient length to allow the interaction of the ligand carried by the marker with the immobi.Iized protein.
The scheme for synthesis of the bifunctional molecule is given in figures 4 to:6. Most of the steps of this synthesis are described in he chemical literature ox~ involve coventional procedures. The molecules are characterized by TR spectroscopy and 1H-NMR.
Sodium p-nitrobenzyloxycarbonyl-2-aminoethane-suTfonate (3) is prepared by addition of two equivalents of p-nitrobenzyl chloroformate to dissolved tsarina (2) one equivalent [sic] of 1N NaOH at 0°C, continuously neutralizing the medium by addition of NaOH, under the Schotten-Haumann conditions. After extraction of the p-nitrobenzyl alcohol with ethyl acetate, the product is freeze-dried. The yield was 66 ~.
The sulfonate {3) was converted to sulfonyl chloride {~j with phosphorous oxychloride according to the method of Fujita (Synthesis, 423-424 (1982)). Hy washing the oil obtained with dry ether, the product was obtained in the form of a white powder with a yield of 97 ~:
In _parallel, 6-aminopenicillanic acid (5) was converted to its methoxymethyl ester (6) according to the method of Manhas et al: (Synthesis, 549-552 (1983)) with the aid of m~thox~rm~thyl chloride after temporary protection with ethyl acetoacetate.
The acid chloride (~) is coupled with the 6-aminoperaicilla.nic acid ester ( 5 ) in dichloromethane in the presence ~f tri~thylamine. The product (7) purified on a silica column {eluantc ethyl acetate-dichloromethane 80 ~ 20 ) , was obtainsc~ with a yield of S0 ~ .
This product was oxidized into a sulfone (8) 2.5 according to the method of Johnson et al > ( J. Org. Cher~i. , 28, 1927-28 ( 1963 ) ) ; 'the product obtained was washed with ether to give a solid white [sic] which was purified on silica (eluants ethyl acetate-dichloromethane 50:50j. The Meld was 75 ~.
On deprotection of the amine functional group by hydrogenation on palladium/carbon according to a conven-tiona3. method, the product (9) was obtained which was coupled directly with sulfosuccinimidyl 2-(biotinamido)-ethyl-1,3--dithioprapionate (10) (marketed by PTERCE~), to 3,5 gave the marker precursor (1-11) which was purified by chromatography on silica {eluant: ethyl acetate-dichloromethane SOt50)a The yield was 25 The protection of the carboxylic functional group in position 3 of the penam ring in (11) was removed with ' y~'.~:~.3ii t .1.
the aid of magnesium bromide in dichloromethane according to the method of Rim et al. (Tetrahedron Letters, 32, 3099- °3100(1991)). The crude product recovered (12) after evaporation of the solvent was used directly to inhibit the enzyme and, to label the phages carrying active enzyme .
Exame,le IV: Immobil.lzation of the phages carrying active (3-lactamase ( fd-bla ) The use of a covalent marker irreversibly binding the active site of the enzyme and making impossible any reuse of the enzyme, dyes not constitute a disadvantage in the method for selecting the enzyme. Tndeed, in this method the desire is only to select and recover the genetic sequence encoding the act~.ve molecule.
Ths optimum conditions for inhibition, by the hifunctional marker, of the phages carrying the active p~-lactaan~se arm first determined. To this end, the rate of disappearance of the ~i-1'actamas~ activity of the phages is measured, by the nitrocefine~test, as a func-Lion of time after addition of the inhibitor. phages are then incubated i,n the presence of the marker.
Then the reaction product is passed through an .sgarose-coupled streptavidin column. The phages are then eluted by cleaving the disulfide functional group of the 25' marker. They are used to'reinfeat an E. coli. culture.
E'xom this culture, the gene encoding the active p-lac~amase can be easily recovered.
