AU764664B2 - Enzyme activity screen with substrate replacement - Google Patents

Description

WO 00/11211 PCT/DK99/00441 ENZYME ACTIVITY SCREEN WITH SUBSTRATE REPLACEMENT TECHNICAL FIELD The present invention relates to a library of catalysts of interest coupled to a substrate by an exchangeable linker pair, X and Y, and a selection method that uses multiple catalytic turnover events to isolate the more active of the catalysts in said library.

BACKGROUND OF THE INVENTION In the past, novel biopolymer DNA, RNA or polypeptide) based catalysts have been created in several different ways. The following paragraphs describe some of these selection schemes.

Binding to transition state analogs Catalytic RNA, DNA and protein (particularly antibodies) have been isolated by this approach. It has mostly been applied to the isolation of catalytic antibodies by immunization of mice with transition state analogs (TSA), also antibodies displayed on phage as well as RNA and DNA libraries have been challenged with TSA. The idea is that a molecule (protein, RNA or DNA) that binds a given TSA is likely to bind the substrate and stabilize the geometry and/or energetics of the transition state. This may result in catalysis.

The method does not select for catalytic activity per se, but rather for binding to a transition state analog (TSA). However, it has been included here as it is currently one of the most used methods to isolate novel catalysts. Problems encountered with this approach include: i) Detailed mechanistic knowledge of the target reaction is required (in order to design an ap- WO 00/11211 PCT/DK99/00441 2 propriate TSA); ii) In many cases a TSA that adequately resembles the transition state is unobtainable or unstable; iii) It is not possible to mimic the structural and electronic dynamics of the reaction coordinate.

Consequently, a rather limited set of reaction types have been successfully targeted by this approach. In most cases the isolated catalysts have poor turn-over numbers.

Functional tagging of active catalysts This selection scheme has been applied to protein and nucleic acids. The substrate is designed so that a reactive product is formed during the reaction (the substrate is called "suicide substrate" or "inhibitor analog"). The reactive product is likely to react with the catalyst that produced it, to form a covalent bond. As a result, active catalysts can be separated from inactive ones by way of the attached label.

Catalytic antibodies displayed on phage have been isolated by this method, and it was shown in a model system that catalytically active and inactive proteins could be separated using this approach. The method should allow the isolation of rare catalysts.

Important limitations with this approach include: i) For many reactions it is not possible to design an appropriate suicide substrate. ii) Successful catalysts need only perform one turn-over during the selective process/round, which is typically on the order of minutes. Hence, there is no selective advantage for efficient catalysts.

Continuous evolution (RNA) RNA libraries have been designed that contain both the substrate and the potentially catalytic domain in the same WO 00/11211 PCT/DK99/00441 3 molecule. RNAs capable of performing the desired reaction (typically ligation) will "activate" themselves for amplification (reverse transcription followed by RNA polymerase transcription). By adequate dilutions and additions of nucleotide precursors this continuous selection can be maintained over several hours, and then analyzed.

The method has two important limitations: i) Both the substrate and the catalyst must be a nucleic acid; ii) As the catalyzed reaction and the amplification of successful enzymes is not separated, the time of the selective step is the sum of the turn-over time of the target reaction and the time of amplification of the "activated" molecules. Thus, as the amplification is on the order of seconds, there is no selective advantage for an efficient catalyst.

4. Substrate-Enzyme-Linked Selection (SELS).

Recently, methods have been described, involving the attachment of the substrate of the target reaction to a protein with potential catalytic activity towards the attached substrate (Pedersen et al., Proc. Natl. Acad. Sci., US, 1998, vol.

pp. 10523-10528; Jestin et al., 1999, Angew. Chem. Int.

Ed., vol. 38, pp. 1124-1127; Demartis et al., 1999, JMB, vol.

286, pp. 617-633; Neri et al., 1997, WO 97/40141). Upon intramolecular conversion of the substrate, the active catalyst can be isolated by means of the attached product.

This scheme is very general. However, since successful catalysts need only perform one turn-over during the selective process/round, which is typically in the order of minutes, there is no selective advantage for efficient catalysts. For the same reason, it presumably is not possible to distinguish WO 00/11211 PCT/DK99/00441 4 enzymes with slightly different specific activity with this selection scheme.

SUMMARY OF THE INVENTION The problem to be solved by the present invention is to provide a method for in vitro selection, from a library of catalyst molecules, of a catalyst molecule of interest having a relatively more efficient specific catalytic activity of interest, as compared to the rest of the catalyst molecules i0 within said library, and wherein said in vitro selection method is characterised by that it allows multiple catalytic activity turn-overs substrate to product catalytic activity turnovers), by the catalyst molecule of interest, before it is finally collected.

The solution is based on using a novel sample comprising a number of individual units in said in vitro selection method.

A summary of the characteristics of said novel sample is given immediately below.

Said novel sample comprises a library of catalyst molecules provided in the form of individual units, wherein the individual units comprise a first type individual unit having the following general structure:

C-XY-S,

wherein C denotes a catalyst molecule, XY an XY exchange pair, and S a substrate which is capable of being catalysed into a product by at least one catalyst comprised within said library of catalyst molecules and thereby providing the possibility of obtaining a second type individual unit comprising the general structure:

C-XY-P,

wherein C and XY has the meaning defined above and P is the product molecule resulting from the catalytic conversion of WO 00/11211 PCT/DK99/00441 the substrate S of the first type individual unit. See figure 1 for a graphic illustration of a suitable example of such an individual unit.

Said novel sample, is then characterised by that it comprises following functionally defined features: Feature i: The substrate S is attached to the catalyst in a configuration that allows catalytic reaction between the catalyst and the substrate within said individual unit; and the nature of said attachment of the substrate and the catalyst provides the possibility, by means of a characteristic of the product, of isolating an entity comprising information allowing the unambiguous identification of the catalyst molecule which has been capable of catalysing the reaction substrate molecule to product molecule.

For illustration reference is made to figure 1, where is shown a suitable example of such an individual unit comprising feature 1 above.

Feature 2: Said sample comprising a number of individual units and comprising feature 1 above is further characterised by that said XY exchange pair allows an asymmetric exchange of the Ymoiety with another Y-moiety Y exchanges with Y, not with whereby said XY exchange pair then allows an exchange reaction between the unit structure: a catalyst an XY exchange pair a product and a "Y substrate" component and thereby generating the unit structure a catalyst an XY exchange pair a substrate.

WO 00/11211 PCT/DK99/00441 6 For illustration reference is made to figure 2, where is shown a suitable example of such an individual unit comprising feature 2 above.

Using such a novel sample in a in vitro selection method as described herein (vide infra), provides then the possibility of selecting a catalyst molecule of interest essentially only based on a characteristic of the product molecule which has been generated by the catalyst molecule of interest (Feature 1 allows this; see figure 1 for an illustration); and it further provides the possibility of selecting a catalyst molecule of interest having a relatively more efficient specific catalytic activity of interest, as compared to the rest of the catalyst molecules within said library, and wherein said selection is characterised by that said catalyst molecule of interest is first finally collected after it has been allowed to perform multiple catalytic activity turn-overs substrate to product catalytic activity turn-overs) (Feature 2 allows this; see figure 2 for an illustration) Accordingly, a first aspect of the invention relates to a sample comprising a number of individual units suitable for use in an in vitro selection system, wherein the purpose of said in vitro selection system is, from a library of catalyst molecules, to select a catalyst molecule of interest having a relatively more efficient specific catalytic activity of interest as compared to the rest of the catalyst molecules within said library, and wherein said in vitro selection system is characterised by that it allows multiple catalytic activity turn-overs substrate to product catalytic activity turnovers), by the catalyst molecule of interest, before it is finally collected and wherein said sample comprises, WO 00/11211 PCT/DK99/00441 7 a library of catalyst molecules provided in the form of individual units, wherein the individual units comprise a first type individual unit having the following general structure:

C-XY-S,

wherein C denotes a catalyst molecule, XY an XY exchange pair, and S a substrate which is capable of being catalysed into a product by at least one catalyst comprised within said library of catalyst molecules and thereby providing the possibility of obtaining a second type individual unit comprising the general structure:

C-XY-P,

wherein C and XY has the meaning defined above and P is the product molecule resulting from the catalytic conversion of the substrate S of the first type individual unit; and the substrate S is attached to the catalyst in a configuration that allows catalytic reaction between the catalyst and the substrate within said individual unit; and the nature of said attachment of the substrate and the catalyst provides the possibility, by means of a characteristic of the product, of isolating an entity comprising information allowing the unambiguous identification of the catalyst molecule which has been capable of catalysing the reaction substrate molecule to product molecule; and said sample comprising a number of individual units is characterised by that said XY exchange pair allows an asymmetric exchange of the Y-moiety with another Ymoiety Y exchanges with Y, not with whereby said XY exchange pair then allows an exchange reaction between the unit structure: WO 00/11211 PCT/DK99/00441 8 a catalyst an XY exchange pair a product and a "Y substrate" component and thereby generating the unit structure a catalyst an XY exchange pair a substrate.

The term "an individual unit", comprised within a sample according to the first aspect as described above, denotes an individual unit comprising the general structure as specified under point in the first aspect of the invention; a i0 substrate molecule attached to a catalyst molecule as specified under point and in the first aspect of the invention; and an XY exchange pair as specified under point in the first aspect of the invention. See figure 1 and 2 for a suitable example of such an individual unit.

The term "an individual unit comprising the general structure: a catalyst an XY exchange pair a substrate; or a catalyst an XY exchange pair a product" denotes that said individual unit comprises at least one molecule of each of said entities, i.e. at least one catalyst molecule, at least one XY exchange pair molecule, and at least one substrate molecule or at least one product molecule. Accordingly, said individual unit may for instance comprise more than one copy of an identical catalyst molecule or may comprise several different catalyst molecules.

Further the term placed between the individual entities within said individual unit denotes that there is a physical connection between said individual entities within said individual unit, i.e. that there is a physical connection between a catalyst an XY exchange pair a substrate or a catalyst an XY exchange pair a product.

Further, "an individual unit" as described herein denotes an individual unit wherein it is possible to physically WO 00/11211 PCT/DK99/00441 9 separate said individual unit from the other different individual units, within said sample, in order to be able to isolate the separate individual unit.

The term "different individual units" denotes different individual units each independently comprising different catalyst molecules, i.e. an example of two different individual units may be catalyst molecule' XY exchange pair substrate; and catalyst molecule 2 XY exchange pair substrate; wherein catalyst molecule' and catalyst molecule 2 denotes two different catalyst molecules.

The term "a sample comprising a number of different individual units" denotes a sample comprising at least two different individual units, preferably at least 100 different individual units, more preferably at least 10.000 different individual units, more preferably at least 106 different individual units, more preferably at least 108 different individual units, more preferably at least 1010 different individual units, even more preferably at least 1012 different individual units, and most preferably at least 10". different individual units. Basically the actual number of different individual units corresponds to the actual size of the library of catalyst molecules.

The term "a sample comprising a number of individual units" and the term "a sample comprising a number of different individual units" may be used interchangeably herein.

The term "a library of catalyst molecules" denotes a library comprising at least two different catalyst molecules, preferably at least 100 different catalyst molecules, more preferably at least 10.000 different catalyst molecules, more preferably at least 106 different catalyst molecules, more preferably at least 108 different catalyst molecules, more WO 00/11211 PCT/DK99/00441 preferably at least 1010 different catalyst molecules, even more preferably at least 1012 different catalyst molecules, and most preferably at least 1014 different catalyst molecules.

The term "a substrate capable of being catalysed into a product molecule by at least one catalyst molecule comprised within said library of catalyst molecules" basically denotes any suitable substrate molecule. Essentially said substrate molecule is chosen according to the specific catalytic activity which it is desired to select for. For instance, if the desired catalytic activity is a protease activity then a suitable substrate may be a peptide molecule and the product will then be a degraded peptide. The terms "substrate" and "substrate molecule" may be used interchangeably.

The term "product" denotes the product obtained by the catalytic reaction substrate to product by a catalyst of interest as specified herein. The terms "product" and "product molecule" may be used interchangeably.

The term "catalyst" denotes any catalyst molecule with a desired catalytic activity, such as organic and inorganic molecules, proteins, enzymes, peptides, nucleic acids, biopolymers and non-biological polymers, small organic or inorganic molecules. The terms "catalyst" and "catalyst molecule" may be used interchangeably.

The term "the substrate is attached to the catalyst in a configuration that allows catalytic reaction between the catalyst and the substrate within said individual unit" denotes a direct or indirect physical connection, within each of the individual units, between substrate and catalyst. This connection should preferably maximize productive interaction of the catalyst and the substrate, within the individual unit, while minimizing the interaction of catalysts and substrates on different individual units.

WO 00/11211 PCT/DK99/00441 11 The term "the nature of said attachment of the substrate and the catalyst provides the possibility, by means of a characteristic of the product, of isolating an entity comprising information allowing the unambiguous identification of the catalyst molecule which has been capable of catalysing multiple times the reaction substrate molecule to product molecule" according to point of the first aspect of the invention denotes that said entity is isolated by means of one or more characteristic of the product.

An example of a suitable characteristic of the product may be that said product does not bind to a matrix and the substrate does bind to a matrix. In this case a suitable selection protocol may be that the individual units are bound to the solid support on the form a catalyst an XY exchange pair a substrate matrix, and released when it is on the form catalyst an XY exchange pair a product. For a detailed description of an example of such a system reference is made to a working example herein (vide infra).

Another example of a suitable characteristic of the product may be that said product binds to a receptor as illustrated in figure i.

The term "an entity comprising information allowing the unambiguous identification of the catalyst molecule which has been capable of catalysing the reaction substrate molecule to product molecule" according to point in the first aspect of the invention, denotes either an entity wherein said information is carried in the catalyst molecule as such or an entity comprising other kind of information providing the possibility of unambiguously identifying the catalyst. Such other kind of information may for instance be an entity comprising a DNA sequence encoding a peptide or a polypeptide when the catalyst molecule of interest is a peptide or a polypeptide. An illus- WO 00/11211 PCT/DK99/00441 12 tration of this may be when the isolated entity is a filamentous phage comprising a DNA sequence encoding a polypeptide of interest attached on the surface of said phage. See e.g. figure 12 and below for further details.

The term "XY exchange pair", comprised within an individual unit as specified above, denotes that the a catalyst is attached to a substrate through an XY exchange moiety, i.e. the individual unit has the following general structure: a catalyst an XY exchange pair a substrate and said XY exchange pair I0 fulfils the criteria according to point in the first aspect of the invention.

Preferably, the XY moiety is stable in the absence of free Y, but allows fast and specific exchange of free Y with Y bound to X (but not exchange of free Y with This exchange reaction can replace product with substrate, if the individual unit: a catalyst an XY exchange pair a product is in contact with a Y-Substrate compound.

The XY exchange pair may for instance have following characteristics: i) X and Y can be covalently or non-covalently bonded; and ii) the XY exchange pair may consist of any kind of molecules, including small organic EDTA) and inorganic (eg. metals, phosphates) molecules as well as macromolecules nucleic acids, peptides) See below for further details and figure 2, 3 and 4 for graphic illustrations.

In a second aspect the invention relates to a method for in vitro selection, from a library of catalyst molecules, of a catalyst molecule of interest having a relatively more efficient specific catalytic activity of interest as compared WO 00/11211 PCT/DK99/00441 13 to the rest of the catalyst molecules within said library and wherein said in vitro selection method is characterised by that it allows multiple catalytic activity turn-overs (i.e.

substrate to product catalytic activity turn-overs), by the catalyst molecule of interest, before it is finally collected and wherein said method comprises following steps, placing a sample comprising a number of individual units according to the invention under suitable conditions where a catalyst molecule of interest performs its catalytic activity of interest and further under conditions wherein said individual units are in contact with an Y substrate compound; (ii) selecting for a catalyst of interest by selecting for one or more individual unit(s) which comprise(s) the product molecule; and (iii) isolating an entity comprising information allowing the unambiguous identification of the catalyst molecule of interest which has been capable of catalysing multiple times the reaction substrate to product, by means of a characteristic of the product; and optionally (iv) repeating step to (iii) one or more times by using the information comprised in said entity of step (iii) to generate the catalyst molecule of interest and construct an individual unit comprising said generated catalyst molecule of interest and then using this individual unit as a starting material in said repetition step.

The term "under suitable conditions where a catalyst molecule of interest performs its catalytic activity of interest" according to step of the second aspect of the WO 00/11211 PCT/DK99/00441 14 invention, denotes any suitable conditions where a catalyst molecule of interest performs its catalytic activity of interest.

Such suitable conditions may be alkaline pH if the purpose of the selection is to identify a catalyst of interest having activity at alkaline pH.

The term "said individual units are in contact with an Y substrate compound" according to point in second aspect of the invention denotes that the individual units and the Y substrate compound are appropriately close that an exchange reaction, as specified under point in the first aspect of the invention, is possible. Said contact may for instance be in a buffer solution wherein the individual units and the Y substrate compound may diffuse together and thereby get in contact with each other. See figure 2 for graphic illustrations.

The term "the catalyst molecule of interest which has been capable of catalysing multiple times the reaction substrate to product" according to step (iii) of the second aspect of the invention denotes that said catalyst molecule of interest has performed the catalytic reaction substrate to product at least two times, more preferably at least 100 times, more preferably at least 10.000 times, even more preferably at least 106 times, and most preferably at least 1010 times.

The term "an entity comprising information allowing the unambiguous identification of the catalyst molecule of interest" denotes either an entity wherein said information is carried in the catalyst molecule as such or an entity comprising other kind of information providing the possibility of unambiguously identifying the catalyst. Such other kind of information may for instance be an entity comprising a DNA sequence when the catalyst molecule of interest is a peptide or WO 00/11211 PCT/DK99/00441 a polypeptide. This is the same definition as described above for the same term in relation to point of the first aspect of the invention (vide supra).

The term "repeating step to (iii) one or more times by using the information comprised in said entity of step (iii) to generate the catalyst molecule of interest and construct an individual unit comprising said generated catalyst molecule of interest and then using this individual unit as a starting material in said repetition step" according to point (iv) in the second aspect of the invention denotes that said repetition may be one time, more preferably 2 times, more preferably more than 5 times, even more preferably more than 10 times, and most preferably more than 25 times.

An advantage of the method for in vitro selection as described above is that it allows the catalyst molecules to perform multiple turn-overs of substrate to product during one selection round before the catalyst molecule(s) of interest is finally collected).

This is fundamentally different from selection protocols previously described in the art, which either involved binding to a transition state analog of the target reaction, wherein there is no turn-over of substrate (see #1 in "Background" above) or a single turn-over of substrate and 3 in "Background" above).

This may provide two advantages, which may be illustrated by following a possible selection scheme using the method of the invention: a) placing, according to point of the method of second aspect of the invention, the sample comprising a number of individual units under suitable conditions where a catalyst molecule of interest exhibits its WO 00/11211 PCT/DK99/00441 16 catalytic activity of interest and further under conditions wherein said individual units are in contact with an Y substrate molecule at one end (starting end) of a product-binding column wherein a receptor specifically binding the product is coupled to the matrix of the product-binding column and with a suitable amount of the Y substrate within the product-column buffer; b) selecting, according to point (ii) of the method of second aspect of the invention, for a catalyst molecule of interest by selecting for one or more individual unit(s) which comprise(s) the product molecule, by collecting the individual unit(s) which arrive(s) latest to the opposite end (collecting end) of the column.

Within this selection scheme the following events, among others, take place within the product-column: 1) individual units comprising a potential catalyst molecule of interest converts the attached substrate to product; 2) said individual units, now comprising the product, are diffusing to and binding to a receptor placed close to the starting end of the product column; 3) the Y substrate molecules, within the productcolumn buffer mediates the exchange reaction catalyst molecule XY exchange pair product and a Y substrate molecule and thereby generating the unit structure catalyst molecule XY exchange pair substrate, and thereby mediates release of said bound individual units of step above; WO 00/11211 PCT/DK99/00441 17 4) said released individual units of step above are now comprising a substrate and a potential catalyst molecule of interests, which has made one catalytic conversion of the substrate to product, and is now regenerated in the original form of the individual units of point 1. Accordingly, said units can therefore once more perform reaction binding to a receptor relatively closer to the collecting end of the product column etc.

The catalyst molecule of interest having most efficient specific catalytic activity of interest will perform most substrate to product conversions in a given time interval, and as a result, will spend more time immobilised on the productbinding receptor. Therefore, the individual unit(s) comprising said most efficient catalyst molecule will arrive latest to the collecting end of said column.

Using this example of a method of the second aspect of the invention, two advantages over the art may be: i) that the possible "selectable" catalytic activity may be much higher than for the prior art selection protocols. A selective step involves performing the target reaction, diffusion to and binding of product to product-binding column, and finally exchange of product and substrate by the XY-exchange reaction. Since diffusion is generally a fast process, the selection stringency can be controlled simply by the amount of product binding receptor on the column or the type/amount of Y-S component in the product-column buffer; moreover, if for example electrophoresis is used as the means of isolating the more active catalysts, diffusion to the receptor may not be a limiting factor, wherefore the WO 00/11211" PCT/DK99/00441 18 selection stringency may be even higher with this system; ii) minor differences in activity can be distinguished; Since the selective step is reiterated many times as the catalysts flow through the column, even minor differences in catalyst activity will result in differential retention on the column, and thus differential enrichments.