A culture of bacteria infected with the fd-bla phag~s is pregared in a 2xYT medium containing 15 ~g per ml of tetracycline and incubated, with stirring, at 37°C
for 16 hours. After centrifugation of the culture for 10 minutes at 10000 rpm .(revolutions per minutej, the supernatant is recovered. The phages are precipitated by additibn of 1/5 , volr~me of 20 ~ polyethylene glycol solution, at 2.5M NaCI. After incubation for 1 hour at ~°C end centrifugation, the phages are resuspended in a 50 mM acetate buffer at pH 5:0.
The marker ( 1-2 ) in solution i.n ~M~O, at a concentration oø l mg/25 ~1, was added at a final ' v;~<~cR~
concentration bf 0.25 mNt to the solution containing the phages (approximately 10" tu/mlj and 1 $ BSA. The inhibi-tion is continued for 30 minutes.
Moreover, a suspension of 250 gel of agarose e~rc~p~avidin (PIEP.CE~j is washed with 20 ml of PHS buffer containing 1 ~ HSA (Bovine Serum Albumin, Sigmaj. 0.3 ml of the solution, containing 1.25 x 109 phages, was loaded onto the column, washed with 6 ml of PBS/1 ~ BSA and 6 ml of PHS. The phages axe then eluted with 1 ml of a 500 mM
dithiothreitol solution in PHS buffer. One aliquot of recovered ghage~ was incubated, after change of buffer, with 10 ml of E, cpli TG1 cells in exponential growth at 37pC. The recovery of the phages was demonstrated by plating various dilutions on I,B-tet medium and counting the colonies. 1.0 x 10° phages carrying active ~i-lactamase, equivalent to 14.4 ppm, were recovered in this manner:
Excmple Vs Tmmobilization oø phages carrying inactive p-lactamase (fd-bla'j ~, . 05 x 10' phagss ( df-bla' j contained in A . 3 mi of acetate buffer at pH 5 were plcced in the presence of the marker (12j according to the invention and the reaction product was passed through an agarc~se-coupled strep-tcvidin column and eluted according to a procedure identical to that described in example 4. 2.0 x 10' phages carrying inactive ~-lactamase, equivalent to 1.9 PPm. wire recovered'in ,this case. .
Comparison of the results of examples IV and V
shows that the active phages are retained better than the 30, inaative.Pha9es by ~ factor of 8.
Example VIc Enrichment, caith active phages, of a mixture containing active (fd-blaj and inactive' ( fd-bla' ) phages a elution with dithiothreitol An experiment of the same type was carried out with a mixture of active phages and inac~ti.ve phages to determine directly the active phage enrichment factor.
A mixture containing F . 4 x 109 ,active phages and 1.4 x 10'° inactivd phages in 0.3 m1 of acdtic buffer at s' a v j .a~ .'; '' pH 5 in the presence of 1 $ BSA was planed in the presence of the marker (12) at.a final concentration of 0.25 mM. Excess marker was removed by ultrafiltration on a unit Ultrafree 30, 000 from Millipore . The solution was Loaded onto streptavidin-agarose. After washing the column with 5 ml of PBS/1 ~ BSA, the phages were eluted with 500 mM dithiothreital. An aliquot of eluted phages was plated at various dilutions on LH-tet dishes; the co?~onies were thus counted: A total of 1.3 x 106 phages were recovered in this manner. The number of phages carrying active ~i-lactamase was determined by directly measuring the ~i-lactamase activity of the colonies by the nitrocefine plate test as described above. This number was also verified by plating an aliquot of the eluate on I5 LB-Amp dishes. The number of eluted active phages was 1.1 x .106. The enrichment factor obtained was therefore 5.5.