In a final aspect the invention relates to a method for producing a catalyst molecule of interest comprising performing the method for in vitro selection according to the invention and the further following step, producing said isolated catalyst molecule of interest in a suitable quantity of interest by a suitable production method.

DRAWINGS:

Figure 1: Graphic illustration of a suitable example of a first type individual unit having the following general structure:

C-XY-S,

wherein C denotes a catalyst molecule, XY an XY exchange pair, and S a substrate. The substrate S is attached to the catalyst in a configuration that allows catalytic reaction between the catalyst and the substrate within said individual unit. The catalytic reaction results in a second type individual unit:

C-XY-P,

wherein C denotes a catalyst molecule, XY an XY exchange pair, and P denotes a product molecule.

The nature of said attachment of the substrate and the catalyst provides the possibility, by means of a characteristic of the product binding affinity to a matrix), of isolating an WO 00/11211 PCTIDK99/00441 19 entity the catalyst itself or a phage displaying it) comprising information allowing the unambiguous identification of the catalyst molecule which has been capable of catalysing the reaction from substrate molecule to product molecule.

Figure 2: Graphic illustration of an individual first type unit wherein the XY exchange pair allows an asymmetric exchange of the Y-moiety with another Y-moiety Y exchanges with Y, not with After the target reation has taken place (figure 1) said XY exchange pair allows an exchange reaction between the second type unit structure: C-XY-P and an unbound Y-S component, thereby generating anew a first type unit structure C-XY-S and an unbound Y-P component, whereafter the catalyst of this unit can perform yet another catalytic reaction on the freshly bound substrate.

A matrix binding the product, P, would effectively keep Y-P components bound as well as C-XY-P type individual units, until an exchange reaction such as described immediately above occured, effectively releasing the catalyst from the matrix in the form of a freshly generated first type unit C-XY-S leaving the Y-P component behind bound to the matrix.

Figure 3: Graphic illustrations of suitable XY exchange pairs of an individual unit according to the invention. The catalytic reaction facilitates dissociation of X and Y X is a DNA strand, Y is a complementary DNA/RNA/DNA hybrid or a DNA/RNA/matrix hybrid, C is an RNase).

Other examples are: Non-covalent bonds between X and Y (His 4 -Fe- IDA and NTA-Fe-IDA), and covalent bond between X and Y (disulfide) Note that symmetric exchange units (XX) like a disulfide bond can be used as exchange units WO 00/11211 PCT/DK99/00441 Figure 4: Graphic illustration of an individual unit, in which the XY exchange unit is another enzyme-substrate pair.

Figure 5: Graphic illustration of the Y-substrate molecule described in example 2 below.

Figure 6: Graphic illustration of the selection scheme described in example 4 below. Optimization of an enzyme that catalyzes aldol formation.

Figure 7-8: Graphic illustrations of suitable selection schemes according to a method for in vitro selection according to the second aspect of the invention.

Figure 9-11: Figures supporting the description in working example 1 herein (vide infra).

Figure 9. Structures and synthesis of base-linker-substrate conjugates.

Figure 10. Covalent attachment of substrate to the pIII protein on phage. DNA encoding the acid peptide sequence and a C-terminal cysteine was fused to the Nterminal end of gene gIII, to form the acid helper phage. A phagemid encodes the protein library in fusion with the pIII protein; Phage production leads to phage particles displaying the phagemid encoded protein; the pIII proteins have acid peptide extensions; Coiled-coil formation of the acid and base peptides noncovalently attaches the substrate to the phage pIII protein; Removal of the reducing agent leads to crosslinking of acid and base peptides through their C-terminal cysteines; In the pres- WO 00/11211 PCT/DK99/00441 21 ent study phages displaying staphylococcal nuclease are attached to streptavidin beads through a biotinylated, single-stranded oligodeoxynucleotide.

Phages displaying active enzyme are released by cleavage of the oligodeoxynucleotide in an intramolecular reaction.

Figure 11:Immobilization and cleavage of phage from solid support. Either no base-linker, the base-linker-pTp or the base linker-oligodeoxynucleotide conjugate was crosslinked to phage displaying SNase or the control protein Fab 39-A11. Columns 1-4 show immobilization on streptavidin beads. Immobilization was either examined by phage titering of the beads directly (columns or after DNase I treatment of the beads (column columns 5-7 show leakage (release in absence of Ca 2 columns 8-10 shows Ca 2 induced release (cleavage). The per cent recovery is shown in parantheses above the columns.

Figure 12: Figure supporting the description in working example herein (vide infra).

Figure 13: Figure supporting the description in working example 6 herein (vide infra).

Figure 14: Figure supporting the description in working example 7 herein (vide infra). Enrichment of cells producing the more active proteases.

WO 00/11211 PCT/DK99/00441 22 Figure 15: Figure supporting the description in working example 8 herein (vide infra). Isolation of hammerhead ribozymes.

Figure 16: Data supporting working example 10 herein (vide infra). Enrichment of wildtype lipase in a background of less active variants.

Figure 17: Figure supporting the description in working example 10 herein (vide infra). Synthesis of Y-substratebiotin conjugates.

Figure 18: Data supporting the description in working example 11 herein (vide infra). Enrichment of wildtype cellulase C6B, in a background of less active cellulase variants.

Figure 19: Figure supporting the description in working example 11 herein (vide infra). The principle of substrate reloading in the context of the phage-displayed cellulase.

Figure 20: Figure supporting the description in working example 12 herein (vide infra). Enrichment of wildtype RNase A in a background of less active RNase A variants.

Figure 21: Data supporting the description in working example 14 herein (vide infra). Measurement of exchange rates by fluorescence polarization spectroscopy.

Embodiment(s) of the present invention is described below, by way of examples only.

WO 00/11211 PCT/DK99/00441 23 DETAILED DESCRIPTION OF THE INVENTION: A sample comprising a number of different individual units according to first aspect of the invention XY exchange pair: As described above, within an individual unit, the catalyst is physically connected to substrate through an XY exchange pair. Thus, the individual unit is having the general structure a catalyst an XY exchange pair a substrate.

Further, as described above the term "XY exchange pair", comprised within an individual unit as specified above, denotes that a catalyst is attached to a substrate through an XY exchange moiety, i.e. the individual unit has following general structure: a catalyst an XY exchange pair a substrate and said XY exchange pair fulfils the criteria's according to point in first aspect of the invention.

Further, said exchange pair is preferably stable in the sense that the unit a catalyst an XY exchange pair a substrate will remain connected in the absence of another Y substrate component, this means that under the conditions of the assay the X moiety should ideally at any given time be loaded with substrate. This may be the case if the Y-substrate concentration is significantly higher than the dissociation constant Kd of the XY interaction under the conditions employed. In most cases, the maximum Y-substrate concentration will be of the order of 1 mM or less. Therefore, the dissociation constant Kd should preferably be less than 10 4 M. The dissociation constant is given by Kd koff/kon. The on-rate kon is generally limited by diffusion, with second-order rate constants for association of around 106-109 M 1 sec 1 Therefore, in order to obtain good exchange rates (more than one exchange per second), the off-rate koff should preferably be at least 1 sec 1 where- WO 00/11211 PCTIDK99/00441 24 fore the Kd in this case should not be less than 10- 9 M. Therefore, in most cases where an exchange rate of more than 1 sec is desired, the dissociation constant should be in the range 4 10- 9

M.

This preferred embodiment provides a system wherein the XY exchange reaction may said to be an associative replacement reaction in the sense that the units C-XY-S or C-XY-P are stable if they are not placed in contact with a Y-S compound.

An XY exchange pair comprises at least one X-moiety and at least one Y-moiety. However it may also comprise more than one copy and/or type of both X- and Y-moieties.

Further the X and Y moiety may be identical molecules.

Such an exchange pair may herein be termed an "XX exchange pair".

Preferably the X and Y moiety are different molecules.

For illustration as a non-limiting example the XY moiety is preferably stable in the absence of free Y, but allows fast and specific exchange of free Y with Y bound to X (but not exchange of free Y with This exchange reaction will replace product with substrate, if the individual units, as described above, are in contact with an Y substrate compound.

The XY exchange pair may have the following characteristics: i) X and Y can be covalently or non-covalently bonded.

The XY exchange pair can consist of any kind of molecules, including small organic EDTA) and inorganic (eg. metal, phosphate) molecules as well as macromolecules nucleic acids, peptides).

ii) The XY exchange reaction is preferably described as an active replacement reaction.

iii) The XY unit is preferably stable in the absence of another

Y.

WO 00/11211 PCT/DK99/00441 iv) The XY unit is preferably asymmetric, i.e. Y will only replace Y, not X.

v) The exchange of Y by Y is preferably very fast.

vi) The association of X and Y is preferably a very fast process.

vii) The exchange unit may be symmetric a XX exchange unit).

Suitable examples of XY exchange pairs are given below.

Further, graphic illustrations of said examples are given in figures 3 and 4.

i) Metal ligands. Preferably, the X moiety consists of a ligand and a metal ion, in a strong interaction described by a slow dissociation rate, basically making a very stable ligandmetal unit. The Y-moiety, on the other hand, is a ligand that coordinates to the same metal ion with high affinity, but with fast dissociation and association rates. Moreover, the exchange of one Y moiety with another Y-moiety is preferably a replacement reaction, for example because the Y-moiety is multi-dentate. Specific examples: a) X: EDDA'-Ca (EDDA, ethylendiaminediacetato, corresponds to EDTA in which the two acetic acids coupled to one of the nitrogens has been removed. EDDA thus makes four-fold coordination to Ca Y: R-N(CH2COO-)2, a bidentate ligand, or Y: R-N(CH2COO-) (CH2)2N(CH2COO-) 2 a tridentate ligand (Ca can make up to nine-fold coordination to certain ligands).

b) X: His6-Ni presumably a four-fold coordination. The histidine tag is used for recombinant protein purification, and the six histidines may be inserted at the N- or C-terminus of proteins, or into exposed loops on the surface of proteins.

WO 00/11211 PCT/DK99/00441 26 Cu, Ni, Zn, Fe, Cd, Co, Mg and other metals, preferably with fast exchange kinetics, may be used with the peptide based chelate. Other peptides that coordinate metals, such as peptides containing 2 or more histidines (Schmidt et al., 1996, Current Biology, vol. 3, pp. 645-653; Kotrba et al., 1999, Applied and Environmental Microbiology, vol. 65, pp. 1092- 1098), or the Cu-binding peptide Diglycyl-L-Histidine(Lau et al., 1974, The Journal of Biological Chemistry, vol. 249, pp.

5878-5884, may be used accordingly.

Y: R-N(CH2COO-) 2 a bidentate ligand.

c) X: His6-Ca+ has fast exchange characteristics with most ligands) Y: Y: R-N(CH2COO-) 2 a bidentate ligand, or R-N(CH2COO-)(CH2)2N(CH 2

COO-)

2 a tridentate ligand.

ii) Macromolecular exchange moieties (nucleic acids).

The Y moiety can consist of two polynucleotides, Y1 and Y2, that bind to overlapping sites on the X-moiety, which is also a polynucleotide. Preferably, the length and sequence of the oligos are adjusted so that the interactions of X with Y1 is stable in the absence of Y2, but Y2 can actively replace Y1 and vice versa.

The principle of overlapping regions of interaction should be generally applicable to exchange units of other chemical substances, such as proteins, polymers, organic or inorganic molecules. The principle is illustrated for polynucleotides: a) X: DNA polynucleotide 5'-GGGGTTGTTCCCC-3' Y: equimolar mix of DNA polynucleotides Yl: and Y2: b) X: DNA polynucleotide 5'-GGAAGGGATGGTCAC-3 WO 00/11211 PCT/DK99/00441 27 Y: Equimolar mix of polynucleotides Y1: and Y2: 3'-TCCCTAAGTG-3'.

iii) Covalent bonds. The formation and splitting of a covalent bond can be a rather fast process. In some cases this is an intrinsic characteristic of the bond in question. In other cases catalysts can speed up the rate at which the bond is formed and broken the. enzymatic transesterification below). Specific examples follow below; symbolises the site of attachment of the individual unit (catalyst), or substrate: a) X: Boric acid, R-B(OH)2 Y: a sugar, or other vicinal diol The "bidentate" nature of the interaction (the concerted formation and splitting of two bonds) should result in the active replacement of one sugar molecule with another.

b) X: R1-COOR2 (an ester) Y: HO-R2 (the corresponding alcohol) Thus, XY is in this case identical to X. To speed up the exchange rate, esterases may be added to the buffer. Likewise, transaminase-, transamylase- and other transferase-reactions can be exploited for the exchange of X and Y. For many of these reactions, it may be possible to employ the relevant transferase enzyme to speed up the exchange reaction.

c) X: R1-SH Y: Comprises two types of molecules, Yl: R2-SH (a free thiol) and Y2: R2-SS-R2 (a disulfide).

In its substrate-attached form, the catalyst is coupled to the substrate through a disulfide bond. Protein-disulfide isomerase may be added to the buffer to speed up the rate of exchange.

WO 00/11211 PCT/DK99/00441 28 d) X: vicinal dithiol, or paired thiols (for example, alphahelical peptide with cysteine at position i and i+l) Y: trivalent arsenic containing compound This type of vicinal dithiol-trivalent arsenic interaction has been used for the chromatographic purification of thiol containing proteins (Gitler et al., 1997, Analytical Biochemistry, vol. 252, pp. 48-55), or for the strong affinity interaction of biarsenical ligands with alpha-helical peptides containing cysteine at position i, i+1, i+4, and i+5 (Griffin et al., 1998, Science, vol. 281, pp. 269-271). The exchange reaction may be accelerated by including reducing agents such as DTT or 2-mercaptoethanol.

iv) Catalyst substrate pairs.

The interaction between a catalyst an enzyme) and a substrate is often described by two constants, the kct (turn-over number) and the Km. The kct can be said to broadly define the lifetime of the productive catalyst/substrate-complex. Therefore, one can obtain XY units with varying exchange rates (stabilities) by using for example different enzyme variants with different kcat's for a given substrate (where the enzyme represents X, and substrate represents Y) Figures 3 and 4.

Catalysts that interact with more than one substrate at a time may be particularly useful as XY exchange units.

v) XY interaction is modified by the reaction substrate to product. In certain cases the structure of the XY-Substrate moiety can be designed so that the reloading process is accelerated by the activity of the catalyst of the individual unit.

For example, if X or Y is both exchange unit and substrate for a cleavage reaction, the cleavage is likely to result in the dissociation of XY, wherefore the reloading process is likely WO 00/11211 PCT/DK99/00441 29 to happen faster. In example 8 below, X and Y are both nucleic acid structures. Y is both exchange unit and the substrate of the desired reaction (cleavage of a ribo-dinucleotide). Upon cleavage of Y the XY unit dissociates, which should speed up the association of X with another Y-substrate unit.

This principle can be applied more generally. For example, the central portion of Y in example 8 (the cleavage target) can be exchanged with the substrate of another desired reaction (see figure 3 Again, the conditions can be chosen so that the association of X and Yleft-Substrate-Yright is strong, but the interaction of X and Yieft-Productl, or X and Product 2 -Yright is weak. Therefore, immediately following cleavage of the substrate, X and Y fall apart. Which speeds up the substrate reloading process.

The substrate does not have to be part of X or Y for this event to take place. For example, if a conformational change, induced by the reaction substrate to product, is transmitted from the product to Y, and this conformational change results in the dissociation of XY, this wil also speed up the reloading process. An example is given in figure 3 B.

vi) Enzymes and other substances in the column buffer may speed up the exchange reaction in other ways. For example, the reaction substrate to product may initiate another reaction (catalyzed by a substance in the column buffer) that dissociates X and Y. For example, if the catalyst of the individual unit cleaves a nucleic acid, this may expose free or accessible to exonucleases. If the nucleic acid represents both the target substrate and the Y unit, the Y unit may be degraded by the nuclease, and therefore the XY complex dissociates. Alternatively, the XY complex (but not uncomplexed X or Y) may be the target for a substance in the column buffer; WO 00/11211 PCT/DK99/00441 if the substance modifies X or Y in the XY complex so that it dissociates faster, the presence of the substance will speed up the exchange rate. For example, if X and Y are complementary DNA and RNA oligos, RNaseH can be included in the buffer.

RNaseH cleaves RNA in duplex complexes; therefore, the RNA (Y) will not be cleaved, until it associates with the DNA to form the duplex. RNaseH will cleave the RNA but leave the DNA intact. Therefore, RNaseH accelerates the dissociation of XY but the individual unit-linker-X unit is left intact, ready to i0 interact with another Y.

Detailed examples of suitable XY exchange pairs are further described in working examples herein (vide infra) A sample comprising a number of different individual units: As specified above said sample may comprise at least two different individual units and up to numerous different individual units.

The actual number of different individual units generally corresponds to the actual size of the library of catalyst molecules.

Besides said specified different individual units said sample may in principle also comprise any other suitable material.

Further said different individual units comprised within said sample may be dissolved in any suitable buffer, such as water.

Even further said sample may also comprise the Y substrate compound which is in contact with the different individual units according to step of the method of the second aspect of the invention.

WO 00/11211 PCT/DK99/00441 31 Different individual units: As described above an individual unit comprises the general structure: a catalyst an XY exchange pair a substrate; or if the substrate has been converted into the product the general structure: a catalyst an XY exchange pair a product.

Further, the term "different individual units" denotes different individual units each independently comprising different catalyst molecules, i.e. an example of two different individual units may be catalyst molecule' XY exchange pair substrate; and catalyst molecule 2 XY exchange pair substrate; wherein catalyst molecule' and catalyst molecule 2 denotes two different catalyst molecules.

Further, "an individual unit" as described herein denotes an individual unit wherein it is possible to physically separate said individual unit from the other different individual units, within said sample, in order to be able to isolate the separate individual unit.

A biologically amplifiable individual unit: An embodiment of the invention relates to a sample comprising a number of individual units according to the invention, wherein the individual unit of point according to the first aspect of the invention is a biologically amplifiable individual unit.

Another embodiment of the invention relates to a sample comprising a number of individual units according to the invention, wherein the individual unit of point according to the first aspect of the invention is a biologically WO 00/11211 PCT/DK99/00441 32 amplifiable individual unit and both said substrate and said catalyst molecule are attached on the surface of said biologically amplifiable individual unit.

The term "a biologically amplifiable individual unit" denotes that within said individual unit either; the catalyst molecule of interest is a biologically amplifiable molecule; or (ii) the catalyst molecule of interest is biologically encoded by the information comprised within the entity allowing the unambiguous identification of the catalyst molecule; providing the possibility of amplifying said catalyst molecule of interest in order to obtain multiple copies of said catalyst molecule.

The term "biologically encoded" in point (ii) above denotes that the information is comprised within a DNA or RNA molecule in the form of the genetic code.

An example of an biologically amplifiable individual unit in relation to point above is an individual unit wherein the catalyst molecule of interest is a DNA or a RNA molecule, since it is well known in the art that DNA or RNA molecules may easily be amplified.

An example of an biologically amplifiable individual unit in relation to point (ii) above is an individual unit wherein the catalyst molecule of interest is a peptide or a polypeptide and wherein said entity comprising information allowing the unambiguous identification of the catalyst molecule is a DNA molecule encoding said peptide or polypeptide.

In relation to the second example above, a physical connection must exist between the peptide and the DNA that encodes it, in order to isolate the DNA with the peptide it encodes.

The connection can be either direct, in which case the peptide WO 00/11211 PCT/DK99/00441 33 is attached directly to the nucleic acid that encodes it, or indirect, where the peptide can be either attached to the surface of for example a cell, or alternatively secreted from a cell and therefore much more abundant in the immediate vicinity of the secreting cell than anywhere else. Such a cell is herein termed a "carrier system" as will be further discussed below.

Flexible linker: An embodiment of the invention relates to a sample i0 comprising a number of different individual units according to the first aspect of the invention, wherein said individual unit of point comprises following structure: catalyst molecule flexible (XY exchange pair) linker substrate.

Preferably the flexible linker is comprising the XY exchange pair.

The term "flexible linker" refers herein to the molecules as a whole connecting the catalyst with substrate. For example, if the substrate is attached to a bead through a flexible molecule, and the catalyst is also attached to the bead through a flexible molecule, "flexible linker" will refer to "flexible molecule-bead-flexible molecule", and the characteristics of the flexible linker will reflect the individual characteristics of the two flexible molecules and the portion of the bead that connects the two. Flexible linkers may for instance consist of flexible polypeptides, polyethylen glycol (PEG), and other polymers of reasonable flexibility.

Further, a flexible linker may also connect the catalyst molecule and a carrier system (see below) Carrier systems An embodiment of the invention relates to a sample comprising a number of different individual units according to WO 00/11211 PCT/DK99/00441 34 the invention, wherein said individual unit of point in the first aspect of the invention comprises the following structure: catalyst molecule carrier system XY exchange pair substrate, or more preferably the structure: catalyst molecule carrier system flexible (XY exchange pair) linker substrate.

The term "carrier system" denotes a system/entity which physically connects the catalyst molecule and the substrate, or alternatively, carries the information allowing the unambiguous identification of the catalyst molecule, and wherein said carrier system does not directly participate in the catalytic reaction substrate to product catalysed by the catalyst molecule.