Exa3a_p1e V'rI: Enrichment, with active phages, of a mixture containing active (fd-bla) and inactive (fd-bla') phages: elution with factor Xa ~,n experiment of the same type was carried out with a mixture of active phages and inactive phages to dixectly determine the enrichment factor; the elution was 25 this time carried out with factor Xa cleaving a peptide linkage between the (i-lactamase and the protein ILT.
mixture containing 2.E x 10'° active phages and 2.2 x 1Q'° inactive phagds in 1.5 ml of acetic buffer 2 ~
BSA was placed in the presence of the marker (12) at a 30 final concentration of 0.75 mM, passed over streptavidin agar~se. After washing the column with 80 ml of PBS, 2 ml of 0.1 M TR1CS buffer at pH 7 containing 0.'02 ~ I~aN3 and 2 ml caf Tris buffer at pH 7; 1 ml of a solution containing 10 ~g of factor Xa (Boeringher) was added to 35 the suspension and it was allowed to stand overnight at room temperature, with gentle stirring. After centrifug~ti.on, the supernatant was analyzed by plating at various dilutions on LH-tet dishes; the colonies were thus counted. A total of 6 x 105 phages were recovered in f ~.~~-'_3~~
this manner. The number of eluted active phages was 5.7 x 105. The enrichment factor obtained was therefore 15.
Example 'IYII: Enrichment, with active phages, of a mixture containing active (fd-blaj and inactive ( fd-bla' j phages :
preimmobilization of the marker Another experiment was carried out with a mixture of active ph~ges and inactive phages, but with prior immobilization of the marker on the streptavidin-agarose column.
250 Nl of streptavidin-agarose suspension are incubated for one hour in an acetic buffer at pB 5 in the presence of l ~ HSA~ after washing wl.tta 2.5 ml of acetic buffer without BSA, 2 ~1 of a 50 mM solution of marker ( 12 ) in DM~O axe added: After 1 minute and 5 cycles of washing and centrifugation with, each time, 1 ml of acetic buffer, 0.8 ml of phages in an acetic buffer 1 ~ BSA containing 9 ~ 6 x 101° active phages and 5 x 10'0 inactive phages were added to the suspension with gentle stixr~.ng, ov~rraight at 4°C. After, washing with 20 ml. of PBS/1 ~ BSA and 50 ml of solution] 0.1 ~ Tween 20 in PBS, the ph~ges ~ ase eluted with factor Xa. and counted as in example VT. 1.15 x lOs active phases and 1.85 x 105 inactive phages were recovered, in this manner. the enrichment factor obtained was therefore 3.2.
Expose gX: Enrichment, with active phages, of a mixtuxe containing active (fd-blaj and inactive ( fd-bla- ) phages : elution by hydrolysis of the aryl-enzyme linkage An experiment similar to that of example VIII was carried out with a mixture of active phages and inactive phages to verify the possibility'of recovering phages by cleaving the linkage between the ~-lactamase and the suicide inhibitor, namely the ester linkage of 'the aryl enzyme. This cleavage was carried out by hydrolysis.
A mixture containing 2.9 x 10'° active phages and 1.05 x 10'° inactive phages wire [sic] loaded onto a column where the marker had been immobilized. After extensive washing as in example ~T11I, the suspension was allowed to stand overnight at room temperature and the supernatant from a centrifugation was analyzed. 1.4 x 105 active phages and 2.5 x 105 inactive phages were recovered in this manner. The enrichment factor obtained was therefore 2.1.
In the examples mentioned, the percentage of immobilized phages is low because the size of the arm between the immobilizable ligand and the inhibitor is quite small. In order to avoid this disadvantage, another marker having a larger-sized arm was prepared.
Example X: Synthesis of a marker of active (i-ldctamase provided with a large-sized arm between suicide inhibitor and ligand Carbobenzyloxy-2-(2-ami.noethoxyjethanol~ (15) ( figure 7 j was prepared by addition of 1 equivalent of benzyl chloraformate (13j to 2-(2~-aminoethoxy)ethanol (1~4) in water saturated by bicarbonate. The product was extracted' in organic phase and purified on a silica column ( el:xant : ethyl acetate-dichloromethane 50 : 50 j . The yield was 5g ~:
Ethyl vinyl-sulfQnate (17), has prepared'from 2-chloroethane-sulfonyl chloride (16j according to the , method of Whitmore and handau (J. Am. Chem. Soc., 68, p.