Such a carrier system is herein further divided into a biologically amplifiable carrier system and a biologically nonamplifiable carrier system.

Examples of biologically amplifiable carrier systems include (carrier system catalyst molecule): phage polypeptide (Boublik et al., 1995, Biotechnol vol. 13, pp. 1079- 1084), filamentous phage peptide (Kay Winter and McCafferty, 1996, "Phage Display of Peptides and Proteins, A Laboratory Manual", Academic Press) retrovirus polypeptide (Buchholz et al., 1998, Nature Biotechnology, vol. 16, pp. 951- 954), plasmid peptide (Schatz et al., 1996, Meth. Enzym., vol. 267, pp. 171-191), polysome peptide (Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA, vol. 91, pp. 9022-9026; He and Taussig, 1997, Nucleic Acids Research, vol. 25, pp. 5132- 5134), bacteria peptide (Brown, 1997, Nature Biotechnology, vol. 15, pp. 269-272) and mRNA peptide (Roberts and Szostak, 1997, Proc. Natl. Acad. Sci. USA, vol. 94, pp. 12297-12302), cDNA peptide (analogous to the mRNA-protein fusion display, except that the protein has been attached to a cDNA of the mRNA WO 00/11211 PCT/DK99/00441 that encodes it, rather than to the mRNA itself) peptidesecreting cell peptide (Kinsella and Cantwell, 1991, Yeast, vol. 7, pp.

4 4 5-454), peptide-secreting artificial microsphere peptide (artificial microspheres containing proteins expressed from the genes contained within the microsphere, see Tawfik and Griffiths, 1998, Nature Biotechnology, vol. 16, pp. 652-656).

Examples of biologically non-amplifiable carrier systems include (carrier system catalyst molecule): bead organic molecule or bead peptide (Brenner and Lerner, 1992, Proc.

Natl. Acad. Sci. USA, vol. 89, pp. 5381-5383), pin inorganic molecule and bead DNA sequence (Geysen et al., 1996, Chemistry and Biology, vol. 3, pp. 679-688).

It should be noted that an individual unit comprising the beads DNA sequence structure is herein a biologically amplifiable unit (se above), however the carrier system, as such (bead) is a biologically non-amplifiable carrier system.

Catalyst and library of catalyst molecules: As stated above the term "catalyst" denotes any catalyst molecule with a desired catalytic activity, such as organic and inorganic molecules, proteins, enzymes, peptides, nucleic acids, biopolymers and non-biological polymers, small organic or inorganic molecules. Further the terms "catalyst" and "catalyst molecule" may be used interchangeably.

Accordingly, a further embodiment of the invention relates to, a sample comprising a number of different individual units according to the invention, wherein said library of catalyst molecules is a library of natural or unnatural peptides or polypeptides, preferably a library of enzymes; WO 00/11211 PCT/DK99/00441 36 (ii) a sample comprising a number of different individual units according to the embodiment immediately above, wherein said library is a library comprising polypeptides having a number of different enzymatic activities; or (iii) a sample comprising a number of different individual units according to the embodiment above, wherein said library is a library comprising polypeptides variants derived from one or more precursor polypeptide(s), wherein said precursor polypeptide(s) exhibit(s) closely related enzymatic activities.

The term "library comprising polypeptides having a number of different enzymatic activities" preferably denotes a library wherein said different enzymatic activities are substantially different activities, e.g. protease, amylase, xylanase, cellulase activities. An advantage of such an library may be that by changing the substrate according to the specific activity of interest, said library may be used to identify a number of polypeptides of interest. If for instance a protease of interest first is isolated by a method for in vitro selection as described herein by use of e.g. a peptide as substrate, then an amylase may be isolated thereafter by chancing the substrate to a e.g. a starch molecule.

The term "natural or unnatural peptides or polypeptides" denotes that the peptides or polypeptides may be build from any of the twenty natural amino acids building blocks, or any unnatural amino acids with other side chains, or any non-amino acid building block that is able to link two peptides together.

Said libraries may be made according to any of the numerous standard processes known for making such libraries.

WO 00/11211 PCT/DK99/00441 37 Accordingly, a further embodiment of the invention relates to a sample comprising a number of different individual units according to the embodiments of the invention mentioned immediately above, wherein said library is a library comprising shuffled/recombined/doped polypeptides.

Another embodiment of the invention relates to, a sample comprising a number of different individual units according to the invention, wherein said library of catalyst molecules is a library of natural or unnatural nucleic acids; (ii) a sample comprising a number of different individual units according to the embodiment immediately above, wherein said library is a library comprising nucleic acids having a number of different catalytic activities; or (iii) a sample comprising a number of different individual units according to the embodiment above, wherein said library is a library comprising nucleic acid variants derived from one or more precursor nucleic acid(s), wherein said precursor nucleic acid(s) exhibit(s) closely related catalytic activities.

The term "library comprising nucleic acids having a number of different catalytic activities" preferably denotes a library wherein said different catalytic activities are substantially different activities, e.g. nuclease, ligase, isomerase, phosphorylase. An advantage of such a library may be that by changing the substrate according to the specific activity of interest, said library may be used to identify a number of nucleic acids of interest. If for instance a DNA ligase of interest first is isolated by a method for in vitro selection as described herein by use of e.g. two DNA oligonucleotides as sub- WO 00/11211 PCT/DK99/0044! 38 strates, then a ribonuclease may be isolated thereafter by changing the substrate to a e.g. a RNA oligonucleotide.

Said libraries may be made according to any of the numerous standard known processes of making such libraries.

Accordingly, a further embodiment of the invention relates to a sample comprising a number of different individual units according to the embodiments of invention mentioned immediately above, wherein said library of nucleic acids is a library comprising shuffled/recombined/doped nucleic acids.

The term "natural or unnatural nucleic acids" denotes that the nucleic acids may contain any of the five natural bases T, G, C, or any unnatural base or backbone structure.

Further embodiments of the invention relate to a sample comprising a number of different individual units according to the invention, wherein said library of catalyst molecules is a library comprising natural polymer molecules, or unnatural polymer molecules, or small organic molecules, or small inorganic molecules or a mixture of said molecules; or (ii) a sample comprising a number of different individual units according to the embodiment mentioned immediately above, wherein said library is made by combinatorial chemistry.

Preferably, the sample may contain a virtual combinatorial library (Proc. Natl. Acad. Sci. USA, 1997, vol. 94, pp.

2106-2110), in the sense that each potential catalyst is made up of more than one subunit, held together by reversible covalent or non-covalent interactions, and the subunits associate and dissociate several times during the multiple turn-over assay.

WO 00/11211 PCT/DK99/00441 39 In the context of virtual combinatorial libraries, the term "unambiguous identification" which is used throughout this text, should be understood in terms of the unambiguous identification of the recovered entities, but not necessarily the composition of the individual catalysts. Such catalysts, isolated from virtual combinatorial libraries, could be catalysts made up of several polypeptide chains, held together by weak interactions (as for example protein subunit association, characterized by low subunit-subunit affinities), or held together by reversible covalent disulfide-bonds, whose exchange rates have been accelerated by the addition of redox-buffers (eg.

oxidized and reduced glutathione) and therefore continuously are associating and dissociating.

The term "natural polymer molecules, or unnatural polymer molecules" denotes that the polymers may be of a kind found in Nature, or of a kind that is artificially produced by Man.

Alternatively, the library members could be assemblies of small organic molecules held together by disulfide bonds; again, the dynamics of the virtual combinatorial library may be speeded up by including a redox buffer.

In an even further embodiment the invention relates to a sample comprising a number of individual units according to the invention, wherein the catalyst molecules and the substrate capable of being catalysed into a product (point in the first aspect) are of a different chemical substance.

The term "catalyst molecules and the substrate capable of being catalysed into a product (point in the first aspect) are of a different chemical substance" denotes that said catalyst molecule and the substrate molecule are of a substantially different chemical substance.

WO 00/11211 PCT/DK99/00441 Accordingly, in this preferred embodiment the situation wherein the catalyst molecules and the substrate molecule are both DNA or RNA molecules are herein said to be a situation wherein the catalyst molecules and substrate molecules are NOT of a different chemical substance.

A method for multiple catalytic activity turn-over in vitro selection, according to the second aspect of the invention: The term "selection" preferably denotes that the selection according to step (ii) in the second aspect of the invention, is performed on more than one, preferably more than 100, or preferably more than 10.000, more preferably more than 1.000.000, even more preferably more than 108, and most preferably more than 1010 individual units comprised within a sample, preferably without interference of the skilled person.

The term "column" denotes herein all kinds of solid support. Examples are: columns, surfaces including BiacoreTM apparatus. In some cases there is no need for a solid support, as is the case if the separation is based on migration in an electric field.

Y substrate compound and isolation of a catalyst of interest according to the method of the second aspect of the invention: As described above, the term "said individual units are in contact with a Y-substrate compound" according to point (i) in second aspect of the invention denotes that the individual units and the Y substrate compound are appropriately close that an exchange reaction is possible. Said contact may for instance be in a buffer solution wherein the individual units and the Y substrate compound may diffuse together and thereby get in contact with each other.

WO 00/11211 PCT/DK99/00441 41 During selection rounds performed according to the method for multiple catalytic activity turn-over in vitro selection, according to the invention, it may be advantageous to perform the first selection rounds less stringently than later selection rounds. This can be done easily, e.g. by using a low concentration of Y-substrate, and/or a low concentration of a product binding receptor, in certain cases it may be desirable in the first round(s) to use no Y-substrate. Additionally, less efficient Y-substrate compounds can be used. In later selection rounds the concentrations are then increased. For certain experimental set-ups, this may not be feasible. For example, if a library consisting of very active enzymes is used, and the method of isolating the catalyst is based on immobilization on a product binding column, the high initial concentration of Ysubstrate may result in high concentration of Y-product. Depending on the capacity of the product binding column, this may saturate the product binding receptors with product and hence, lead to lower selection stringency. However this problem can be solved, e.g. by (in the case of cleavage reactions) attaching the catalyst to the column via a linker that contains the substrate (see figure To further increase the stringency of the selections, one may add excess substrate S (not connected to Y) to a column buffer (this will serve as "competitor substrate"). Other ways to modify selection stringency include variation of the length of the linker that connects enzyme and substrate, addition of factors to the column buffer which have affinity for the substrate or enzyme, or addition of factors that affects subtrate-enzyme interaction (eg. receptors/antibodies binding the enzyme's active site, enzyme inhibitors, receptors/antibodies with affinity for the substrate) WO 00/11211 PCT/DK99/00441 42 In order to limit the time available to the catalysts for substrate turn-over, pulses of for example voltage or light may be applied during the selection. Appropriately separated pulses could create for example transient pH- or ionic gradients that would initiate the reaction substrate to product, performed by the catalyst. The pulses should be separated enough in time that it allows plenty of time for a catalyst in solution to become immobilized on a receptor before the puls initiates the next reaction. In this way, the dead time of the selection performed in the column format the time the catalyst spends diffusing from one receptor to the next) can be drastically reduced, and very high stringencies obtained.

Generally speaking, it is within the skilled persons general knowledge to optimize the specific experimental set-up.

Performing an enrichment step prior to step of the second aspect of the invention Certain proteins are difficult to display on filamentous phage. In particular large proteins or proteins which have a toxic or growth inhibiting effect on E. coli often have low display efficiency, i.e. the majority of phage particles produced carry no displayed proteins on the surface. Display efficiencies as low as one out of a thousand phages displaying the protein of interest have been reported (Jestin et al., 1999, Angew. Chemi. Int. Ed., vol. 38, pp. 1124-1127; Demartis et al., 1999, JMB, vol. 286, pp. 617-633). In such cases, a high non-specific background is expected, because of the large excess of phage particles carrying the DNA encoding the protein of interest, but not displaying said protein on the surface.

To circumvent this potential problem, an affinity tag may be coupled to the C-terminal end of the protein of interest, al- WO 00/11211 PCT/DK99/00441 43 lowing the purification of phages displaying full-length tagged protein column chromatography.

A non-limiting variety of affinity tags that may be used in this manner is: histidine tag (see Example intein-chitin binding domain fusion (Chong et al., 1997, Gene, vol. 192, pp 271-281), FLAG peptide (Slootstra et al., 1997, Molecular Diversity, vol. 2, pp. 156-164), and the maltose binding protein (Pryor and Leiting, 1997, Protein expression and Purification, vol. 10, pp. 309-319).

Accordingly, embodiments of the invention relate to a method for in vitro selection according to the second aspect of the invention, wherein the catalyst molecules of interest are enzymes or proteins that have been coupled to an affinity tag, and wherein an optional step is performed prior to step the optional step comprising an enrichment for individual units displaying (full length) enzyme or protein through a purification in which the units displaying the enzyme or protein are isolated by means of the affinity tag.

(ii) a method for in vitro selection according to the second aspect of the invention, wherein the individual units displaying (full length) enzyme or protein are purified by the means of an anti-affinity-tag antibody column in which the units displaying the tagged enzyme or protein are isolated by means of the tag.

(iii) a method for in vitro selection according the second aspect of the invention, wherein the affinity tag comprises six histidine residues that are coupled to the C-terminal end of the enzyme or protein of interest, and the individual units displaying (full length) enzyme or protein are purified on a Ni-NTA column, or WO 00/11211 PCT/DK99/00441 44 on a anti-histidine antibody column, in which the units displaying the tagged enzyme or protein are isolated by means of the tag.

Means of isolating an active catalyst of interest according to a method of the invention: The separation of active and less active catalysts preferably involves a selective step during which the catalyzed reaction leads to either release or attachment of the catalyst i0 through the linker-substrate attached to it.

When using the column set-up, there are at least four means to separate active from inactive catalysts, i) The active catalysts can be isolated by immobilization of the product on a product binding column (or more generally, by means of the attached product). ii) The inactive catalysts can be removed by immobilization on a substrate binding column. iii) Prior to the target reaction the catalyst may be attached to support; when a cleavage reaction occurs, the catalyst is released from support and can be collected. iv) The active catalysts may attach themselves to solid support upon reaction of substrate 1 (attached to the catalyst) and substrate 2 (attached to support). See figure 7 and 8 for graphic illustrations.

The product and substrate specific columns may immobilize the product and substrate through binding to a receptor molecule with specificity for the product and substrate, respectively. Alternatively, immobilization may be mediated by a product- or substrate-specific reaction between functional groups on the column and the product or substrate attached to the catalyst.

Other means of isolating the product or substrate (and with these, the active or inactive catalysts, respectively) include partitioning between different phases, mass spectrometry, WO 00/11211 PCT/DK99/00441 precipitation, electric or electromagnetic separation etc. In particular, electrophoresis of various kinds may be performed, especially in cases where product formation results in a significant change in the electric charge of the individual unit.

A significant change in charge may result, for example, if the reaction is a ligation that ligates two substrates, one of which is charged and free in solution prior to the reaction.

Accordingly, embodiments of the invention relate to a method for in vitro selection according to the second aspect of the invention, wherein the selecting for a catalyst molecule of interest, in step is done by specific immobilization resulting in said product molecule; (ii) a method for in vitro selection according to the second aspect of the invention, wherein the selecting for a catalyst molecule of interest, in step is done by the following strategy, (a)constructing a system wherein substantially each of the individual units in step of the second aspect comprising the substrate molecule and the catalytic molecule is bound to a matrix and wherein the unit is released from said matrix when the substrate is converted into the product; and (b)selecting for the unit(s) which are released from said matrix; (iii) a method for in vitro selection according to the second aspect of the invention, wherein the selecting for a catalyst molecule of interest (step is done by one of the following strategies, constructing a product-column wherein a receptor specifically binding the product is placed along the matrix of the product-column; and WO 00/11211 PCT/DK99/00441 46 adding the sample of individual units at one end of the product-column and selecting for the catalyst molecules of interest by isolating the individual unit(s) which arrive(s) latest to the opposite end on the column; (iv) a method for in vitro selection according to the second aspect of the invention, wherein the isolation of an entity comprising information which allows the unambiguous identification of the catalyst molecule of interest (step is done by physical or chemical procedures; or a method for in vitro selection according to the immediately above aspect, wherein the physical procedure is electrophoresis.

Repeating step to (iii) one or more times, according to point (iv) of the second aspect of the invention: As stated above, the term "repeating step to (iii) one or more times by using the information comprised in said entity of step (iii) to generate the catalyst molecule of interest and construct an individual unit comprising said generated catalyst molecules of interest and then using this individual unit as a starting material in said repetition step" according to point (iv) in the second aspect of the invention denotes that said repetition may be one time, more preferably 2 times, more preferably more than 5 times, even more preferably more than 10 times, and most preferably more than 25 times.

A method for producing a catalyst molecule of interest, according to the final aspect of the invention: As stated above, in a final aspect the invention relates to a method for producing a catalyst molecule of interest WO 00/11211 PCT/DK99/00441 47 comprising performing the method for in vitro selection according to the invention and the further following steps, producing said isolated catalyst molecule of interest in a suitable quantity of interest by a suitable production method.

As described above, in the method for in vitro selection, according to the second aspect the invention step (iii) reads: "(iii) isolating an entity comprising information allowing the unambiguous identification of the catalyst molecule of interest which has been capable of catalysing multiple times the reaction substrate to product, by means of a characteristic of the product." Accordingly, the information comprised within said entity provides the possibility of producing said catalyst molecule of interest by any standard production strategy known to the skilled person.

If said catalyst molecule of interest for instance is a polypeptide of interest said standard production strategy may be a standard protocol for recombinant production of said polypeptide of interest, or if said catalyst molecule of interest for instance is an organic molecule of interest said standard production strategies may be a standard protocol for production of such an organic molecule.

EXAMPLES:

Example 1: An example of an individual unit comprising the features of the first aspect of the invention, except the XY exchange pair.

In this example 1, the catalyst of interest is a Staphylococcal DNase (SNase); the substrate is a single WO 00/11211 PCT/DK99/00441 48 stranded oligonucleotide (ssDNNA); and product is the ssDNA cleaved by a SNase of interest.

Further, a filamentous phage is used as a carrier system and an acid/base linker is used as a flexible linker.

Accordingly, the individual units in this example has following general structure: SNase fil. Phage acid/base link. ssDNA Catalyst Carrier system flexible linker substrate.

See figure 10 for an illustration.

In this example the "selection characteristic" of the product cleaved ssDNA) is that said product does not bind to a matrix and the substrate (ssDNA) does bind to a matrix.

Accordingly, in this example a SNase molecule of interest is isolated by selecting for individual units which are released from said matrix. See figure 10 for an illustration.

MATERIALS AND METHODS.

Synthesis of compounds. Fmoc-S-(2-nitro-4,5-dimethoxybenzyl) L-cysteine 1 was synthesized by a variation of the method of Merrifield Briefly, 605 mg L-cysteine (5 mmol) was suspended in 100 mL degassed ethanol/water and 1.39 mL triethylamine (10 mmol) and 1.39 g l-(bromomethyl)-2-nitro-4,5dimethoxybenzene (5 mmol) were added. The mixture was stirred for 10 h at 23 °C in the dark under nitrogen and filtered. The filter cake was washed with ethanol and recrystallized from ethanol/water to provide 0.95 g S-(2-nitro-4,5dimethoxybenzyl)-L-cysteine (3 mmol). The recrystallized product (0.8 g) was suspended in 20 ml water; 0.53 ml triethylamine (3.8 mmol) was added followed by a solution of 0.9 g 9fluorenylmethoxycarbonyl succinate ester (2.7 mmol) in 12 mL acetonitrile and the mixture stirred for 10 h at 25 oC under nitrogen. The product precipitated upon acidification to pH 2- WO 00/11211 PCT/DK99/00441 49 3 with 1 M HC1 and evaporation of the acetonitrile. The precipitate was collected on a frit and washed with water and ethylacetate to remove excess HC1 and reagent. The resulting crude product 1 (1.13 g) was extensively dried under vacuum, and used directly in the synthesis of the base-linker peptide C(GGS)4AQLKKKLQALKKKNAQLKWKLQALKK-KLAQGGC (base sequence underlined, photoprotected cysteine in bold). Compounds 2, 3 and 4 were synthesized on an ABI DNA synthesizer on a 1 mmole scale with a 3'-biotin group (BiotinTEG CPG, Glen Research) and a thiol (5'-Thiol-Modifier C6, Glen Research) and purified by reverse phase HPLC following removal from the resin (Rainin Microsorb C18 column, flow 1 mL/min.; solvent A: 50 mM triethylammonium acetate (TEAA), pH 7, solvent B: acetonitrile, linear gradient from 5 to 50 solvent B over 40 min); the trityl protecting group on the thiol was removed according to the protocol of Glen Research. The products were lyophilized and dissolved in water (1.0 mM final concentration). The conjugate of 2 with the base-linker peptide was prepared as follows: 2 mg (415 nmole) base-linker peptide was reacted with a 20 fold molar excess of N,N'-bis(3-maleimidopropionyl)-2-hydroxy-l,3propanediamine (3.2 mg) in 1 mL of 50 mM sodium phosphate buffer, pH 5.5, for 10 h under nitrogen at 4 Compound 5 was purified from the reaction mixture by reverse phase HPLC (Vydac RP-18 column, flow 2 mL/min; solvent A: 0.1 TFA in water, solvent B: 0.1 TFA in acetonitrile; linear gradient from to 55 solvent B over 35 minutes), and the product fractions concentrated to approximately 0.3 mL (OD280 6, compound should not be concentrated to dryness). To 100 mL (138 nmoles) of this solution was added 75 ul water, 75 mL of aqueous 1 M aqueous sodium phosphate, pH 7, 30 mL of aqueous 5 M NaC1, and 22 mL (22 nmoles) of compound 2, and the reaction incubated for h under nitrogen at 23 oC (to avoid precipitation the rea- WO 00/11211 PCT/DK99/00441 gents should be added in this order). The product was purified by anion exchange FPLC (Mono Q HR 5/5 column (Pharmacia), flow 0.75 mL/min solvent A: 20 mM Tris-HC1, pH 7, solvent B: 20 mM Tris-HC1, pH 7, 2 M NaCl; linear gradient from 20 to 60 B in 7.5 min); on a 10 denaturing polyacrylamide gel the product ran as a single band. Fractions of OD260 0.3-1 were used directly for the photo-deprotection step (vide infra). The conjugates of 3 and 4 with the base-linker-peptide were prepared as follows: approximately 200 nmoles of either 3 or 4, and a fold excess of bismaleimide were incubated in 1 mL of aqueous mM phosphate buffer, pH 5.5, at 4 OC for 15 hours. After purification by reverse phase HPLC and lyophilization, the identity of compounds 6 and 7 was verified by Maldi-ToF MS.

Either 6 or 7 (150 nmoles) was then incubated with 100 nmoles base-linker-peptide in 100 mL of 10 mM TEAA, pH 6.5, 100 mM NaCl for 15 hours at 4 The products were purified by reverse phase HPLC (Vydac RP-18 column, conditions as described above), lyophilized and analyzed by Maldi-ToF MS The 2protecting group on the C-terminal cysteine of the three conjugates was removed by photolysis to afford compounds 8, 9 and 10 as follows: for compound 8, 100 mL of the FPLC purified fraction containing the protected conjugate (vide supra) was degassed thoroughly with argon for min, and then exposed to a mercury lamp (450 W high pressure mercury lamp, Ace-Hanovia; Pyrex TM filter, cutoff 2 300 nm) in a septum capped glass vial for 30 min For compounds 9 and 10 nmole of the conjugate was dissolved in 100 mL of 10 mM DTT, degassed and photolyzed as described above. After 30 min of irradiation no remaining starting material could be detected by MALDI-ToF MS. The reaction mixture was separated by HPLC (Vydac RP-18 column, conditions as described above) and the WO 00/11211 PCT/DK99/00441 51 product fractions were lyophilized. The conjugates were stored frozen, and used within a week after photo-deprotection, to ensure efficient attachment to phage.

Construction of acid helper phage. A NarI restriction site was introduced between the third and fourth codon of mature pIII protein of M13K07 helper phage (Promega) by Kunkel mutagenesis with the primer K07-NarI-prim ACAACTTTCAACGGCGCCAGTTTCAGCGG-3') to give NarI-helper phage.

DNA encoding the amino acids GAAQLEKELQALEKENAQLEWELQALEKELAQ- GGCPAGA (acid peptide sequence underlined, GGC motif in bold) with a NarI restriction site at both ends, was produced by polymerase chain reaction (PCR) with the plasmid pCRII acid (Ellis L. Reinherz, Dana Farber Cancer Institute, Boston) with the primers NarIfwd (5'-ACTACAAATTGGCGCCGCTCAGCTCGAAAAAGAGC-3') and Narlbck AATTATAGGCGCCAGCCGGGCAACCGCCCTGAGCCAGTTCCTTTTCC-3'). The PCR product was digested with NarI and inserted into NarI digested NarI-helper phage to afford acid helper phage.

Construction of phagemids encoding the staphylococcal nucleasepIII fusion and 39-All Fab-pIII fusions. To make the SNase-pIII fusion, PCR was performed on the plasmid pONF1 carrying the gene encoding SNase, with primers CGCGAATTGGCCCAGCCGGCCATGGCCGCAACTTCAACTAAA-3' (SfiI restriction site underlined) and 3' (NotI restriction site underlined). The product was digested with SfiI and NotI and inserted into SfiI-NotI digested pFABa derivative of plasmid pFAB-5c to give phagemid pII78-6. As a negative control the phagemid pComb3H.DA was employed. This phagemid (12) carries the 39-All Fab antibody (13) fused to the pIII protein. The expression of both the SNase and control protein is driven by the lac promoter.

WO 00/11211 PCTIDK99/00441 52 Production of phage particles. Phage particles were produced with minor modifications according to Orum et al. (11).

Briefly, E. coli XL1-blue was transformed with p1178-6 or pComb3H.DA, and shaken at 37 OC in 2xYT broth and 100 mg/mL ampicillin. At an OD600 of 0.5, acid helper phage was added to a final concentration of 1.5 x 108 cfu/mL, and incubated at 37 °C for 20 min. The cells were pelleted and resuspended in 2xYT, 100 mM IPTG, 100 mg/mL ampicillin, 50 mg/mL kanamycin, and shaken for 14 hours at RT. Cells were pelleted and phage partides in the supernatant were PEG precipitated, followed by resuspension in TBS (25 mM Tris-HC1, pH 7.4, 140 mM NaCl, mM KC1). Phage titrations were performed with E. coli XL1-blue using standard procedures (14).

Covalent attachment of base-linker-substrate conjugates to phage.

Approximately 108 phage particles were incubated in 40 mL buffer A (TBS, 10 mM EDTA, 0.1 BSA), supplemented with 1 mM mercaptoethylamine (MEA) and 1 nmole of either base-linkeroligodeoxynucleotide base-linker-pTp or base-linkerpTpTp at 37 °C for 60 minutes, then PEG precipitated twice and resuspended in buffer A.

Phage immobilization and release from solid support. Approximately 108 phage particles, covalently attached to the baselinker-substrate conjugates, were incubated with 50 mL magnetic streptavidin beads (Boehringer Mannheim, biotin binding capacity: 1.5 nmole/mL) in 1 mL buffer A for 15 minutes at 23 oC; eight 1 min washes were performed in buffer A with 0.1 Tween followed by two 1 min washes in buffer A. The number of phage immobilized on the beads was determined by suspending the beads in buffer A, and then either directly infecting E. coli XLl-blue with the bead suspension and titering or alterna- WO 00/11211 PCT/DK99/00441 53 tively, infecting after treatment of the beads with DNase 1 (lunit/mL DNase 1, 10 mM MgCI2, 20 mM Tris-HC1, pH 8, 23 °C for min). Calcium-dependent release (cleavage) from solid support was examined by suspending beads in buffer B (TBS, 10 mM CaC12, 0.1 BSA), incubating at 23 °C for 5 min, and titering the supernatant. Calcium-independent release from the beads (leakage) was determined by resuspending the beads in buffer A, incubating for five minutes at 230C, and titering the supernatant.

Enrichment of active enzymes from a library-like ensemble.

Phage particles displaying SNase or 39-All Fab were mixed in a 1:100 ratio and the base-linker-oligodeoxynucleotide conjugate was covalently attached. Phage were then immobilized on magnetic streptavidin beads, washed in buffer A, and incubated in buffer B as described above, E. coli XLl-blue were infected with the supernatant and the cells plated on a LA plate containing 100 mg/mL ampicillin. Randomly picked colonies were identified as SNase- or control clones by PCR or restriction enzyme digestion.

RESULTS DISCUSSION.

Selection scheme. To test the above strategy for directedenzyme evolution in a phage-display format, it was first necessary to develop a general method for selectively attaching a given substrate to or near a phage-displayed enzyme. Importantly, the substrate must be attached so that it can bind productively in the active site of the conjugated enzyme. Moreover, the substrate should be covalently linked to the phage to ensure that there is no crossover of reaction product between members of the library. One possible strategy involves selective chemical modification of the enzyme or a nearby phage coat protein pill protein) with substrate by a disulfide ex- WO 00/11211 PCT/DK99/00441 54 change reaction. For example, a cysteine residue introduced near the active site of staphylococcal nuclease through sitedirected mutagenesis has been used to selectively introduce unique chemical functionality by a disulfide exchange reaction To apply this method to proteins expressed on filamentous phage, the three single cysteines of the pVI, pVII and pIX coat proteins were first mutagenized to alanine. The eight buried cysteine residues in the pIII protein were left unchanged, as they likely form structurally important disulfide bridges Unfortunately, repeated attempts to selectively modify unique cysteine residues introduced near the active site of several enzymes displayed on phage, by either disulfide exchange, maleimide addition or alkylation reactions, resulted in significant nonspecific labelling of phage coat proteins. No conditions or reagents were found that made possible selective labelling of the pIII fusion protein containing the unique surface cysteine residue. It is likely that the thousands of proteins constituting the phage coat make the specificity requirement for a chemical reaction too great; also, the probability of cysteine misincorporation due to the intrinsic error rate in protein biosynthesis becomes significant for such a large ensemble of proteins. Alternatively, the cysteine residues in the pill protein may be accessible to crosslinking reagents.

To circumvent these problems, a two step process was developed in which chemical crosslinking is preceded by the selective formation of a noncovalent complex at the site of modification (Figs. 9 and 10). The complex is a heterodimeric coiled-coil consisting of a synthetic basic peptide B C(GGS)4AQLKKKLQALKKKNAQLKWKLQALKKKLAQGGC, to which substrate is covalently coupled before heterodimerization, and an acidic peptide A, GAAQLEKELQALEKENAQLEWELQALEKELAQGGCPAGA that is expressed as an N-terminal fusion to the pIII coat protein of WO 00/11211 PCT/DK99/00441 filamentous phage. The acid and base peptides (underlined) were chosen as dimerization domains because of their small size (thirty amino acids) and high tendency to form stable, parallel heterodimeric coiled-coil structures the acid-acid and basebase homodimers form 10 5 fold less efficiently than the heterodimer Heterodimerization of the synthetic and phage-encoded peptides should bring the substrate into close proximity of the displayed enzyme, and lead to spontaneous disulfide bond formation between cysteines on each of the peptides (figure 10). The tripeptide Gly-Gly-Cys was added to the C-termini of the acid and base peptides to facilitate formation of a disulfide bridge between the two helices The substrate is covalently linked to the basic peptide B through a flexible linker to facilitate productive binding of substrate to enzyme (figure The acidic peptide A is fused to the pIII protein of the phage rather than to the displayed enzyme itself for the following reasons: insertion of the acid peptide sequence into an enzyme might interfere with enzyme function; (ii) the flexible linker of the base-linker-peptide as well as hinges in the pill protein and a peptide linker inserted between pill and the displayed enzyme, should allow many possible orientations of the substrate relative to the enzyme active site; and (iii) it should be possible to use a single helper phage bearing the acid peptide extension to display many enzyme-substrate pairs, rather than having to engineer into each enzyme a functional conjugation site.

Generation of the acid helper phage and base-linker-substrate conjugate.

To attach the base-linker-substrate conjugate to phage we introduced the acidic peptide A at the N-terminus of pill protein in the M13K07 helper phage. The enzyme library is fused to the N-terminus of the pill coat protein; this construct is WO 00/11211 PCT/DK99/00441 56 carried in the phagemid. Upon superinfection by helper phage, phage particles are produced that contain the phagemid DNA but whose coat consists (with one exception) of proteins encoded by the helper phage genome. The one exception is the pIII protein, present in 4-5 copies at one tip of the phage. During packaging of the phage, both enzyme-pIII fusions and acid peptide A-pIII fusions are produced; the phage particles obtained from a typical preparation carry either one or zero enzyme-pill fusions plus three to five copies of acid peptide A-pIII fusion.

To generate phages bearing an acid peptide-pIII fusion, DNA encoding the acidic peptide A with a C-terminal extension containing a cysteine residue, was introduced into the of gene III of the M13K07 helper phage. The resulting acid helper phage particles were immoblized more than hundred fold more efficiently than M13K07 on an ELISA-plate coated with basic peptide B, indicating that the mutant helper phage carry accessible acid peptide extensions on their pIII proteins.

Likewise, when E. coli containing a phagemid encoding a pIII fusion protein were superinfected with the acid helper phage, the resulting phage particles displayed modified pill extensions in addition to the pill fusion protein (figure 10). The insertion of the acid peptide did not appear to change the titer or rescue efficiency of the helper phage significantly.

The synthetic base-linker-peptide to which substrate is attached consists of the twelve residue (GlyGlySer)4 linker followed by the thirty amino acids constituting the base sequence (figure The base-linker peptide also contains cysteine residues at the N-and C-termini that allow efficient, selective coupling of the peptide to substrates and disulfide bond formation to phage, respectively (figures 9 and 10). The C-terminal cysteine of the synthetic peptide is initially pro- WO 00/11211 PCT/DK99/00441 57 tected with the photochemically removable 2-nitro-4,5dimethoxybenzyl protecting group. This allows substrate to be selectively conjugated by a thiol specific reaction by disulfide exchange, alkylation, or Michael addition reactions) to the free thiol group of the N-terminal cysteine. After substrate conjugation, the C-terminal cysteine is photochemically deprotected in high yield to generate a free thiol available for crosslinking to the acid peptide extension on phage. Because the chemical conjugation of substrate and base-linker peptide, and the crosslinking of this conjugate to phage are carried out separately, many different chemistries and reaction conditions can be used to couple the base-linker peptide and substrate. Moreover, the composition of the conjugate can be purified and characterized by mass spectrometry) before it is crosslinked to phage.

Staphylococcal nuclease as a model system. The enzyme staphylococcal nuclease is a well-characterized enzyme consisting of a single polypeptide chain 149 amino acids in length The enzyme preferentially hydrolyzes the phosphodiester bonds of single-stranded RNA (ssRNA), ssDNA, and duplex DNA at A,U- or A,T- rich regions to generate 3'-phosphate and 5'-hydroxyl termini Ca2+ is required for enzymatic activity, providing a mechanism for modulating enzyme action. In addition, SNase has successfully been displayed as a pill fusion protein on phage (19).

Because no reagent, antibody or receptor is available that can easily distinguish between a single-stranded oligodeoxynucleotide substrate and its cleavage product (a complementary oligonucleotide would be degraded), a selection scheme was developed in which enzymatic cleavage of ssDNA substrate results in release of phage from solid support. In this scheme, WO 00/11211 PCT/DK99/00441 58 one round of selection involves the following steps: i) attachment of phage displaying SNase to solid support through a single-stranded oligodeoxynucleotide (in the absence of Ca 2 to inactivate SNase); ii) removal of unbound phage by washing; iii) initiation of the cleavage reaction by addition of Ca 2 and iv) isolation of eluted phage. In later rounds of selection, elution can be done under increasingly stringent conditions, eg., shorter reaction time, lower temperature and altered pH. Attachment of phage to solid support is carried out by coiled-coil formation between 5'-biotinylated oligodeoxynucleotide-peptide B conjugates and acid peptide A extensions on phage, followed by disulfide crosslinking of the two peptides and immobilization on streptavidin beads (figure This scheme, in which the phage is attached to solid support through the substrate, requires that the enzyme or substrate be maintained in an inactive state during attachment to phage, and then be activated by a change in reaction conditions. Such changes can include modulation of pH, addition of cofactors or co-substrates, and photochemical or chemical activation of the substrate. In the case of biomolecular condensation reactions in which bond formation results in phage immobilization on solid support, it is not necessary to initiate the reaction; the same is true if capture of active enzymes is by a productspecific reagent, antibody or receptor.

Covalent attachment of the substrate to phage. Phage displaying either SNase or a control protein (antibody 39-All Fab fragment) were prepared by superinfection with the acid helper phage. To evaluate the efficiency of the attachment of baselinker-substrate conjugates to phage, an excess of a control conjugate, "pTp"-peptide B (compound was incubated with the phage. The base-linker-pTp conjugate consists of a biotin moi- WO 00/11211 PCT/DK99/00441 59 ety, followed by deoxythymidine-3',5-diphosphate (pTp), the flexible peptide linker and base peptide sequence, and a Cterminal cysteine. The base-linker-pTp conjugate is not a substrate for wildtype SNase in solution (pTp is a potent inhibitor of SNase) Phage and the substrate-peptide B conjugate were first incubated with the reducing agent mercaptoethylamine (MEA) to reduce disulfide bonds between cysteines on the phage acid peptide or the synthetic peptide. Then, MEA and free base-linker-pTp were removed by PEG precipitation, and magnetic streptavidin beads were added. After ten washes, the number of phage that were immobilized was determined by infection of E.

coli XLl-blue with the beads, and titering phage. When measured this way, the efficiency of phage immobilization was approximately 10%, for both phage displaying SNase and 39-All Fab (figure 11).

Next it was determined whether an oligodeoxynucleotide substrate attached to phage displaying SNase would be stable in the absence of Ca 2 The base-linker-oligodeoxynucleotide conjugate was attached to phage displaying SNase (in the presence of EDTA), and the immobilization efficiency determined as above. The efficiency of immobilization was again approximately 10% (figure 11), indicating that the tethered oligodeoxynucleotide substrate is not cleaved by SNase in the absence of Ca 2 It is possible that the true immobilization efficiency is higher than observed if some of the phage are rendered non-infective when attached to the beads. This notion was tested by addition of DNase I, which should cleave the tethered oligodeoxynucleotide substrate and release the immobilized phage. As can be seen in figure 11, most of the immobilized phage are non-infective, but become infective upon addition of DNase I, indicating that the true immobilization effi- WO 00/11211 PCT/DK99/00441 ciency is about 80 (figure 11). If the oligodeoxynucleotidepeptide B conjugate is not included, less than 0.01% of the phage become immobilized; if the wildtype M13K07 helper phage is used to superinfect, about 0.3% of phage are immobilized.

It thus appears that the two-step protocol for attachment of substrate to phage pill protein is efficient and highly sitespecific.

Enzyme dependent cleavage of phage from solid support and enrichment. To determine whether phage-displayed SNase is capable of specifically cleaving the tethered oligodeoxynucleotide substate in an intramolecular reaction, Ca 2 was added to the immobilized phage to activate the enzyme. Approximately 15 of the phage were released (figure 11), in contrast to release of only 0.2% of the control phage displaying Fab 39-All (figure 11). This experiment demonstrates that SNase cleaves and releases phage from the solid support much more efficiently than the control protein, as expected. However, it appears that a small but significant fraction of the phage leak off the support during the assay (this background leakage is observed without Ca 2 for both the base-linker-oligodeoxynucleotide and base-linker-pTp conjugates, and for both displayed proteins, figure 11. Addition of Ca 2 leads to an initial burst of phage release from support; however, the release of phage quickly declines to a level corresponding to the leakage observed without Ca 2 This result demonstrates that phage released into solution by intramolecular cleavage events do not release other phage from support as a result of intermolecular cleavage reaction. Cross-reactivity therefore does not appear to be significant, even with a very active enzyme like SNase.

The above analysis suggests that it should be possible to enrich phage displaying SNase from a library-like ensemble of WO 00/11211 PCT/DK99/00441 61 phage displaying catalytically inactive proteins. To test this, phage displaying SNase and the Fab 39A-11 control protein were mixed in a ratio of 1:100, crosslinked to the oligodeoxynucleotide-peptide B conjugate and immobilized. After incu- 2+ bation with Ca 2 the ratio of recovered phage was 22:18, which corresponds to an enrichment factor slightly higher than 100.

This degree of enrichment should be sufficient to isolate an active catalyst from a library of 1010 members after five rounds of selection and amplification.

The enrichment factor can likely be increased by minimizing background leakage of phage from support. This leakage may result from release of streptavidin from support, or alternatively, reduction or incorrect formation of the disulfide bridge between the synthetic and phage encoded peptides. We are currently exploring these possibilities. Alternatively, the enrichment factor can be increased by increasing the extent of the enzyme-catalyzed cleavage reaction. Under the conditions of phage production, the ratio of pill expressed from the helper phage relative to the pIII fusion protein expressed from the phagemid is such that most of the phage carry only wildtype pill proteins; only a minor fraction of the phage carry the protein-pIII fusion. The number of phage that can cleave themselves off can be increased simply by increasing the number of phage that display the enzyme. For the phagemid/helper phage combination described here, we estimate that only about 15 of the phage are monovalent. By appropriate vector design and phage preparation, it should be possible to increase the average display to about one protein per phage. This should increase the cleavage to leakage ratio 7 fold, and hence, increase the enrichment factor of active versus inactive enzymes from the present -100 to about 700.

WO 00/11211 PCT/DK99/00441 62 To examine whether the selection scheme described here can be used for reactions that involve small molecule substrates, a pTpTp-peptide B conjugate (compound 10) was attached to phage displaying SNase or the control protein. Phage were carried through the enrichment routine described above, and again SNase displaying phages were enriched. MALDI-ToF mass spectrometry was used to show that the pTpTp substrate was cleaved at the phosphodiester bond between the two thymidines; no side products were detected. It thus appears that the methodology is applicable to both macromolecular and small molecule substrates. We are currently exploring the possibilities for isolating novel catalysts from libraries of enzyme or antibody origin.

Most enzyme libraries displayed on phage require superinfection by a helper phage like M13K07. The selection protocol described here can therefore be applied directly to these libraries one simply needs to prepare phage after superinfection of the phagemid encoded library with the acid peptide helper phage, and conjugate the substrate of choice to the basic peptide B. Likewise, this methodology can be applied to populations of structurally diverse proteins. The collection of proteins encoded by a genome is one such population. For example, it should be possible to isolate natural kinases with predefined substrate specificity from a genomic protein library using this selection scheme. This type of functional cloning in which a natural enzyme (and the gene that encodes it) is isolated on the basis of its catalytic activity should be applicable to many reactions catalyzed by natural enzymes.