1'i97-1798 (1946j). It eaas purified bY horizontal distil-lation at 8g°C under 0.6 mm of mercury to give a colorless o~.l (Yield 45 ~j. One equivalent of the protected amino alcohol (1~5j was' added onto (17) in dry a~etonaarile to which finely ground potassium bicarbonate was added After heating, for 3 days at 80°C, filtration and evaporation of the solvent, the product (18j was purified on silica (eluant: ethyl acetate-dichlorometh~ne 50:50). ThE yield was 60 ~: The sul.fone functional group was deprotected according to the method of Tipson et al, (J. Org. Chern., 12, 133-137 (1947)) to give, after 24 hours o~ reaction, ( 1-9 ) in the form of a white powder after washing with hexane (yield 86 ~). This product was converted to an acid chloride (20) according to the method of Fujita (Synthesis, 423-424 (i1982j).
The acid chloride (20) (figure 7) was coupled directly with the ester of of [sic] 6-aminopenicillanic acid (6) in dichloromethane in the presence of triethylamine -for one hour. The product obtained (21), pur.ifi.ed on silica (eluant: ethyl acetate-dichloromethane 50:50) was obtained with a yield of 34 $. It was oxidized into a sulfone ( 22 ) according to the method of Jotmson et al. (J. Org. Ohem., 28, 1927-28 (1963)), the product obtained was washed with ether to give a white solid which was purified on silica (eluant: ethyl acetate-dichloromethane 50:50). The yield was 72 ~.
pn deprotection of the amine functional group by hydrogenation on palladiumlcarbon in ethanol according to the conventional anethod, the product (23) (figure 8) was obtained which was coupled directly with sulfosuccinimidyl 2-(biotinamido)ethyl-1,3 dithiopropionate (10) (marketed by PIERCE~) in order to obta~:n the marker precursor ( 24 ) which was purified by chr~matography on silica (eluant: ethyl acetate dichloromethan~ 50:50). The yield was 13 ~.
The protection of the carboxylic functional group in positian-3 of the penam ring in (2~,4) was removed with the aid of magnesium bromide in dichloromethane according to the'method of lKim et al. (Tetrahedron Letters, 32, 3099-310U (1991)). , .
Exa~ngle xL: Enrichment, with active phages, of a gn~.xture containing active ( fd-bla ) and inactive (fd-bla') phages: labeling with the inhibitor (24), elution with factor Xa An enrichment experiment was carried out with labeling with the inhibitor (24) of a mixture of active phages and inactive phages in order to determine the-effect o.f the size of the arm connecting the biotin 2igand with the suicide inhibitor module.
A mixture containing l.2 x 10'° active phages and 3.3 x 10'° inactive phages in 1.5 ml of acetic buffer 1~
BSA was placed in the presence of the marker at a final concentration of 0.75 mM, passed over streptavidinagarose. After washing the column, elution as described in example VTT and analysis, 6.8 x 106 activo phages and 1.9 x 10~ inactive phages were found. The.
enrichment factor obtained was therefore 90.
EXA~ipLE KIT : synthesis of a second active ~9-lactaxnase marker provided with a large-sized arm between suicide inhibitor az~d ligand In order to have a general method for the synthesis of a marker provided with a large-sized arm between the ligand and the inhibiting head, a precursor carrying an activated ester functional group and there-fQre capable of being coupled to numerous inhibitors and containing itself a large-si.zed arm waa prepared. It was coupled to the suicide inhibitor of the (i-lactamase.
1,5 The activated ester of the N-hydroxysucci.nimide with biotin-N-e-aminocaproic (26j was prepared as described (Wilchek and Bayer, Methods Enz. (1990), .~04, 123-160) , It was dissolved in DMF and added to a solution of 3 equivalents of cystamine (26) [sic] in DMF (figure 9j.
The crude product, isolated by precipitation with ether (10 times the volume ~f the so2ution) was purified on ~ Sepharose ~ (Pharmacia~ j coluann equilibrated with 1'ythl.um chloride, using as eluant 25 ~ ethanol. The addition product ( 20 j caws eluted with 50 mM LriCl . The solution was freeze-dried: The yield was 79 ~.
Three equivalents of glutaric anhydride (29j, dissolved in acetonitrile, were added in portions to the purified prodr~et (containing 7Gi:CI) dissolved at a 30' concentration o~ the arde~ of 0.01 M in buffered aqueous medium { K~iCO,-KzCO" 5 0 / 5 0 , 10 mM at pI3 9 . 6 j containing 10 ~k ethar~ol. The acid obtained (30.j was precipitated by acidification with 1N HC1 up to pH 2. The product was filtered and dried, the yield was 43 ~.
The acid (3a) was converted .into an activated ester under standard conditions by coupling with Id-hydroxysuccinimide' (31j with the aid of c~icyclohexylcarbodii.mide (DCCj in the DMF. After 72 hours, the reaction.medium was precipitated with dry - 2~ -ether. The precipitate was dried in a desiccator (yield:
B? ~D, this product (32) was used as sucks for the coupling reaction with the deprotected ester of 6-aminopenicillanic acid (6) in dry ethanol.
E_xampl~ XI~~e Enrichment, with active phages, of a mixture containing active (fd-bia) and inactive ( fd-'bla' ) phages : immobilization on paramagnetic particles.
.An experiment similar to experiment VI was I0 carried out, the only di~fesenae being that the streptavidin is carried by paramagnetic particles. The washes are carried out by resuspension in fresh buffer.
Tlxe elutioh of the ~.mmobilized phages is carried out with 50 mM dithiothrei.tol; The enrichment factor is determined as in example V'I.
Ea~ample XIVa Enrichment, with acti~re phages, of a mixture aontaini-~g active (fd-bla) and inactive ('fd-bla'j phages: immobilization by "panni.ng't ire a polystyrene tube 2~ An' experiment similar to experiment VII is carried out, the only difference berg that the streptavidin is immobilized by "panning" at the surface of a polystyrene .tube . following a technique conventionally used for EL~ISA tests. The washes are 25 carried out by resuspensic~n in-fresh buffer. The elution of the iman~bilized phages is carried out with factor Xa.
The enrichment f aotor i.s detern2ined a~ in example III I .
Given that, in'examples XIII and XIV, the washes can be carried out more rapidly than in the examples 30 using the chromatographic technique and the phages less easily entrapped here than in a gel, these variants of the enrichment experianents are more suitable for the observation of high enrichmea~t factors Example ' XIIL: ( i.c ] Sel.ec'tiora of phages carrying an 3~ abzyme In this case, the covalent inhibitor will consist of a suicide a.mhibitor characteristic of the reaction to be catalyzed by the abzyme or by an analog of the specific ~rarasition state.
~%~'~ ~~~
~- Zs -The detection and selection of these phages are then carried out in .a rraanner identical to the detection and selection of the phages which expose active enzymes at their surface.
Claims (25)
1. Method for selecting recombinant micro-organisms containing at their surface at least one molecule having enzymatic activity, characterized in that the recombinant microorganisms are immobilized on a solid support by a covalent inhibitor of the active site of the molecule having enzymatic activity, connected to the solid support by an arm; in that the recombinant microorganisms or all or part of their genome are recovered and in that all or part of their genome is isolated.
2. Method of selection according to claim 1, characterized in that prior to their immobilization on the solid support, the recombinant microorganisms are labeled with the covalent inhibitor of the active site of the molecule having enzymatic activity, connected by an arm to a ligand which can be immobilized on the solid support.