References and Notes used in example 1.

1. Schultz, P.G. Lerner, R.A. (1995) Science 269, 1835- 1842.

wo 00/11211 PCT/DK99/00441 63 2. Marks, Hoogenboom, Bonnert, McCafferty, Griffiths, A.D. Winter, G. (1991) J. Mol.

Biol. 222, 581-597. Barbas, III, Bain, J.D., Hoekstra, D.M. Lerner, R.A. (1992) Proc. Natl. Acad.

Sdi. U.S.A. 89, 4457-4461. Griffiths, et al.

(1994) EMBO J. 13, 3245-3260.

3. Janda, K. Lo, C-H. L. Li, Barbas, C. F. III, Wirsching, P. Lerner, R.A. (1994) Proc. Natl. Acad. Sci.

U.S.A. 91, 2532-2536.

4. Soumillion, Jespers, Bouchet, Marchand- Brynaert, Winter, G. Fastrez, J. (1994) J. Mol.

Biol. 237, 415-422. Janda, Lo, Lo, C- Sim, Wang, Wong, C-H. Lerner, R.A.

(1997) Science 275, 945.

5. Gao, Lin, Lo, Mao, Wirsching, P., Lerner, R.A. Janda, K.D. (1997) Proc. Natl. Acad. Sci.

U.S.A. 94, 11777-11782.

6. Erickson, B.W. Merrifield, R.B. (1973) J. Am. Chem. Soc.

3750-3756.

7. Piles, Zi~rcher, Schar, M. Moser, H.E. (1993) Nuci. Acids. Res. 21, 3191-3196.

8. Marriott, G. Heidecker, M. Biochem. (1994) 33, 9092- 9097.

9. Kunkel, Roberts, J.D. Zakour, R.A. (1987) Methods in Enzymology 154, 369.

Hibler, Barr, Genlt, J.A. Inouye, M. (1985) J. Biol. Chem. 260, 2670-2674.

11. 0rum, Andersen, Oster, Johansen, L. Riise, Bjornvad, M. Svendsen, I. Engberg. J. (1993) Nuci. Acids Res. 21, 4491-4498.

12. Schultz, P.G. Rornesberg, F.E. unpublished results.

WO 00/11211 PCT/DK99/00441 64 13. Romesberg, Spiller, Schultz, P.G. Stevens, R.C. (1998) Science 279, 1929-1933.

14. Sambrook, Fritsch, E.F. Maniatis, T. (1989) "Molecular Cloning: A Laboratory Manual," 2nd Ed. Cold Spring Harbor Lab., Cold Spring Harbor, NY.

Pei, Corey, D. R. Schultz, P.G. (1990) Proc. Natl.

Acad. Sci. 87, 9858-9862.

16. Lubkowski, Hennecke, Plockthun, A. Wlodawer, A. (1998) Nature Structural Biology 5, 140-147. (b) Kremser, A. Rasched, I. (1994) Biochemistry 33, 13954- 13958.

17. O'Shea, Rutkowski, Kim, P.S. (1989) Science 243, 538-542. O'Shea, Rutkowski, Stafford, W.F. III Kim, P.S. (1989) Science 245, 646-648. (c) O'Shea, Klemm, Kim, P.S. Alber, T. (1991) Science 254, 539-544. xZhou, Kay, C. M. Hodges, R.S. (1993) Biochemistry 32, 3178-3187.

18. Cotton, Hazen, Jr., Legg, M.J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555. Tucker, Hazen, Cotton, F.A. (1978) Mol. Cell. Biochem. 22, 67-77. Sondek, J. Shortle, D. (1990) Proteins 7, 299-305. Hale, Poole, L.B. Gerlt, J.A. (1993) Biochemistry, 32, 7479-7487. Hynes, T.R.

Fox, R.O. (1991) Proteins 10, 92-105. Loll, P.J., Quirk, Lattman, E.E. Gravito, (1995) Biochem.

34, 4316-4324. Judice, Gamble, Murphy, de Vos, A.M. Schultz, P.G. (1993) Science 261, 1578-1581.

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Tucker, Hazen, Jr. Cotton, F.A. (1979) Mol.

Cell. Biochem. 23, 3-16.

WO 00/11211 PCT/DK99/00441 Example 2: Isolation of lipase variants, using phage-displayed lipase as the catalyst and DNA oligos as the XY exchange pair.

This is an example of the selection scheme depicted in figure 8: Phagemid construction.

The PCR-product obtained by using the Humicola lanuginosa lipase gene (SP400) as template and the primers Fwd: GTCACA-

GATCCTCGCGAATTGGCCCAGCCGGCCATGGCCGAGGTCTCGCAGGATCTGTTTAACCAGTTC

and Rev: CAGTCACAGATCCTCGCGAATTGGTGCGGCCGCAAGACATGTCCCAAT- TAACCCGAAGTACC, was digested with SfiI and NotI restriction enzymes and inserted into SfiI and NotI digested phagemid (0rum et al., 1993, NAR 21:4491-4498). The resulting phagemid, pFab-SP400, carries a gene fusion shown SEQ ID NO: 1 which comprises (reading from the N-terminal end) the pelB signal sequence (underlined), the gene encoding mature lipase, and the pIII gene from amino acid residue 198 (underlined). The pFab- SP400 lipase library comprises a number of such phagemid constructions, differing in the lipase amino acids sequence.

Production of phage particles.

Phage particles is produced with minor modifications according to Orum et al. (see above). Briefly, E. coli ToplOF' is transformed with the pFab-SP400 lipase library, and shaken at 37 OC in LB medium containing 100 pg/mL ampicillin. At an OD600 of 0.5, acid helper phage (a M13K07 derivative, carrying a 30'meric acid peptide extension at the N-terminus of pIII, see Pedersen et al., PNAS (1998), in press) is added to a final concentration of 1.5 x 108 cfu/mL, and incubated at 37 °C for min. The cells are pelleted and resuspended in LB, 5 pM IPTG, 100 pg/mL ampicillin, 50 pg/mL kanamycin, and shaken for 6-8 hours at 37 OC. Cells are pelleted and phage particles in WO 00/11211 PCT/DK99/00441 66 the supernatant PEG precipitated, followed by resuspension in TBS (25 mM Tris-HCl, pH 7.4, 140 mM NaC1, 2.5 mM KC1). PEG precipitation and resuspension is repeated, and finally the phage are steril filtrated.

Synthesis of base-linker-DNA conjugate ("base-linker-X").

The photoprotected and F-moc protected compound, Fmoc-S-(2- 1 is synthesized. The resulting crude product is extensively dried under vacuum, and used directly in the synthesis of the base-linker peptide C(GGS)4AQLKKKLQALKKKNAQLKWKLQALKKK-LAQGGC (base sequence underlined, photoprotected cysteine in bold). A DNA oligo of approximate length and sequence 5'-SH-GGGAAGAACCC-3' is synthesized with a 5'-thiol (5'-Thiol-Modifier C6, Glen Research) and purified by reverse phase HPLC following removal from the resin; the trityl protecting group on the thiol is removed according to the protocol of Glen Research. The products are lyophilized, dissolved in water and conjugated to the base-linker peptide as follows: Base-linker peptide is reacted with a fold molar excess of N,N'-bis(3-maleimidopropionyl)-2-hydroxy- 1,3-propanediamine in sodium phosphate buffer, pH 5.5, for approximatelylO hours under nitrogen at 4 OC. The resulting product is purified from the reaction mixture by reverse phase HPLC. The buffer is adjusted to sodium phosphate, pH 7, NaCl is added to avoid precipitation, and the DNA oligo described above is added, and the solution incubated at RT for approximately h under nitrogen. The product is purified by anion exchange FPLC, and product fractions are used directly for the photodeprotection step. In the photodeprotection step the 2-nitroprotecting group on the C-terminal cysteine is removed by photolysis as follows: The protected conjugate (vide supra) is degassed thoroughly with argon and then exposed WO 00/11211 PCT/DK99/00441 67 to a mercury lamp in a septum capped glass vial for 30 min, to yield the deprotected base-linker-DNA conjugate.

Synthesis of "DNA-pnp butyrate ester-column matrix" conjugates.

The Y-substrate-column moiety to be used in this selection is prepared by coupling 5'-thiol-modified DNA oligos of approximate length and sequence 5'-SH-CCCTTCTT-3' and 5'SH-TTCTTGGG to a p-nitro-phenyl-butyrate ester or p-nitro-phenyl-palmitate ester, eg. by reaction of the thiolated DNA with a maleimide unit on the butyrate or palmitate. The product is then attached to the column matrix through either the ortho or meta position by specific reaction with a functional group on the matrix.

Covalent attachment of base-linker-DNA conjugate to phage.

Approximately 101 0 phage particles are incubated in 4 mL TBS, 10 mM EDTA, 0.1 BSA, 1 mM mercaptoethylamine (MEA) and 100 nmole of base-linker-DNA, at 37 °C for 60 minutes, then PEG precipitated twice and resuspended in lipase buffer (50 mM Tris-HCl pH 7.5, 0.3 mM CaCI, 0.1 mM MgC12, 0.1 Triton X- 100).

Pre-sorting phages to which the base-linker-DNA conjugate has been attached.

To get rid of phages to which has not been attached a baselinker-DNA conjugate, phages that have gone through the covalent attachment procedure described above are loaded on an affinity-column carrying DNA oligos of a sequence complimentary to the DNA in the base-linker-DNA-conjugate the sequence GGGAAGAACCC). Phage that are retarded on the column are collected.

Isolation of the most active lipases in the library.

A column to which the "DNA-pnp butyrate" has been coupled to the matrix, is equilibrated in lipase buffer. Then the phage particles to which the base-linker-DNA conjugate has been at- WO 00/11211 PCT/DK99/00441 68 tached (see above) are loaded on the column. A titer determination is performed on each fraction collected; the fastest moving fraction that contains a significant number of phage should contain the phage carrying the more active lipases.

Example 3: Isolation of RNA-cleaving deoxy-ribozyme variants, using a PCR-amplifiable DNA library and DNA oligos as the XY exchange pair (the Y exchange unit consists of two oligos, Y1 and Y2, that have overlapping binding sites on the X oligo).

DNA library construction.

A PCR-reaction is performed with the primers "sense": GGAAGGGATGGTCACATGCA-3' and "antisense": AGCTTACCG-3', on the template TTAGGCTAGCTACAACGATTTTTCCcggtaagcttggcaactgac-3' (priming sites in lower case, deoxy-ribozyme catalytic motif italicized, substrate binding sequence underlined, X exchange moiety in bold).

The PCR-product is immobilized by repeated passing through streptavidin affinity columns (Genosys, The Woodlands, Texas), washed with several column-volumes of wash buffer (1 M NaCI, 0.1 mM EDTA, 50 mM Tris-HCl pH and the non-biotinylated strand eluted with 100 mM NaOH. The single-stranded DNA molecules are ethanol precipitated, and used in the column-based selection described below. The ssDNA molecules of the ("wildtype") sequence shown above cleave RNA molecules of sequence 5'-r(GGAAAAAGUAACUAG)-3' in a Mg++ dependent reaction. The deoxy-ribozyme library (see below) comprises a number of such ssDNA-molecules, differing in the sequence between the priming sites.

Preparation of Y-RNA target-column matrix.

An equimolar solution of two mixed DNA/RNA oligos of sequences -r(GGAAAAAGUAACUAG) -d(TTAGTGTCTCACCATCAT- WO 00/11211 PCT/DK99/00441 69 CC)-3' and 5'-biotin-d (TACACG)-r(GGAAAAAGUAACUAG)-d(TTAGTGT- CTCGTGAATCCCT)-3' (Oligos Etc., USA) are immobilized by repeated passing through streptavidin affinity columns, and washed with several column-volumes of wash buffer (1 M NaC1, 0.1 mM EDTA, 50 mM Tris-HCl pH The Y exchange moieties Y1 and Y2 are in bold.

Isolation of the most active RNA-cleaving deoxy-ribozymes in a ssDNA library.

A ssDNA library (see above) is loaded on a streptavidin column on which the "Y1-RNA-biotin" and "Y2-RNA-biotin" target substrates (see above) have been immobilized in wash buffer. After equilibration with several column-volumes of wash buffer, flow of a reaction buffer (10 mM MgC12, 1 M NaC1, 50 mM TrisHCl pH 7.5) is maintained for a while. Fractions are collected, and the DNA content measured by UV detection). The fastest moving fraction that contains a significant amount of DNA should contain the DNA carrying the most active magnesiumdependent RNA-cleaving molecules.

Example 4: Optimization of an enzyme that catalyzes aldol formation. In this example a polyhistidine loop and an associated metal ion comprise the X unit; a bidentate ligand (ethylenediamine) constitutes the Y unit.

A ketone substrate is coupled to an ethylenediamine (EDA) moiety, the enzyme carries a polyhistidine loop. When the ketone and enzymes are mixed in the presence of an appropriate metal ion the polyhistidine and the metal ion will form a kinetically and thermodynamically stable interaction.

The EDA-ketones will compete for the remaining two coordination sites of the polyhistidine-bound metal. Enzymes with aldolase activity will promote the reaction of the ketone attached to WO 00/11211 PCT/DK99/00441 the enzyme with an aldehyde in the column buffer, to form the aldol product. Typically, this aldol with some frequency undergoes dehydration, to form an an a,P-unsaturated ketone. This conjugated olefin can undergo a Michael addition reaction with the free thiols on the column, thereby immobilizing the enzyme.

However, EDA-ketone conjugates in the buffer will exchange with the immobilized enzyme, and release the enzyme in its original substrate bound form. In this set-up, the His*metal-EDA represents X-Y, and the product binding moiety is a nucleophile that traps the product after water elimination. The depicted selection scheme should thus select for enzymes that efficiently catalyze the aldol and dehydration reactions.

Example Examples of XY-exchange pairs suitable to be incorporated in a phage-display system as described in example 1: XY "embodiments" in the phage display system as describe in example 1: Solution 1: Synthesize base-linker-X following the guidelines from example 1, except that now X is coupled to the base-linker peptide, not the substrate (see example 2 above). Crosslink the base-linker-X conjugate to acid extensions on pIII (on the helper phage), as described in example 1 above (see figure 12 Couple Y to the substrate, and perform the selection (see example 2 above).

Solution 2: Insert four, five or six histidines at the Nterminus of pill of the helper phage, or into an exposed loop of pIII (the His4 and a metal ion comprise the X moiety). To substrate attach a metal ligand bidentate or tridentate) through a flexible linker a polyethylene glycol). Mix WO 00/11211 PCT/DK99/00441 71 phage and metal ligand-substrate in the presence of a metal ion of choice, then perform selection (See figure 12 B).

Solution 3: Insert the acid or His4 peptide sequence into the enzyme-pIII fusion on the phagemid, preferably in the linker connecting pIII and the enzyme. Proceed as described above.

XY "embodiments" for proteins in general: Solution 1: Insert the acid peptide or His4 sequence into an exposed loop, or at the N- or C-terminal terminus of the enzyme. Proceed as described above.

Solution 2: Introduce (at the DNA level) a unique cysteine into the enzyme, couple the X-moiety by a thiol specific reaction disulfide bond formation, addition reaction to maleimide), and proceed as described above.

Example 6: Example of an exchange pair especially suitable to be incorporated in a plasmid-peptide. polysome-peptide or mRNA-peptide system, In the following it is described how the exchange pairsubstrate may be attached to an enzyme in a plasmid peptide system (Schatz et al., 1996, Meth. Enzym., vol. 267, pp. 171- 191), polysome peptide system (Mattheakis et al., 1994, Proc.

Natl. Acad. Sci. USA, vol. 91, pp. 9022-9026; He and Taussig, 1997, Nucleic Acids Research, vol. 25, pp. 5132-5134) or mRNA peptide system (Roberts and Szostak, 1997, Proc. Natl. Acad.

Sci. USA, vol. 94, pp. 12297-12302). Here examplified with the mRNA-peptide fusion system.

Design the template DNA so as to make a portion of the mRNA in the mRNA-protein fusion become a X unit (eg. include the DNA sequence 5'-GCCGAAGCGCAATGAAGGGCAACCCG-3' in the template DNA) See figure 13A. Attach the desired substrate to a DNA Y-unit (eg. attach substrate to a mix of the the two oligos WO 00/11211 PCT/DK99/00441 72 Y1: 3'-TTACTTCCCGTTGGGC-5' and Y2: Then after preparation of the mRNA-peptide fusion, mix the mRNA-peptide fusion (carrying the X unit) and the substrate conjugate (carrying the Y unit), and perform substrate reloading selection, for example by applying the mix to a product binding column (see figure 2).

Alternatively, the X unit can be part of the linker that connects the mRNA and the encoded peptide (eg. include the DNA sequence 5'-GCCGAAGCGCAATGAAGGGCAACCCG-3' in the linker that connects the mRNA and peptide), see figure 13B. Then mix with the substrate conjugates and perform substrate reloading selection as described above.

Example 7: Enrichment of cells producing the more active proteases in a background of cells producing less active protease variants.

In this example, the individual unit consists of a cell, a substrate attached to the surface of that cell through an XY exchange unit, and enzymes produced and secreted by the cell (figure 14). Free Y-substrate in the surrounding media continuously replace substrate attached to the cell surface. Consequently, as the local concentration of secreted enzyme is much higher near the substrate attached to the cell from which it was secreted than near any of the other cells' attached substrates, there will be more product attached on a cell secreting an active enzyme than on a cell secreting a less active enzyme.

The substrate can be attached to the cell surface in many ways. For example, phospholipids, fatty acids, sterols, cholesteryl esters may be derivatized with the substrate of the target reaction. When incubated with cells, these molecules readily localize in the membrane interior, and expose the substrate WO 00/11211 PCT/DK99/00441 73 on the surface of the cell. Alternatively, the substrate may be derivatized with crosslinking reagents that react with the surface constituents. Finally, the substrate may be derivatized with structures proteins, antibodies) that bind to membrane components such as polysaccharides or membrane proteins.

The principle is here examplified in the case where the individual unit consists of a cell (for example Bacillus), attached substrate (peptide), and secreted enzyme (protease). The His6-metal-IDA or His6-metal-NTA complex is used as XY exchange unit. The selection is performed in the column format. The column matrix is coated with peptides carrying a target sequence for the protease. The peptides are attached to the column matrix at one end, and carry a polyhistidine (His6) tag at the other.

The experiment is performed as follows. Bacillus cells, secreting the protease of interest (for example c-component from Bacillus licheniformis or the commercial Savinase protease), are harvested in the exponential growth phase, resuspended in apropriate buffer and incubated with a bifunctional molecule that will crosslink to cell surface components. The bi-functional molecule may be a Nhydroxysuccinimide (NHS) moiety, linked to an iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA) moiety. NHS reacts with primary amines on the cell surface, which covalently anchors the IDA or NTA moiety to the cell surface.

A suitable column matrix for the separation of cells (for example Sepharose or Sephadex) is coated or derivatized with a peptide target for the protease of interest (for example, in the case of the c-component, the sequence IELSEPIGNTVCHHHHHH).

At one end the peptide carries a polyhistidine extension. At the other end it is attached to the column matrix (for example WO 00/11211 PCT/DK99/00441 74 through reaction of the N-terminal amine with NHS-activated Sepharose, Pharmacia Biotech).

The IDA- or NTA-modified cells are loaded on the peptidemodified column, in appropriate buffer (for example 2xYT or LB medium, with added Ni", Zn", Cu", or Co"; temperature 35-50 0

C)

The flow-rate is kept below 0.5 mL/min.

The complex His6-metal-IDA (or His6-metal-NTA) forms, and thus the protease substrate. becomes attached to the cell. If the cell secretes active proteases, these cleave the target, and release the cell from the column matrix; also, the His6metal-IDA (or His6-metal-NTA) complexes are in rapid equilibrium, resulting in continuous replacement of the substrate or product with new substrate. Consequently, the cells secreting the more efficient enzymes, or cells that secrete more of an active enzyme, elute first at the bottom of the column.

The principle is tested in the following way. Cells secreting a number of protease variants, or that secretes the same protease in varying amounts, are treated as described above, and the substrate reloading protocol performed as described. Cells secreting the more efficient protease, or secreting most of the protease, will elute first.

The stringency of the selection is controlled by the density of substrate immobilized on the column matrix, and on the IDA- (or NTA-) coupling density on the cell surface.

Variants with improved expression or activity at different conditions, such as salt concentration, pH or temperature may be isolated by this method.

Example 8: WO 00/11211 PCT/DK99/00441 Isolation of hammerhead ribozymes. using a DNA/RNA hybrid duplex as the XY exchange pair. and part of the Y-unit as the target substrate.

The hammerhead is a naturally occurring ribozyme that cleaves at a specific phosphodiester to produce 3'-cyclic phosphate and termini with a rate (kcat) approaching 1 min-' (Stage-Zimmermann et al., 1998,RNA vol. 4, pp. 875-89). The specificity is mediated by simple Watson-Crick base pairring between the .ribozyme and the target and because the only requirement in the target sequence for cleavage is a U followed by A,U or C, the hammerhead can be designed to cleave virtually any RNA molecule. However, for therapeutic use the hammerhead suffers from one major drawback. When the two duplexes are extended to increase the specificity, the off-rate decreases significantly and the ribozyme fails to turn over. To search for hammerhead variants exhibiting increased cleavage rates others have performed in vitro selections from a RNA pool containing stretches of randomised sequence e.g. (Vaish et al., 1997, Biochemistry, vol. 36, pp. 6495-501). However the selection protocol has in all reported studies been based on a single round of cleavage. Using the substrate reloading strategy the ribozyme must release itself multiple times from an immobilised matrix to be eluted from the column. The salt concentration, temperature and duplex length in the experiment described below is optimized in order for the ribozyme to rapidly liberate the substrate after cleavage. The liberated ribozyme quickly binds a new target stabilised by two coaxial stacked 5 bp helices. This rapid X-Y exchange is accomplished by the fact that Y is cleaved into half by the ribozyme, thereby destabilizing the X- Y interaction (see figure 3 When applying a continuos flow of buffer through the column the faster cleaving ribozyme elutes first.

WO 00/11211 PCT/DK99/00441 76 To eliminate the potential selection of RNA molecules, that due to competing internal structures or misfolding, exhibits a slow substrate binding (and therefore a rapid elution profile), a pre-selection for molecules that efficiently will interact with an uncleavable DNA-substrate oligo, can optionally be performed (See figure To demonstrate the principle we have chosen to randomise 7 positions within the conserved core of the ribozyme (See figure This corresponds to 16384 different molecules, one of which corresponds to the wild type hammerhead exhibiting a cleavage rate of approximately 1 min' (Stage-Zimmermann et al., 1998, RNA, vol. 4, pp. 875-89). Since our library in the experiment described below contains more than 1013 RNA molecules we expect to isolate the wild type ribozyme or a ribozyme with at least the rate of the wild type.

Outline of the experiment: Materials: 1. RNA library (approximately 100 pg) as shown in figure 15 is produced by conventional techniques (in vitro transcription by T7 RNA polymerase using a randomised synthetic DNA oligo as template). The library is purified by PAGE and redissolved in column buffer and stored in 10 tg aliquots at -80 oC.

2. NHS activated Sepharose for coupling of the substrate oligo (through 2 -amino-l-(2-pyrridyldithio)-ethane) and a siliconized glass column( 0.2 cm x 10 cm) 3. Substrate RNA oligo (figure 15) is synthesised by conventional chemistry and HPLC purified. A spacer is incorporated at the 5'-end. HEGspacer- AGCUGUCACUCC-3') WO 00/11211 PCT/DK99/00441 77 4. Counter selection DNA oligo (Fig 15 B) based on deoxynucleotides is synthesised by conventional chemistry thiophosphate-HEGspacer-AGCTGTCACTCC-3') Column buffer (10 mM MgCl 2 50 mM Tris/HCl pH Experimental procedure: (The DNAcounter selection step 2 4 is optional) 1. 100 nmol of RNA substrate oligo or 100 nmol of counter selection DNA oligo is loaded on a 1 ml bed volume NHS activated Sepharose, derivatized with the activated disulfide, in a 0.2 cm diameter, 10 cm long siliconized glass column and equilibrated with 5 volumes of column buffer immediately before use.

2. The RNA library is loaded onto the DNA oligo column and washed with 10 volumes of column buffer at 20 OC and a flow speed of 1 ml/min.

3. The column temperature is adjusted to 60 OC and the RNA is eluted in two column volumes of H 2 0.

4. The buffer is adjusted to 0.25 M NaOH pH 6.0 and the RNA is precipitated with 2.5 Vol EtOH and redisolved in 20 ml of column buffer.

This sample is incubated at 37 OC for 5 mins to renature the RNA and carefully loaded on the RNA column. The temperature should be adjusted in the range 25 50 OC for optimisation of the subsequent selection. The flow rate should be kept below 0.5 ml/min to allow substrate reloading of the ribozymes.

6. Samples are collected from the bottom of the column and tested individually for ribozyme activity by standard methods (Vaish et al., 1997, Biochemistry, vol. 36, pp. 6495- 501).

7. Ribozymes from the earliest collected pools exhibiting ribozyme activity is reverse transcribed using a 3'end compli- WO 00/11211 PCT/DK99/00441 78 mentary primer and PCR amplified using up- and downstream matching primers. A T7 promoter is included in the upstream primer which enables subsequent transcription. The DNA pool is cloned in a plasmid and individual clones are analysed by sequencing. Ribozymes from individual clones are produced by T7 transcription and tested.

Example 9: Pre-enrichment of phages displaying His-taaged proteins.

Certain proteins are difficult to display on filamentous phage.

In particular, large proteins or proteins which have a toxic or growth inhibiting effect on E. coli often have low display efficiency, i.e. the majority of phage particles produced carry no pIll-fusion on the surface. Display efficiencies as low as one out of a thousand phages displaying the fusion protein have been reported (Jestin et al., 1999, Angew. Chemi. Int. Ed., vol. 38, pp. 1124-1127; Demartis et al., 1999, JMB, vol. 286, pp. 617-633). In such cases, a high non-specific background is expected, because of the large excess of phage particles carrying the DNA encoding the pIII fusion, but not displaying the fusion on the surface. To circumvent this potential problem, we inserted a histidine tag between the pIII coat protein and the enzyme, allowing the purification of phages displaying Histagged protein by Ni-NTA column chromatography.

Other tags that could have been used in a similar manner as described below for the Histidine tag include the intein-chitin binding domain fusion (Chong et al., 1997, Gene, vol. 192, pp 271-281), FLAG peptide (Slootstra et al., 1997, Molecular Diversity, vol. 2, pp. 156-164), and the maltose binding protein (Pryor and Leiting, 1997, Protein expression and Purification, vol. 10, pp. 309-319).

WO 00/11211 PCT/DK99/00441 79 Ni-NTA column purification of phages displaying the lipase- His6-pIII or cellulase-His6-pIII fusion protein.

A Ni-NTA spin column (Qiagen Spin Kit was equilibrated with 600 AL "50 mM sodium-phosphate buffer pH 8, 300 mM NaC1, 1 mM Imidazole, 0.05% BSA" (centrifuged 2 minutes at 700 To 400 tl phage preparation (see example 10 and 11) (approximately 1012 phage particles) was added .100 .L "250 mM sodium-phosphate buffer pH 8, 1.5 M NaCl, 0.25% BSA" and 4 AL 100 mM Imidazole, the solution loaded onto the pre-equilibrated column, and centrifuged for 4 minutes at 200 G. The column was washed twice with 600 pL "50 mM sodium-phosphate buffer pH 8, 300 mM NaC1, mM Imidazole, 0.05% BSA" (centrifugation 200 G for 4 minutes and 700 G for 2 minutes, respectively). Then the phages were eluted with 3 x 333 pL "50 mM sodium-phosphate buffer pH 8, 300 mM NaCl, 250 mM Imidazole, 0.05% BSA" (700 G, 2 minutes), and the 999 iL eluate PEG precipitated and resuspended in 400 pL mM sodium-phosphate buffer pH 8, 300 mM NaCl, 1 mM Imidazole, 0.05% BSA". The solution was loaded on a fresh spin column, and the procedure repeated, except that the final PEG precipitate was dissolved in 50 .L TE buffer pH 8. This procedure enriches approximately 500 fold for phage displaying His-tagged protein.

Example 10: Enrichment of wildtype lipase in a background of excess. less active lipase variants, using phage-displayed lipase and DNA oligos as the XY exchange pair.

This is an example of the selection scheme depicted in figure

B.

Phagemid construction.

WO 00/11211 PCT/DK99/00441 Phagemid ph8 (wildtype Lipase) and phl8 (Lipase S146A mutant): DNA oligos "Not-His6-sense" TCACCATCACTC-3') and "Not-His6-antisense" TGGTGATGGTGATGTGATCCTCCTGGTGC-3') were annealed, and the double-stranded product ligated into NotI-digested pFab-SP400 (described above) and pFab-SP400-S146A (identical to pFab- SP400, except that it carries a serine to alanine mutation at position 146, lowering its activity at least 100-fold). The resulting phagemids, ph8 (wildtype Lipase) and phl8 (Lipase S146A mutant), carry the amino acids sequence shown in SEQ ID NO 2 (wildtype shown) The resulting gene fusion comprises (reading from the N-terminal end) the pelB signal sequence (underlined), the gene encoding mature lipase (wt or S146A mutant), the insert (italics) with the six histidines (bold), and the pIII gene from amino acid residue 198 (underlined).

Production of phage particles.

Phage particles were produced with minor modifications according to Orum et al. (see example Briefly, E. coli XLlblue were transformed with either the phagemid ph8 (Lipase wt) or phagemid phl8 (Lipase S146A mutant), or phl3 (a negative control; carries a His-tagged cellulase instead of Lipase, but is otherwise identical to the ph8 and phl8 constructs). The transformed cells were shaken at 37 OC in 2xYT medium containing 100 gg/mL ampicillin, 5 pg/mL tetracyclin and 2 glucose. At an 0 of 0.5, acid helper phage (a M13K07 derivative, carrying a 30'meric acid peptide extension at the N-terminus of pill, see Pedersen et al., PNAS (1998), 95, pp. 10523-10528) was added to a final concentration of 1.5 x 108 cfu/mL, and incubated at 37 °C for 20 min. The cells were pelleted and resuspended in 2xYT, 5 pM IPTG (100 mM IPTG for phl3), 100 gg/mL am- WO 00/11211 PCT/DK99/00441 81 picillin, 50 pg/mL kanamycin, and shaken for 3 hours at 30 OC.

Cells were pelleted and phage particles in the supernatant PEG precipitated three times and resuspended in 400 pL TE buffer mM Tris-HCl pH 8; 1 mM EDTA).

Before covalent coupling of base-linker-X to phage (see below), a pre-enrichment step for the phages displaying the Lipase variant was performed, following the protocol of example 9. The procedure generally led to a recovery of 0.04-0.2 of the input phage; we estimate that more than 90 of the recovered phage display lipase.

Active. histidine-tagged Lipase is displayed on phage.

In order to verify that the prepared phages display the LipasepIII fusion proteins, separate wells of a microtiter plate were coated with antibodies against the histidine tag (Penta-His antibody, Qiagen), antibodies against the Lipase protein, or, as a negative control, an antibody against an unrelated amylase enzyme. Approximately 109 phages displaying non-His-tagged wildtype Lipase (ph3), His-tagged wildtype Lipase (ph8) or Histagged Lipase S146A mutant (phl8) were added to separate wells.

The results shown in figure 16 show that ph8 and phl8 both display the His tag as expected; the non-His-tagged wildtype Lipase is not significantly bound to the anti-His antibody. Both mutant and wildtype Lipase are immobilized on anti-Lipase antibody, as expected. Finally, none of the Lipase variants are immobilized on the unrelated antibody. It is therefore concluded that the ph8 and phl8 phagemids encode Lipase-Histidine tagpIII fusions that are folded properly on the surface of phage.

The His-tagged phages (ph8 and phl8), were taken through the Ni-NTA column purification step described in example 9. The enrichment step is expected to yield a phage population almost WO 00/11211 PCT/DK99/00441 82 entirely consisting of phages displaying the His-tagged protein. The procedure involves two consecutive Ni-NTA column purifications; the first run reproducibly recovered 0.2-0.3 of the input, the second run recovered 10-20% of the input. These numbers are taken as an indication that the resulting phage population have been enriched dramatically with regard to displayed protein, and that the final phage population consists of phages that nearly all display the protein. Finally, in a Brilliant Green plate assay, cells containing the wildtype Lipase- His-pIII fusion but not cells containing the Lipase S146A-pIII fusion, exhibited Lipase activity. It is therefore tentatively concluded that ph8 display active, properly folded wildtype Lipase; the phl8-phages display properly folded Lipase S146A mutant with decreased Lipase activity.

Synthesis of base-linker-DNA conjugate ("base-linker-X").

The base-linker peptide C(GGS)4AQLKKKLQALKKKNAQLKWKLQALKKK- LAQGGC was conjugated to the 5'-thiol of the DNA oligos "X- 26mer" (HS-5'-ATTAAATTAGCGCAATGAGGGCAAC-3') and photodeprotected, following the protocol described in Pedersen et al., PNAS (1998), 95, 10523-10528. The underlined sequence constitutes the X moiety. The resulting conjugate is called "base-X- 26mer".

Synthesis of Y-substrate-biotin conjugates.

The heterofunctional molecule (39) (see figure 17) containing a maleimide moiety at one end, a biotin at the other, and an ester that serves as a substrate for the Lipase in the middle, was prepared. o-Aminododecanoic acid was first protected as its methyl ester (40) which was coupled with biotin-NHS followed by hydrolysis to give biotin-acid Convergently, Maleimide alcohol (42) was prepared by reacting maleimide-NHS with 6- WO 00/11211 PCT/DK99/00441 83 hydroxyhexylamine. Esterification between (41) and (42) afforded the target substrate Finally, compound (39) was conjugated to either Y1 DNA oligo CCCTTCATT-3') or Y2 DNA oligo -TTCATTGCGCTTCGGCAAATAAATAA- The underlined sequences constitute the Y1 and Y2 exchange moieties.

Covalent attachment of base-linker-DNA conjugate to phage.

The X-26mer conjugate (see above) is covalently attached to the Ni-NTA purified phages (see above), for example by following the guidelines in example 1 above. The phages from the coupling reaction may optionally be taken through another purification step, in order to assure a high degree of coupling. This involves annealing of the phages to streptavidin coated beads, to which a biotinylated oligo complementary to the X-26mer, has been immobilized. Following several washes to remove phages that have not been covalently attached to the X-26mer, the temperature is increased to melt the DNA duplexes and release the coupled phages.

Isolation of the more active lipase.

The Y1-substrate-biotin and Y2-substrate-biotin conjugates are mixed with a streptavidin-derivatized matrix (for example streptavidin immobilized on 4 agarose, Sigma), at a concentration of 1-10 gM, and incubated at room temperature 1-2 hours. The column is washed, and phages to which the X-26mer conjugate has been coupled (see above) are added in a buffer that allows Lipase activity as well as efficient annealing (contains MgCl 2 and CaCl 2 at 20-35 0 C. Alternatively, the coupling step is performed directly on the column. The X-26mer DNA coupled to phage will anneal to the Yl- or Y2-substrate-biotin molecules and become immobilized on the matrix through the sub- WO 00/11211 PCT/DK99/00441 84 strate. Phages displaying catalytically active Lipase will cleave the substrate and continue the migration through the column; upon interaction with another Y1- or Y2-substrate, an exchange reaction may take place, which will immobilize the phage again. A less catalytic Lipase will spend more time bound to a given substrate. Therefore, the catalytically more active phages will migrate faster than the less active phages, and can therefore be collected first at the bottom of the column.

Example 11: Enrichment of wildtvpe cellulase C6B. displayed on filamentous phage, in a background of less active mutant cellulases, using the cellulose binding domain (CBD) of the cellulase as the X unit. and Avicel (cellulose) matrix as both substrate and Y-unit.

Phagemid construction, Phagemid ph7 (cloning vector): The DNA oligos "Not-His6-sense" (see above) and "Not-His6antisense" (see above) were annealed, and the double stranded product ligated into NotI digested pFab5c.His (Orum et al., 1993, Nucleic Acids Research, vol. 21, pp. 4491-4498), to form the phagemid ph7. Basically, this results in the addition of the amino acids sequence APGGSHHHHHHS to the N-terminal end of the pIII coat protein (the His-tag is underlined).

Phagemid phl3 (cellulase wt): A PCR reaction was performed on a synthetic DNA construct encoding wildtype cellulase C6B of Humicola insolens, using the primers: NcoI-Cellulase-fwd: Not I Cellulase-bck: 5' -CCTTTAGAGCCTGCGGCCGCGCCTCCTGGGAGGCACTGGCT

GTACCAC.

WO 00/11211 PCT/DK99/00441 The product was digested with NcoI and NotI, and inserted into NcoI- and NotI-digested ph7 (see above), to give phl3.

Phagemid phl4 (cellulase D316A): Construction as for phl3, except that a DNA molecule with the mutation D316A was used as template.

Phagemid 16 (cellulase D139A, D316A): Two PCR reactions were first performed. In one, the template containing the mutation D316A (see above) was employed with the primers: D139A-fwd:

GTACCAC.

In another PCR, the same template but the primers: NcoI-Cellulase-fwd: (see above) D139A-bck: was used. The two PCR products were purified on an agarose gel, and used as template for a PCR reaction using the primers "NcoI-Cellulase-fwd" (see above) and "NotI-Cellulase-bck" (see above). The resulting PCR product was digested with NcoI and NotI and inserted into NcoI- and NotI digested ph7 (see above).

The resulting gene fusions have the sequence shown in SEQ ID NO 3 (wildtype shown; starting with the first codon of pelB and ending with the STOP codon of pIII). This corresponds to the following protein fusion (reading from the N-terminus): PelB peptide, Cellulase C6B, Histidine linker, pIII coat protein.

Phagemid phl7 (cellulose binding domain, CBD): A PCR reaction was performed using a synthetic DNA encoding wildtype Cellulase C6B from Humicola insolens (see above) and the primers "NcoI-CBD-fwd" GCGCTGCCGGTTC-3') and "NotI-Cellulase-bck" (see above). The product was digested with NcoI and NotI, and inserted into NcoI- and NotI-digested ph7 (see above), to give phl7. The re- WO 00/11211 PCT/DK99/00441 86 suiting protein fusion has the sequence shown in SEQ ID NO 4.

This corresponds to the following protein fusion (reading from the N-terminal: pelB leader peptide, CBD domain, Histidine tag, pIII coat protein.

Preparation of phage particles.

Phages were prepared as described in example 10, except that 100 iM IPTG was added after super-infection with Helper Phage.

Active. histidine-tagged cellulase is displayed on phage.

In order to verify that the prepared phages display the Cellulase-pIII or CBD-pIII fusion proteins, separate wells of a microtiter plate was coated with antibodies against the histidine tag, antibodies against the Cellulase C6B protein, or, as a negative control, an antibody against an unrelated lipase enzyme (SP400, see example Approximately 108 phages displaying Cellulase wt (phl3), Cellulase D316A (phl4), Cellulase D316A,D139A (phl6), CBD (phl7) or an unrelated Lipase enzyme (ph3) were added to each well. The results shown in figure 18 show that phl3, phl4, phl6, and phl7 all display the His tag.

The full-length cellulases (phl3, phl4, phl6) bind the anticellulase antibody, the CBD domain does not bind this antibody.

As expected, the negative control (ph3) does not bind the anti- His or anti-cellulase antibodies. Also as expected, the cellulase and CBD displaying phages do not bind the anti-lipase antibody; the lipase-displaying phage (ph3) does, as expected.

It is therefore concluded that the phl3, phl4 and phl6 phagemids encode cellulase-Histidine tag-pIII fusions, and that these most likely are folded properly on the surface of phage.

The His-tagged phages (phl3, phl4, phl6, and phl7), were taken through the Ni-NTA column purification step described in example 9. The enrichment step is expected to yield a phage popula- WO 00/11211 PCT/DK99/00441 87 tion almost entirely consisting of phages displaying the Histagged protein. The procedure involves two consecutive Ni-NTA column purifications; the first run reproducibly recovered less than 0.1 of the input, the second run recovered approximately 20% of the input. These numbers are taken as an indication that the resulting phage population have been enriched dramatically with regard to displayed protein, and that the final phage population consists of phages.that nearly all display the protein. Finally, in a liquid CMC-Congo Red cellulase activity assay it was further verified that cell extracts of XL1 blue carrying phagemid expressing wildtype Cellulase, but not cells expressing mutant D316A Cellulase, exhibited cellulase activity (data not shown). It is therefore proposed that phl3-phages displays active, properly folded wildtype Cellulase; the phl4and phl6-phages display properly folded cellulase mutants with decreased cellulase activity.

The principle of substrate reloading in the context of the phage-displayed cellulase.

The Cellulase C6B from Humicola insolens consists of two domains, a core domain with catalytic activity and a cellulose binding domain, CBD, connected to the core via a linker of approximately 25 amino acids. We therefore reasoned that conditions might exist where the affinity of CBD for cellulose was appropriately weak that a reasonable exchange of CBD for different sites on cellulose could be achieved, and it might be possible to set up a colum-based selection based on this exchange. Under such conditions, the CBD would bind a site on the cellulose, and thereby hold the core enzyme in the vicinity of this binding site (figure 19). An active enzyme might cleave the cellulose string to which its CBD domain was attached, and WO 00/11211 PCT/DK99/00441 88 hence release itself. Whereas a less active enzyme would be immobilized on the cellulose until the CBD associated from its binding site. At one extreme, if the binding of CBD to cellulose was extremely strong, the cellulase would release itself from the column by cleavage, and elute from the column as a CBD-cellulose string complex. In this case, only one turn-over of substrate would be required, and hence, there would be a small selective pressure for the better cellulase. At the other extreme, the CBD-cellulose interaction would be so weak that the half-life of the CBD-cellulose complex, rather than the turn-over rate of the enzyme, would determine the time spent immobilized on the column. In this case there would therefore be little selective pressure for the more active enzyme.