3. Method of selection according to any one of claims 1 or 2, characterized in that the microorganisms are viruses, preferably phages such as filamentous phages.
4. Method of selection according to any one of claims 1 or 2, characterized in that the microorganisms are phagemids.
5. Method of selection according to any one of the preceding claims, characterized in that the solid support consists of a chromatographic column on which avidin, streptavidin and/or an antibody is immobilized.
6. Method of selection according to any one of the preceding claims, characterized in that the molecules having enzymatic activity are chosen from the group consisting of enzymes, abzymes, catalytic peptides or a mixture thereof.
7. Method of selection according to any one of the preceding claims, characterized in that the microorganisms are recovered by detachment from their solid support.
8. Method of selection according to any one of the preceding claims, characterized in that the recombinant microorganisms are recovered by cleavage of the arm connecting the covalent inhibitor to the solid support.
9. Method of selection according to any one of claims 1 to 7, characterized in that the recombinant microorganisms are recovered by detaching the ligand from the solid support with an eluting solution, preferably a solution at an acidic pH.
10. Method of selection according to any one of claims 1 to 7, characterized in that the recombinant microorganisms are recovered by cleavage of the bond between the covalent inhibitor and the molecule having enzymatic activity.
11. Method of selection according to any one of claims 1 to 7, characterized in that the recombinant microorganisms are recovered by cleavage of the peptide linkage between the molecule having enzymatic activity and the microorganisms.
12. Method of selection according to claim 11, characterized in that the cleavage is performed with a protease.
13. Method of selection according to claim 11, characterized in that the cleavage is performed with cyanogen bromide.
14. Method of selection according to claim 11, characterized in that the cleavage is performed with hydroxylamine.
15. Method of selection according to any one of claims 1 to 7, characterized in that the recombinant microorganisms are destroyed and that all or part of their genome is recovered, isolated and cloned.
16. Marker used in the process according to any of the preceding claims characterized in that it consists of a covalent inhibitor of the active site of a molecule having enzymatic activity, connected by an arm to a ligand which can be immobilized on a solid support.
17. Marker according to claim 16, characterized in that the covalent inhibitor is an irreversible inhibitor.
18. Marker according to claim 16 or 17, characterized in that the ligand is a biotin or an iminobiotin.
19. Marker according to any one of claims 16 to 18, characterized in that the arm contains a cleavable functional group, preferably a disulfide functional group.
20. Marker according to any one of the preceding claims 16 to 19, characterized in that the covalent inhibitor is an analog of the specific transition state of the enzymatic reaction performed by the molecule having enzymatic activity.
21. Method for preparing the marker according to any one of claims 16 to 20, characterized in that a covalent inhibitor of the active site of a molecule with enzymatic activity is coupled by an arm to a ligand which can be immobilized on a solid support.
22. Solid support used in the process according to any of the preceding claims, characterized in that it comprises a covalent inhibitor of the active site of a molecule having enzymatic activity, connected to a solid support by an arm.
23. Solid support according to claim 22, characterized in that the covalent inhibitor connected to the solid support consists of the marker according to any one of claims 17 to 20.
24. Solid support according to claim 22, characterized in that the arm is connected to the solid support by an ester linkage or contains a vicinal diol.