CBD of Cellulase C6B has high affinity for acid-treated Avicel

M

(microcrystalline cellulose, Merck) at neutral pH, and low affinity at high pH. Cellulase can be eluted from Avicel' at pH above 11.6. We therefore set out to define conditions where the Cellulase would be active and the CBD binding appropriately low to obtain a multiple turn-over system that would distinguish between cellulases of different specific activity.

The CBD domain of the displayed cellulase binds the Avicel column material.

We wanted to use the CBD-cellulose interaction as a simple model for a XY-exchange unit. Therefore, the ability of the CBD domain to reversibly bind phosphoric acid swollen Avicel M was examined. Approximately 1 mg acid-swollen Avicel M was mixed with Sephacryl to obtain a column bed volume of approximately 2 mL. Ni-NTA purified phl7-phage (see above) were loaded on the gel in Naphosphate buffer pH 7.5, and washed with several column volumes at increasing pH (same conditions as below). Con- WO 00/11211 PCT/DK99/00441 89 trol phages displaying an unrelated lipase enzyme were collected from the first fractions; the phl7-phages were not eluted until after several column volumes of Naphosphate buffer pH 11.6 (data not shown). Therefore, the CBD domain displayed on phage binds acid-swollen Avicel at neutral pH; at pH 11.6 the affinity is decreased and CBD is eluted.

Active cellulase is enriched over less active cellulase.

Next, we loaded an approximately 1:1 mix of Ni-NTA purified phages displaying wt cellulase (phl3) or D139A,D316A mutant cellulase (phl6) on an AvicelM/SephacrylM column, prepared as described above. In this experiment the temperature was 50 0

C.

Again, the loading buffer was 20 mM Na-phosphate buffer pH 0.05 BSA, 0.05 Tween. After washing with 1 volume 20 mM Naphosphate buffer pH 7.5, 0.05 BSA, 0.05 Tween, 500 mM NaC1 and 1 volume 20 mM Tris-HCl pH 8.5, 0.05 BSA, 0.05 Tween, 200 mM NaC1, the pH was increased to pH 10.6 for the next 6 column volumes. Finally, the pH was increased to 11.6.

Fractions were collected and the phage titer in each fraction determined. Also, the ratio of phl3:phl6 was determined by PCR reaction followed by PstI digestion and agarose gel analysis.

The D316A mutation introduces a PstI site, wherefore PstI digestion gives rise to two, smaller fragments. The intensity ratio of the upper and middle band on an agarose gel therefore correlates with the relative number of wildtype cellulase (phl3) to mutant cellulase phage (phl6) in a given fraction.

The ratio of phl3:phl6 in the input was approximately 0.7. In fractions 1-2 (corresponding to pH 7.5) and fractions 4-11 (corresponding to pH 10.6/11.6) the pH16 phage was in 1-10 fold excess, as estimated from the gel. In fraction 3, the ratio of pH13:pH16 was approximately 1.5. Therefore, an approximately 2fold enrichment of the more active (wildtype, phl3) cellulase WO 00/11211 PCT/DK99/00441 in a background of less active (double-mutant, phl6) cellulase had been achieved. It should be noted that 52% of the output phage was recovered in fractions 2-4, 17% in fractions 1 and 7- 11, and 31% of the phage remained immobilized on the column after 30 column volumes wash at pH 11.6.

In similar experiments we have not been able to enrich the wildtype cellulase in a background of the pH14 single-mutant.

We have reproducibly observed the two phases of the selection in the first fractions phl6 is recovered in excess, then phl3 is recovered in excess over one or two fractions, and then phl6 is recovered in excess in the remaining fractions). The fact that there are these two phases corroborate the proposal that at least two events take place as the phage displaying the cellulase migrates through the column: binding of the CBD domain to cellulose, and cleavage of the cellulose.

In the experiment the CBD-Avicel interaction represents an XYexchange unit. It was expected that this very simple XY unit would be sub-optimal: Firstly, most macromolecular interactions are described by slow kinetics, which would lead to slow substrate reloading in this example. Secondly, the binding of CBD and cellulose with respect to on- and off-rates was sought optimized through appropriate choice of pH. As the pH also affects the catalytic activity of the cellulase this is not ideal. Possibly, the binding may be adjusted by other means that do not interfere significantly with cellulase activity, for example salt concentration or the inclusion of high concentrations of detergents. If the affinity and/or the kinetics of the CBD-cellulose interaction can be optimized further this way, this should increase the stringency of the selection, and thus allow a separation of enzymes with only slightly different levels of activity (represented in the experiment above by the wildtype and single-mutant cellulase).

WO 00/11211 PCT/DK99/00441 91 Alternatively, the approach of example 10 may be applied to the phage-displayed Cellulase: )Rescue the phages (phl3, phl4, phl6, phl7 etc.) with the acid helper phage, iiNi-NTA purify phages displaying cellulase variants, "icouple the base-X-26mer conjugate to phages, i" immobilize Y-substrate conjugates on a column (substrate here denotes cellulose, for example 2 or 6 glucose units linked by 0-1,4 bonds), v)perform the substrate reloading protocol. This might increase the rate of substrate reloading, and allow separation of slightly different cellulase activities.

We conclude that even with this simple CBD-cellulose exchange unit it was possible to enrich for the more active cellulase (phl3) in a background of less active cellulases (phl6).

Example 12: Enrichment of wildtype RNaseA peptide on beads, in a background of excess, less active RNase A peptide variants, using a polyhistidine tag as the X unit, and iminodiacetic acid as the Y unit: In order to test the substrate reloading principle in the field of synthetic combinatorial libraries, we have designed a simple experiment involving the RNase A peptide. Two Cterminally biotinylated peptides, Peptidel and Peptide2, are synthesized, for example as outlined in (Gutte et al., 1971, Journal of Biological Chemistry, vol. 246, pp. 1922-1941). Peptidel carries wildtype RNase A sequence, except that six histidines have been added to the N-terminus. Peptide 2 has the same sequence as Peptidel, except that mutation(s) have been introduced that fully or partly eliminates the ribonuclease activity of the peptide (for example by replacing one or both of the two active site histidines, H12 and H119, with Alanine).

After deprotection, cleavage from synthesis support, and re- WO 00/11211 PCT/DK99/00441 92 folding as described in (Gutte et al., 1971, Journal of Biological Chemistry, vol. 246, pp. 1922-1941) the peptides are immobilized through the C-terminal biotin moiety to streptavidin coated beads of approximate diameter 10-100 nm. Such beads may be prepared by immobilizing streptavidin to Nhydroxy-succinimide-activated Latex beads (Polysciences Inc.).

A column (eg. Sepharose or Sephadex) is prepared, carrying RNA molecules immobilized at one. end to the matrix, and at the other end coupled to iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA). This may be done by synthesis of an RNA oligo (for example 20 nt long), functionalized with a thiol at one end, and biotin at the other end (Oligos Etc., Inc.). Then the thiol is used to specifically couple the RNA and iminodiacetic acid (or nitrilotriaceetic acid), by standard protocols.

A substrate reloading experiment is now performed as follows (figure 20). The beads carrying either Peptidel or Peptide2 are mixed in a buffer that allows RNase A activity (for example Tris-HCl pH 7 or 8) containing metal ions (for example Zn" or and loaded on the column described above. The column is washed with the same buffer, and fractions collected.

During their migration through the column, the Peptide beads become immobilized to the column matrix through the interaction of the six N-terminal histidines, Zn" or Ni", and the IDA coupled to the RNA molecule. Thus, a His6-metal-IDA complex is formed, which in effect attaches the bead carrying the peptides to the RNA, the substrate of RNase A. The active RNase A (Peptidel) will cleave the attached RNA and release itself, and thus continue its migration through the column. Peptide2, however, is catalytically less active than Peptidel, and will stay immobilized for a longer period of time, and hence, after a number of turn-overs the Peptidel beads will have separated from the Peptide2 beads as a result of their differential cata- WO 00/11211 PCT/DK99/00441 93 lytic activity towards the RNA substrate. The beads carrying the more active RNase A variant can therefore be collected first at the bottom of the column.

Using unnatural amino acids the catalytic machinery of RNase A may be examined, and potentially, new improved variants of RNase A evolved (Jackson et al., 1994, Science, vol. 266, pp. 243-247), using the substrate reloading selection outlined above. Alternatively, totally. random peptide libraries may be searched for novel catalysts. The sequence of the peptides on the recovered beads can be determined by Edamn degradation or mass spectrometry.

Example 13: Enrichment of the more active Stahyloccoal nuclease (SNase) variants in a background of less active Snase variants. This selection employs electrophoresis to isolate the more active enzymes.

A His6-tag is introduced either into the linker connecting SNase and pill coat protein in the phagemid p1178-6 (Pedersen et al., 1998, Proc. Natl. Acad. Sci. USA, vol. pp. 10523-10528), or alternatively, the His6-tag is cloned at the N-terminal end of gpIII gene of the M13K07 Helper Phage (following the cloning procedure outlined for the cloning of the acid extension into M13K07, described in Pedersen et al., 1998, Proc. Natl. Acad. Sci. USA, vol. 95, pp. 10523-10528).

Phage particles are produced, using regular M13K07 Helper Phage and the His6-tagged SNase phagemid, or using the p1178-6 phagemid and the His6-tagged Helper Phage. Both methods yield phages displaying SNase and a His6-tag on their surface.

A DNA oligo of approximately 20 nucleotides is derivatized at the 5'-end with iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA), using standard procedures. For example, the oligo may be synthesized with a 5'-thiol, which is then deri- WO 00/11211 PCT/DK99/00441 94 vatized with a bifunctional IDA-maleimide moiety. The derivatized oligo is now used for PCR, together with another primer, and a template that gives rise to a PCR product of 100-1000 base pairs.

The DNA fragment, derivatized with IDA or NTA at one end, is incubated with phages produced above, and loaded on a gel for electrophoresis, for example an 0.5-0.7 agarose gel in a suitable buffer. The buffer (Tris-HCl pH 5-8) contains Ca" (for SNase activity) and Zn", Ni", Co", or Cu" (for coordination to His6 and the IDA or NTA moiety), and a high concentration of the IDA- or NTA-derivatized DNA fragment described above.

The His6-tag, displayed on phage, binds DNA through formation of the complex His6-metal-IDA (or His6-metal-NTA). If the SNase displayed on the same phage is active, it cleaves the associated DNA. At the same time, a rapid exchange between different DNA fragments results in the constant reloading of fulllength fragment on phage. Therefore, the phage displaying the more active SNase will on average be associated with smaller fragments of DNA, and therefore migrates differently than phages displaying inactive SNase. The experiment is performed by mixing phages displaying a number of different SNase variants, and isolating the more active variants by electrophoresis as described above.

Example 14: DNA .olicos as XY exchange units: Measurement of exchange rates by fluorescense polarization spectroscopy.

A fundamental feature of the indirect substrate reloading protocol is the XY exchange unit that links the substrate and enzyme. Ideally, the XY unit should provide a means for the dynamic, fast and efficient substrate reloading on the enzyme.

WO 00/11211 PCT/DK99/00441 We wanted to design nucleic acids that would fulfill (at least partly) the requirements of the ideal XY unit: fast exchange, yet intrinsically stable XY complexes. Therefore, two sets of oligos, Set#1 and Set#2, were designed. The XY complexes of the two sets have expected melting temperatures around 60 and respectively. To provide a dynamic exchange, the DNA oligos were designed so that the two DNA oligos, Y1 and Y2, bound to different but overlapping targets on X (see Materials and Methods). With the expectation that the overlap would provide a faster exchange between the X-Y1 and X-Y2 complexes.

The exchange rates of the two sets of oligos were analyzed by fluorescense polarization spectroscopy at various temperatures.

The dynamic range (the temperature range at which the oligos exchange relatively fast yet where X is complexed) was slightly lower than the predicted melting temperature of the XY duplexes. The time required to obtain a 90% exchange of free Y for complex bound Y ("t(exchange)") varied between 30 and 500 seconds. It is expected that with other designs of oligos and optimized conditions (in particular Mg" concentration and temperature), it should be possible to obtain exchange rates for nucleic acids faster than one per second, possibly 10-100 per second.

Materials and Methods.

DNA oligo sequences.

Set #I: X#1: 3' -TGCTAGCATGGCCCAACGGGAAGTAACGCGAAGCCGATGCTAGCATGC Y1-Fl#1: 5'-Fam-CGGGTTGCCCTTCATT-3' Yl#l: 5'-CGGGTTGCCCTTCATT-3' Y2#1: 5'-TTCATTGCGCTTCGGC-3' WO 00/11211 PCT/DK99/00441 96 Set#II: X#2: Y1-Fl#2: 5'-Fam-TGCCCTTTT-3 Y1#2: 5'-TGCCCTCA-3' Y2#2: 5'-TTCATTGCGCT-3' Sequences that are complementary in X and Y are in bold; the region of Y1 and Y2 that overlap is underlined. FAM denotes the fluoresccent moiety.

Flourescense polarization spectroscopy.

The measurements were done on a Perkin Elmer LS50 spectrophotometer. Excitation was at 485 nm, emission was recorded at 525 nm. All measurements were done in Buffer A (10 mM Tris-HCl pH 9, 100 mM NaC1, and 1 mM MgC1, unless noted otherwise).

A

thin tubing was connected to the sample cuvette; therefore, it was not necessary to open the lid during addition of extra material. With this set-up, it is not possible to measure rates of reactions that proceed to near-completion within 30 seconds.

Results.

Design of XY exchange units based on nucleic acids.

For the final application, the XY exchange unit in the enzymelinker XY linker-substrate structure should be in rapid equilibrium with an excess of Y-substrate molecules in the buffer, thus facilitating a rapid exchange of product (or substrate) "attached" to the enzyme, through an exchange of Y-product (or Y-substrate) with Y-substrate in the buffer.

In order to accomplish this rapid equilibrium, we designed two oligos, Y1 and Y2, with overlapping (but not identical) binding sites on the X oligo (see materials and methods). Thus, Y2 would be able to transiently interact with X in the X:Y1 com- WO 00/11211 PCT/DK99/00441 97 plex through the portion of X that does not anneal to Y1, and vice versa. When annealing to the full complementary sequence on X, Y1 and Y2 form the same number of AT and GC base pairs, and are therefore expected to have near-identical affinity for X, and presumably similar on- and off-rates. Therefore, Y1 should replace Y2 from the X:Y2 complex as fast as Y2 replaces Y1.

We designed two sets of oligos, with different length of annealing site and different relative length of annealing and overlapping regions. The sequence of the overlapping region (5'-TTCATT-3') was chosen so as to avoid triplex formation; moreover, in the experiments (and in example 10) very low concentrations of X and Y are used. Therefore, triplex formation is highly unlikely.

Exchange of Y2 with Y1 of the X-Y1 complex.

Fluorescense polarization was used to analyze the exchange rates of each of the two sets of oligos. Fluorescense polarization of a fluorescently labelled molecule in solution is proportional to the molecule's rotational relaxation time. If viscosity and temperature is held constant, the fluorescense polarization value is directly proportional to the molecular volume. Changes in molecular volume may result from binding or dissociation of two molecules, as used in this study.

First oligo Set#1 was analyzed. Fluorescein-labelled Y1 oligo (Y1-Fl#1, see Materials and Methods) at a concentration of 5 nM and temperature 46 OC gives a fluorescense polarization value of 0.028 (see figure 21, upper panel). Upon addition of a 200fold excess (1 pM) of oligo X#1 at time t=660sec, the polarization rapidly increases to a plateau at about 0.037, indicating the formation of the X:Y1-Fl#1 complex. When Y2#1 is added in a 20-fold excess to X (20gM) at t=2400, the polarization rap- WO 00/11211 PCT/DK99/00441 98 idly drops, indicating the release of Y1-Fl#1 from the X:Y1- Fl#l complex. Most likely, the X:Y2#1 complex is formed, displacing Y1-Fl#1 from X#l.

The displacement of Y1-Fl#1 from X#l by Y2#1 is a relatively fast process; within 30 seconds, 90% equlibrium is observed.

Formation of the X#1:Yl-Fl#1 complex is a slower proces (approximately 600 seconds for 90 equilibrium to be obtained). However, this is explained by the fact that the concentration of the oligo in excess is 20 fold lower.

Next, the same binding reactions were analyzed at 50 oC. Less time (300 sec) is required for formation of the X:Y1 complex; on the other hand, the exchange goes to 90 completion a little slower (40 sec, see table 1).

We wanted to challenge the idea that overlapping targets can speed up the exchange rate. Therefore, the X#1:Y-Fl#1 complex was formed under identical conditions (46 OC), but now oligo Y1#l (instead of Y2#1) was added in a twenty-fold excess to X#l. As can be seen from (figure 21, lower panel) and (table the displacement of Y1-Fl#1 by un-labelled Y1#l is slow.

About 500 seconds are now required to obtain 90 equilibrium.

We conclude that the principle of overlapping targets for Y1 and Y2 on X in this case accelerates the exchange by a factor of approximately 17.

Oligo Set#2 was analyzed in the same way (see table At reference conditions of 24 oC and 1 mM MgCI 2 the exchange reaction goes to 90 completion within 200 seconds. Increasing the temperature to 30 oC speeds up the exchange 4-fold; likewise, increasing the Mg" concentration to 10 mM increases the exchange 4-fold. The present set-up has a response time of WO 00/11211 PCT/DK99/00441 99 about 30 seconds. Therefore, we did not ,attempt to measure the exchange rate at 10 mM MgC1, and 30 OC.

Finally, the principle of overlapping targets was again challenged. Under less than optimal conditions (24 OC, 1 mM MgC12), the exchange of Y1 for Y1-F1 complexed to X goes to 90 completion within 500 seconds; this is 2.5-fold slower than for the exchange of Y2 for Y1. Therefore, it is advantageous to include two rather than one. "Y-unit", eventhough under these conditions it speeds up the exchange only In figure 21, lower panel, the fluorescense polarization signal does not come down to the base line (the signal level for free Y1-Fl) This observation was done several times, for both Set#1 and Set#2 oligos, for exchange of Y1 for Y1 and Y2 for Y1, as well as with different MgC1, concentrations. We have no explanation for this phenomenon; however, it did not seem to influence the measured exchange rates. Addition of a large excess of Y2 to free Y1-F1 in the absence of X has no effect on the fluorescense polarization signal.

Discussion.

The exchange rates of both sets of oligos showed a strong dependence on the temperature; the temperature at which the selection experiment is performed should be held within a relatively narrow window of about 5 oC around the optimal temperature, in order for the exchange to be efficient.

The MgC12 concentration had a strong effect on the exchange rate. A similar effect may have been observed by (Shimayama et al., (1995), FEBS Letters 368, 304-306), for a so-called "DNA-armed hammerhead ribozyme". They found that the catalytic activity of the hammerhead ribozyme, in which the hybridizing arms had been replaced with deoxyribonucleotides, depended strongly on the Mg" concentration, even at high magnesium con- WO 00/11211 PCT/DK99/00441 100 centrations where the active site (requiring Mg++ for activity) is believed to be saturated with Mg'+ Their results might therefore be interpreted in terms of high Mg concentrations speeding up the exchange of the nucleic acids arms on the RNA target.

Design of XY exchange units based on nucleic acids should be a very general way to produce fast and efficient exchange units.

Appropriate choice of length and composition of annealing sites and conditions under which the selection is performed, should allow the use of DNA oligos as XY exchange units under different conditions of pH, salt, temperature, pressure etc. In the present study it was shown that optimization of either Mg++ concentration or temperature could bring the exchange rate down to the limit of the apparatus (tens of seconds). A combination of these conditions, potentially combined with optimization of other conditions, should bring the exchange rate down to approximately 1 per second. Finally, tuning of the relative length and composition of the annealing sites and overlapping regions of the Y1 and Y2 oligos should provide further improvements.

The design of Y1 and Y2 oligos with overlapping binding sites on the X oligo accelerated the exchange rate. Presumably the overlapping targets mediate active displacement of one oligo by the other. More sophisticated designs of XY exchange units, based on this concept and on the structural and mechanistic features of antisense RNA, should improve the dynamics of the system even more.

WO 00/11211 WO 0011211PCT/DK99/00441 101 Mg++ temp. t (complex t (exchange) exchange (mM) (OC) formation) (sec) (sec) Y1/Y2 1 46 600 Y1 /Y2 1 50 300 Y1/Y1 1 46 800 500 Mg++ temp. t (complex t (exchange) exchange (mM) (OC) f ormation) (sec) (sec) Y1/Y2 1 24 80 200 Y1 /Y2 1 27 300 100 Y1/Y2 1 30 100 YI/Y2 1 33 80 200 Y1/Y2 5 24 70 Y1/Y2 10 24 90 Y1 /Y2 500 24 400 lY1/Y1 1 24 200O 500 .awlJJ Time of 90%' complex formation and 90% exchange, at various conditions of. temperature and MgCl 2 First oligo (Y-F1) was added to 5 rLM; second oligo was added to 1 .LM; third oligo (Y or Y1) was added to 20 JiM.

EDITORIAL NOTE APPLICATION NUMBER 51540/99 The following Sequence Listing pages to are part of the description. The claims pages follow on pages '102' to '110'.

WO 00/11211 PCT/DK99/00441 1 SEQUENCE LISTING <110> Novo Nordisk A/S <120> Enzyme activity screen with substrate replacement <130> Enzyme activity screen with substrate <140> <141> <160> 4 <170> PatentIn Ver. 2.1 <210> 1 <211> 379 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Phagemid, pFab-SP400 <400> 1 Met Lys 1 Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Ala Gin Pro Ala Met Ala Glu Val Ser Gin Asp Leu 25 Asn Leu Phe Ala Gin Tyr Ser Ala Ala Ala Tyr Cys 40 Asp Ala Pro Ala Gly Thr Asn lie Thr Cys Thr Gly 50 55 Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Gly Asn Leu Leu Ala Asn Gin Phe Lys Asn Asn Ala Cys Pro Phe Glu Asp Ser WO 00/11211 WO 0011211PCT/DK99/00441 Gly Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu 100 Asn Phe Arg Gly Gly Asn Leu 115 Cys Arg Gly 130 Phe Asp Leu Lys 120 Thr Ser Arg Ser 105 Giu Ile Asn Ser Ser Trp, Ala Val Arq His Asp Gly Phe 135 Giu Ile Glu Asn Trp Ile 110 Asp Ile Cys Ser Gly 125 Arg Ser Val Ala Asp 140 Glu His Pro Asp Tyr 160 Ala Leu Ala Thr Val 175 Thr 145 Arg Leu Arg Gin Lys Val i50 Gly Asp 155 Gly Val Val Phe Thr 165 Leu His Ser Leu Gly 170 Tyr Ala Gly Ala Asp 180 Pro Arg Gly Asn Gly 185 Arg Asp Ile Asp Tyr Gly Ala 195 Val Gin Thr Arg Val Gly Asn 200 Ala Phe Ala Giu 205 Thr Val Phe Ser 190 Phe Leu Thr Asn Asp Ile Gly Gly Thr 210 Val Pro Leu Tyr Arg 215 Ile Thr His 220 Ser Arg Leu Pro 225 Glu Pro 230 Ser Arg Gly Tyr Trp Ile Lys 245 Glu Giu Phe Gly Tyr 235 Thr Leu Val Pro 250 Asp Ala Thr Gly 265 His Ser Ser Pro 240 Val Thr Arg Gly Asn Asn 270 Asn Asp 255 Gin Pro Ile Val Lys Ile 260 Gly Ile WO 00/11211 Asn Ile Pro 275 PCT/DK99/00441 Asp Ile Pro Ala His 280 Leu Trp Tyr Phe Gly Leu Ile Gly 285 Pro Phe Val Cys Thr Cys 290 Leu Ala Ala Ala Gly 295 Ser Lys Asp Ile Glu 305 Tyr Gin Gly Gin Ser 310 Ser Asp Leu Pro Gin Pro Pro Val 315 Gly Ser Glu Giy Asn Gly Gly Giy Ser Gly 325 Gly Gly Ser Gly Gly 330 Giy Gly 335 Ser Glu Gly Gly Gly Gly 355 Giy 340 Giy Ser Glu Gly Gly 345 Gly Ser Giu Giy Gly Gly Ser 350 Met Ala Asn Ser Gly Ser Gly Asp 360 Phe Asp Tyr Giu Lys 365 Ala Asn 370 Lys Gly Ala Met Thr 375 Glu Asn Ala <210> <211> <212> <213> <220> <223> 2 391

PRT

Artificial Sequence Description of Artificial Sequence: Phagemid, ph8 <400> 2 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu 1 5 10 Leu Ala Ala Gin Pro Ala Met Ala Giu Val Ser Gin Asp Leu Phe Asn Gin Phe 25 WO 00/11211 WO 0011211PCT/DK99/00441 Asn Leu Phe Ala Gin Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp Ala Pro 50 Glu Val Glu 40 Ile Asn Ala Gly Thr Lys Ala Asp 70 Asp Val Thr Thr Cys Thr Gly Ser Ala Cys Pro Thr Phe Leu Gly Tyr Leu Phe Giu Asp Val Gly Gly Phe Leu Asp Asn Thr Ser Asn Lys Leu Ile Val Leu 100 Asn Phe Arg Gly Ser 105 Glu Ser Ile Glu Gly Asn Leu 115 Cys Arg Gly Phe Asp Leu Lys 120 Thr Ile Asn Asp Ile 125 Ser Asn Trp Ile 110 Cys Ser Giy Val Ala Asp His Asp Gly Ser Ser Trp 130 Thr Leu Arg 140 Glu Arg Gin Lys 145 Arg Val 150 Gly Asp Ala Val Arg 155 Gly His Pro Asp Val Val Phe Thr 165 Leu His Ser Leu Giy 170 Tyr Ala Leu Ala Thr Val 175 Ala Gly Ala Asp 180 Pro Arg Gly Asn Gly 185 Arg Asp Ile Asp Tyr Gly Ala 195 Arg Vai Gly Asn 200 Tyr Ala Phe Ala Giu 205 Thr Val Phe Ser 190 Phe Leu Thr Asn Asp Ile Val Gin 210 Val Pro 225 Thr Gly Gly Thr Leu Pro Pro 230 Leu 215 Arg Arg Ile Thr His 220 Ser Arg Giu Phe Giy Tyr 235 His Ser Ser Pro 240 WO 00/11211 PTD9/04 PCT/DK99/00441 Giu Tyr Trp Ile Lys 245 Ser Gly Thr Leu Val 250 Pro Val Thr Arg Asn Asp 255 Ile Val Lys Asn Ile Pro 275 Ile 260 Giu Gly Ile Asp Ala 265 Thr Gly Gly Asn Asn Gin Pro 270 Leu Ile Gly Asp Ile Pro Ala His 280 Leu Trp Tyr Phe Gly 285 Thr Cys 290 Leu Ala Ala Ala Pro 295 Gly Gly Ser His His His His His 300 Val Cys Giu Tyr His Ser 305 Ala Ala Gly Ser Gin Ser Ser Asp 325 Asp Ile Arg Pro Phe 315 Gin 320 Gly Leu Pro Gin Pro Pro 330 Val Asn Ala Gly Gly Gly 335 Ser Gly Gly Gly Gly Ser 355 Gly 340 Ser Gly Gly Gly Ser 345 Glu Gly Gly Gly Ser Glu Gly 350 Gly Gly Gly Glu Gly Gly Gly Ser 360 Giu Gly Gly Gly Ser Gly 370 Ser Gly Asp Phe Tyr Glu Lys Met Ala 380 Asn Ala Asn Lys Gly 385 Ala Met Thr Glu Asn Ala 390 <210> 3 <211> 2031 <212> DNA <213> Artificial Sequence wo 00/11211 WO 0011211PCT/DK99/00441 6 <220> <223> Description of Artificial Sequence: Phagemid, ph7 <400> 3 atgaaatacc tattgcctac atggcgcagt ccggcaatcc tccaagttgg accagactcg aaggtgaagt acgttcagga ttgcgtgata tcgacgttgc cccattgttg gtttggtcct agtggcgagc tgaagctgag cccttcgctc agaagcttaa gatgccatcg gcaacatqgt cagcaagagg ctatcggcta ctggacgtgg ccaacggcgg gaggtggcta ctatcctgca aacgtgagca actacaatcc ccgtctcctg acgagtcccg ttgcccactc agttcattat tggggacagt ggtgcaacgt aacaatccta acgtggacgc tgcggtatgg gcggtgctcc actcagaatg ctcacgacga ggcggaggta acaatccgaa aaccctggag gcggtaactg ggacccacct gttgcgaagc tgcctcccag gaggcgcggc gcaggctcta aagatatcag cctcaacctc ctgttaatgc ggtggtggct ctgagggtgg ggtggctctg gttccggtga atgaccgaaa atgccgatga gtcgctactg attacggtgc aatggtaatg gtgctactgg gacggtgata attcaccttt tcggttgaat gtcgcccttt tgtgacaaaa taaacttatt atgtatgtat tttcgacgtt ggcagccgct gttctctgga acaggctttc gaaggtcggt tatccagaac gtacaacttg ccagaacggt ggctgcatcc gaccggcacc cgcgatcagc ttggctcggt gaaggctggt ctacagcacc ctacgctacc cgatcagagc gaacccggct gatcgtctgg cgctgccggc gat cgcgaga tcctactccc cgcatccaag tggaagcact cgcaccagga accattcgtt tggcggcggc cggttctgag ttttgattat aaacgcgcta tgctatcgac tgattttgct aatgaataat tgtctttggc ccgtggtgtc tgctaacata ggattgttat cgtaccttgc ctgagccgag accttctact gcacgtgctg cctgatcgag ctcaac cggt gacgtgcagt agtgctttct cagttgcagg tgggctgaca aacaatgcga tccaaccctc aatatcgcta cgcgtcgctc ggtttcggtc gtcaagcctg atgtggt tcg ggcgctgccg accaatccca tggggtcagt tgcacccgtc ggatcacatc tgtgaatatc tctggtggtg ggtggcggct gaaaagatgg cagtctgacg ggtttcattg ggctctaatt ttccgtcaat gctggtaaac tttgcgtttC ctgcgtaata tactcgcggc tcgt taacag gtgatcagac ggattagcaa ccaaggcccg attgcagcgc acaagaacga tcgctgtgat gccgaaatgc cctcccacat agctcgagcc agat ccgcgg Cgccctacac acgccatgcg tgtccggagc agccgttcac gaggcgaatc acgcgtatgc gttccggtgg cgaatcccgg gcggaggcca agaacgagtg accatcacca aaggccaatc gttctggtgg ctgagggtgg caaacgctaa ctaaaggcaa gtgacgtttc cccaaatggc atttaccttc catatgaatt ttttatatgt aggagtctta ccagccggcc cgactacagt 120 caacgctgcc 180 catcttcctc 240 tggagagaat 300 tggtgagagc 360 gtacgtgaat 420 tctggaaccc 480 acggggccct 540 tcacctgtac 600 cactgctcag 660 cttcagttcg 720 tagcggttct 780 ccagcgaggc 840 ccgtagcgaa 900 taccaacacg 960 tgacggtcaa 1020 ccaaatgctc 1080 aggcaacaat 1140 tcctacttct 1200 aggatgggca 1260 gtacagccag 1320 tcactcggcc 1380 gtctgacctg 1440 cggctctgag 1500 cggttccggt 1560 taagggggct 1620 acttgattct 1680 CggCcttgct 1740 tcaagtcggt 1800 cctccctcaa 1860 ttctattgat 1920 tgccaccttt 1980 a 2031 WO 00/11211 WO 0011211PCT/DK99/00441 <210> 4 <211> 993 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Phagemid, ph17 <400> 4 atgaaatacc atggcggcga aatcctactc tgcgcatcca gctggaagca gccgcaccag agaccattcg gctggcggcg ggcggttctg gattttgatt gaaaacgcgc gctgctatcg ggtgattttg ttaatgaata tttgtctttg ttccgtggtg tttgctaaca tattgcctac gaggcgctgc ccaccaatcc agtggggtca cttgcacccg gaggatcaca tttgtgaata gctctggtgg agggtggcgg atgaaaagat tacagtctga acggtttcat ctggctctaa atttccgtca gcgctggtaa tctttgcgtt.

tactgcgtaa ggcagccgct cggttccggt cacgaatccc gtgcggaggc tcagaacgag tcaccatcac tcaaggccaa tggttctggt ctctgagggt ggcaaacgct cgctaaaggc tggtgacgtt ttcccaaatg atatttacct accatatgaa tcttttatat taaggagtct ggattgttat ggaggcaaca ggtcctact t caaggatggg tggtacagcc catcact cgg tcgtctgacc ggcggctctg ggcggttccg aataaggggg aaacttgatt tccggccttg gctcaagtcg tccctccctc ttttctattg gttgccacct taa tactcgcggc atggcggagg ctaaccctgg caggacccac agtgcctccc ccgcaggctc tgcctcaacc agggtggtgg gtggtggctc ctatgaccga ctgt cgctac ctaatggtaa gtgacggtga aatcggttga attgtgacaa ttatgtatgt ccagccggcc taacaatccg aggcggtaac ctgttgcgaa aggaggcgcg taaagatatc tcctgttaat ctctgagggt tggttccggt aaatgccgat tgattacggt tggtgc tact taattcacct atgtcgccct aataaactta attttcgacg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 993

Claims (34)

1. A sample comprising a number of individual units suitable for use in an in vitro selection system, wherein the purpose of said in vitro selection system is, from a library of catalyst molecules, to select a catalyst molecule of interest having a relatively more efficient specific catalytic activity of interest as compared to the rest of the catalyst molecules within said library, and wherein said in vitro selection system is characterised by that it allows multiple catalytic activity I0 turn-overs substrate to product catalytic activity turn- overs), by the catalyst molecule of interest, before it is finally collected and wherein said sample comprises, a library of catalyst molecules provided in the form of individual units, wherein the individual units comprise a first type individual unit having the following general structure: C-XY-S, wherein C denotes a catalyst molecule, XY an XY exchange pair, and S a substrate which is capable of being catalysed into a product by at least one catalyst comprised within said library of catalyst molecules and thereby providing the possibility of obtaining a second type individual unit comprising the general structure: C-XY-P, wherein C and XY has the meaning defined above and P is the product molecule resulting from the catalytic conversion of the substrate S of the first type individual unit; and the substrate S is attached to the catalyst in a configuration that allows catalytic reaction between the catalyst and the substrate within said individual unit; and the nature of said attachment of the substrate and the catalyst provides the possibility, by means of a WO 00/11211 PCT/DK99/00441 103 characteristic of the product, of isolating an entity comprising information allowing the unambiguous identification of the catalyst molecule which has been capable of catalysing the reaction substrate molecule to product molecule; and said sample comprising a number of individual units is characterised by that said XY exchange pair allows an asymmetric exchange of the Y-moiety with another Y- moiety Y exchanges with Y, not with whereby said XY exchange pair then allows an exchange reaction between the unit structure: a catalyst an XY exchange pair a product and a "Y substrate" component and thereby generating the unit structure a catalyst an XY exchange pair a substrate.

2. The sample comprising a number of individual units according to claim 1, wherein the individual unit of point in claim 1 is a biologically amplifiable individual unit.

3. The sample comprising a number of individual units according to claim 1, wherein the individual unit of point in claim 1 is a biologically amplifiable individual unit and both said substrate and said catalyst molecule are attached on the surface of said biologically amplifiable individual unit.

4. The sample comprising a number of individual units according to any of the preceding claims, wherein said individual unit of point comprises following structure: catalyst molecule flexible (XY exchange pair) linker substrate.

WO 00/11211 PCT/DK99/00441 104 The sample comprising a number of individual units according to any of the preceding claims, wherein said individual unit of point in claim 1 comprises following structure: catalyst molecule carrier system XY exchange pair substrate, or more preferably the structure: catalyst molecule carrier system flexible (XY exchange pair) linker substrate.

6. The sample comprising a number of individual units according I0 to claim 3 and 5, wherein said carrier system of claim within said biologically amplifiable individual unit of claim 3 is a phage.

7. The sample comprising a number of individual units according to claim 5, wherein said carrier system is a bead particle.

8. The sample comprising a number of individual units according to any of claims 1 to 7, wherein said library of catalyst molecules is a library of natural or unnatural peptides or polypeptides, preferably a library of enzymes.

9. The sample comprising a number of individual units according to claim 8, wherein said library is a library comprising polypeptides individually having a number of different enzymatic activities. The sample comprising a number of different individual units accordingto claim 8, wherein said library is a library comprising polypeptides variants derived from one or more precursor polypeptide(s), wherein said precursor polypeptide(s) exhibit(s) closely related enzymatic activities.

WO 00/11211 PCT/DK99/00441 105

11. The sample comprising a number of individual units according to any of claims 8-10, wherein said library is a library comprising shuffled/recombined/doped polypeptides.

12. The sample comprising a number of different individual units according to claims 1 to 7, wherein said library of catalyst molecules is a library of natural or unnatural nucleic acids.

13. The sample comprising a number of different individual units according to claim 12, wherein said library is a library comprising nucleic acids having a number of different catalytic activities.

14. The sample comprising a number of different individual units according to claim 12, wherein said library is a library comprising nucleic acid variants derived from one or more precursor nucleic acid(s), wherein said precursor nucleic acid(s) exhibit(s) closely related catalytic activities.

The sample comprising a number of different individual units according to any of claims 12 to 14, wherein said library of nucleic acids is a library comprising shuffled/recombined/doped nucleic acids.

16. The sample comprising a number of individual units according to any of claims 1 to 7, wherein said library of catalyst molecules is a library comprising natural polymer molecules, or unnatural polymer molecules, or small organic WO 00/11211 PCT/DK99/00441 106 molecules, or small inorganic molecules or a mixture of said molecules.

17. The sample comprising a number of individual units according to claim 16, wherein said library is made by combinatorial chemistry.

18. The sample comprising a number of individual units according to any of the preceding claims, wherein the catalyst molecules and the substrate capable of being catalysed into a product (point in claim 1) are of a different chemical substance.

19. A method for in vitro selection, from a library of catalyst molecules, of a catalyst molecule of interest having a relatively more efficient specific catalytic activity of interest as compared to the rest of the catalyst molecules within said library and wherein said in vitro selection method is characterised by that it allows multiple catalytic activity turn-overs substrate to product catalytic activity turn-overs), by the catalyst molecule of interest, before it is finally collected and wherein said method comprises following steps, placing a sample comprising a number of individual units according to any of claims 1-18 under suitable conditions where a catalyst molecule of interest performs its catalytic activity of interest and further under conditions wherein said individual units are in contact with an Y substrate compound; (ii) selecting for a catalyst of interest by selecting for one or more individual unit(s) which comprise(s) the product molecule; and WO 00/11211 PCT/DK99/00441 107 (iii) isolating an entity comprising information allowing the unambiguous identification of the catalyst molecule of interest which has been capable of catalysing multiple times the reaction substrate to product, by means of a characteristic of the product; and optionally (iv) repeating step to (iii) one or more times by using the information comprised in said entity of step (iii) to generate the catalyst molecule of interest and construct an individual unit comprising said generated catalyst molecule of interest and then using this individual unit as a starting material in said repetition step.

The method for in vitro selection according to claim 19, wherein the catalyst molecules of interest are enzymes or proteins that have been coupled to an affinity tag, and wherein an optional step is performed prior to step of claim 19, the optional step comprising an enrichment for individual units displaying (full length) enzyme or protein through a purification in which the units displaying the enzyme or protein are isolated by means of the affinity tag.

21. The method for in vitro selection according to claim wherein the individual units displaying (full length) enzyme or protein are purified by the means of an anti-affinity-tag antibody column in which the units displaying the tagged enzyme or protein are isolated by means of the tag.

22. The method for in vitro selection according to claim wherein the affinity tag comprises six histidine residues that are coupled to the C-terminal end of the enzyme or protein of interest, and the individual units displaying WO 00/11211 PCT/DK99/00441 108 (full length) enzyme or protein are purified on a Ni-NTA column or on a anti-histidine antibody column, in which the units displaying the tagged enzyme or protein are isolated by means of the tag.

23. The method for in vitro selection according to claim 19, wherein the selecting for a catalyst molecule of interest, in step (ii) of claim 19, is done by specific immobilization to said product molecule.

24. The method for in vitro selection according to claim 19, wherein the selecting for a catalyst molecule of interest, in step (ii) of claim 19, is done by the following strategy, constructing a system wherein substantially each of the individual units in step of claim 19 comprising the substrate molecule and the catalytic molecule is bound to a matrix and wherein the unit is released from said matrix when the substrate is converted into the product; and (ii) selecting for the unit(s) which are released from said matrix.

The method for in vitro selection according to claim 19, wherein the selecting for a catalyst molecule of interest (step (ii) of claim 19), is done by one of the following strategies, constructing a product-column wherein a receptor specifically binding the product is placed along the matrix of the product-column; and adding the sample of individual units at one end of the product-column and selecting for the catalyst WO 00/11211 PCT/DK99/00441 109 molecules of interest by isolating the individual unit(s) which arrive(s) latest to the opposite end on the column.

26. The method for in vitro selection according to claim 19, wherein the isolation of an entity comprising information which allows the unambiguous identification of the catalyst molecule of interest (step (iii) of claim 19), is done by physical or chemical procedures. i0

27. The method for in vitro selection according to claim 26, wherein the physical procedure is electrophoresis.

28. The method for in vitro selection according to any of claims 19 to 27 and the further following step, producing said isolated catalyst molecule of interest in a suitable quantity of interest by a suitable production method. 110

29. A sample comprising a number of individual units suitable for use in an in vitro selection system, said sample substantially as hereinbefore described with reference to any one of the examples.

30. An individual unit suitable for use in an in vitro selection system, said unit substantially as hereinbefore described with reference to any one of the examples.

31. An individual unit suitable for use in an in vitro selection system, said unit substantially as hereinbefore described with reference to the accompanying drawings.

32. A method for in vitro selection, from a library of catalyst. molecules, of a catalyst molecule of interest, said method substantially as hereinbefore described with reference to the accompanying drawings.

33. A catalyst molecule of interest prepared by the method according to any one of claims 19 28 and 32.

34. An individual unit comprising a generated catalyst molecule (as set out in claim prepared by the method according to any one of claims 19 28 and 32. Dated 1 July 2003 Novozymes A/S Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON *°eo *ot *•oo *oo *o *ee [R:\LIBUU]02636.doc:jin