25. Method for preparing a solid support according to any one of claims 22 to 24, characterized in that a covalent inhibitor of the active site of a molecule having enzymatic activity is connected by an arm to a solid support.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| BE9101106 | 1991-11-29 | ||
| BE9101106A BE1006312A3 (en) | 1991-11-29 | 1991-11-29 | Selection process for recombinant microorganisms with their area at least one molecule enzymatic activity. |
| PCT/BE1992/000052 WO1993011242A1 (en) | 1991-11-29 | 1992-11-30 | Method for selecting recombinant micro-organisms of which the surface comprises at least one molecule having enzymatic activity |
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| EP (1) | EP0617737B1 (en) |
| JP (1) | JP4125364B2 (en) |
| KR (1) | KR100243997B1 (en) |
| AT (1) | ATE147783T1 (en) |
| AU (1) | AU671089B2 (en) |
| BE (1) | BE1006312A3 (en) |
| BR (1) | BR9206970A (en) |
| CA (1) | CA2124580C (en) |
| DE (1) | DE69216853T2 (en) |
| DK (1) | DK0617737T3 (en) |
| ES (1) | ES2098555T3 (en) |
| FI (1) | FI109131B (en) |
| HU (1) | HU220876B1 (en) |
| NO (1) | NO313593B1 (en) |
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| IL104829A (en) * | 1992-02-24 | 1999-04-11 | Igen Inc | Reaction-based selection for expression of and concentration of catalytic moieties |
| DE60031965D1 (en) * | 1999-04-14 | 2007-01-04 | Us Gov Health & Human Serv | HIGHLY SENSITIVE DETECTION OF BIOMOLECULES BY "PHAGE DISPLAY" |
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| JPS5325030B2 (en) * | 1972-12-19 | 1978-07-24 | ||
| US4165258A (en) * | 1975-10-08 | 1979-08-21 | University Of Pennsylvania | Plasminogen activating enzyme-specific competitive inhibitor |
| AU1043788A (en) * | 1986-12-01 | 1988-06-30 | Mcdonnell Douglas Corporation | Method of identifying unknown organisms |
| DE3833628A1 (en) * | 1988-10-03 | 1990-04-12 | Genlux Forschungsgesellschaft | METHOD FOR DETECTING AND IDENTIFYING TOXIC SUBSTANCES USING CLONED MICROORGANISMS |
| EP0437537B1 (en) * | 1988-10-04 | 1997-02-26 | DNA Plant Technology Corporation | Bacterial detection by phage transduction of detectable phenotype |
| JP2810138B2 (en) * | 1989-08-23 | 1998-10-15 | 昭和電工株式会社 | Filler for measuring enzyme activity, method of using the same and apparatus therefor |
| ATE102074T1 (en) * | 1989-09-29 | 1994-03-15 | Rohm & Haas | LIGAND-CONTAINING MEDIUM FOR CHROMATOGRAPHIC SEPARATION, PROCESS FOR ITS PREPARATION AND ITS USE FOR ISOLATION OF SYNTHETIC OR NATURAL MOLECULES FROM A FLUID MIXTURE. |
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| HU220876B1 (en) | 2002-06-29 |
| FI942469A (en) | 1994-05-26 |
| EP0617737A1 (en) | 1994-10-05 |
| NO313593B1 (en) | 2002-10-28 |
| ES2098555T3 (en) | 1997-05-01 |
| CA2124580A1 (en) | 1993-06-10 |
| AU2937392A (en) | 1993-06-28 |
| BR9206970A (en) | 1995-12-19 |
| NO941731L (en) | 1994-06-21 |
| FI942469A0 (en) | 1994-05-26 |
| EP0617737B1 (en) | 1997-01-15 |
| DK0617737T3 (en) | 1997-07-07 |
| HUT70310A (en) | 1995-09-28 |
| HU9401593D0 (en) | 1994-08-29 |
| AU671089B2 (en) | 1996-08-15 |
| KR100243997B1 (en) | 2000-02-01 |
| JPH07505999A (en) | 1995-07-06 |
| SG48094A1 (en) | 1998-04-17 |
| DE69216853T2 (en) | 1997-06-26 |
| FI109131B (en) | 2002-05-31 |
| JP4125364B2 (en) | 2008-07-30 |
| NO941731D0 (en) | 1994-05-10 |
| WO1993011242A1 (en) | 1993-06-10 |
| DE69216853D1 (en) | 1997-02-27 |
| ATE147783T1 (en) | 1997-02-15 |
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