EP1326970A2 - Rapid profiling of the interactions between a chemical entity and proteins in a given proteome - Google Patents

Rapid profiling of the interactions between a chemical entity and proteins in a given proteome

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Publication number
EP1326970A2
EP1326970A2 EP01958221A EP01958221A EP1326970A2 EP 1326970 A2 EP1326970 A2 EP 1326970A2 EP 01958221 A EP01958221 A EP 01958221A EP 01958221 A EP01958221 A EP 01958221A EP 1326970 A2 EP1326970 A2 EP 1326970A2
Authority
EP
European Patent Office
Prior art keywords
proteins
protein
array
dna
tagged
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01958221A
Other languages
German (de)
English (en)
French (fr)
Inventor
Jonathan Michael Blackburn
Michelle Anne Mulder
Mitali Reddy's Lab Ltd Biotech. Divis. SAMADDAR
Roland Kozlowski
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sense Proteomic Ltd
Original Assignee
Sense Proteomic Ltd
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Filing date
Publication date
Priority claimed from GB0020357A external-priority patent/GB0020357D0/en
Application filed by Sense Proteomic Ltd filed Critical Sense Proteomic Ltd
Publication of EP1326970A2 publication Critical patent/EP1326970A2/en
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to novel methods of producing proteins in which one or more domains are full length and correctly folded and which are each tagged at either the N- or C-terminus with one or more marker moieties and arrays containing such proteins, as well as the use of such arrays in rapid screening.
  • Protein-protein interactions are being increasingly recognised as being of critical importance in governing cellular responses to both internal and external stresses. Specific protein-protein interactions therefore represent potential targets for drug- mediated intervention in infections and other disease states.
  • yeast two- hybrid assay is the only reliable method for assessing protein-protein interactions but in vivo assays of this type will not be readily compatible even in a non-high throughput format with the identification of specific agonists or antagonists of protein- protein interactions.
  • Functional proteome expression arrays, or "proteome chips” will enable the specificity of protein-protein interactions and the specificity of any drug- mediated effect to be determined in an in vitro format. They will therefore have enormous potential because they will simply revolutionise this area of research.
  • a typical bacterial genome is ⁇ 5Mbp and a small number have now been completely sequenced (for example Helicobacter pylori, Escherichia coli, and Mycobacterium tuberculosis); fungal genomes are typically ⁇ 40Mbp, mammalian genomes at ⁇ 3Gbp and plant genomes at ⁇ 10Gbp.
  • Current estimates are that the human genome sequence will be finished around 2003, although how much of this information will be in the public domain is very much open to question.
  • model organisms are of only limited value.
  • the Inventors have now developed a novel approach which solves the problems described above by providing methodology which allows each protein in a proteome to be tagged with a common marker at a defined position within the protein without requiring any prior knowledge of the DNA sequence of the corresponding genes.
  • This 'tag' can then be used to impart a commonality and specificity to downstream immobilisation and purification procedures, which in turn enables the creation of spatially defined arrays in which many thousands of proteins from a given proteome are displayed.
  • the methodology described here allows the tag to be inserted in the correct reading frame either precisely at the N- or C-terminus of each protein, or within a region close to either terminus which is unimportant in the folding and function of the protein, such that the individual tagged proteins fold correctly and hence retain function when specifically immobilised in the array.
  • the methodology described here also allows insertion of the tag within the overall coding sequence but outside specific domain boundaries such that the individual tagged domains fold correctly and hence retain function when specifically immobilised in the array.
  • each protein in the array will be fully functional, the arrays can then be screened directly to identify the targets of drugs and other biologically relevant molecules.
  • the spatial definition of the arrays will allow the phenotype of each protein to be related directly to its genotype to allow the identification of 'hits'.
  • the present invention provides a method producing one or more proteins in which one or more domains are full length and correctly folded and which are each tagged at either the N- or C-terminus with one or more marker moieties, said method comprising:
  • step (e) cloning the fragments generated by step (d) into an expression vector containing a coding sequence for one or more 5' or 3' marker moieties;
  • the amplification of the DNA molecule or molecules statistically incorporates a single.
  • ⁇ -S-dNTP more preferably eiither ⁇ -S-dTTP or ⁇ -S-dATP.
  • the marker moiety can be either a peptide sequence, eg a hexa-histidine tag, an antibody epitope or a biotin mimic, or indeed a complete protein, or protein domain, eg the maltose binding protein domain.
  • the marker moiety itself can be post- translationally modified, eg by addition of a biotin or lipid molecule. In a preferred embodiment, the marker moiety would also allow purification of "tagged" proteins.
  • the methods of the present invention allow the specific modification, in one pot, of every member of a cDNA library in a manner which does not rely on any knowledge of the sequence of individual genes. Instead, it relies on non-processive truncation of each cDNA by a nuclease such that either the 5'- or the 3'- untranslated region of each cDNA is removed. Additional known DNA sequence encoding a known marker moiety is then appended to the resultant set of nested deletions of each cDNA.
  • each resultant genetically modified cDNA produced according to the methods of the present invention will thus encode an individual protein which now has a common moiety, eg. a polypeptide "tag" fused to either its N- or C-terminus.
  • a screen for correctly folded, tagged proteins then allows all truncations which cross a domain boundary and affect the folding (and hence function) of the individual protein and all out-of-frame fusions to the tag to be discarded.
  • the proteins expressed from the cDNA library will be "tagged" and can be readily identified and isolated. Once purified they can be attached to microarrays, for example. Attachment can be effected by means of the tag itself, or alternatively, by means of another moiety which is first attached to the proteins.
  • Arrays formed by the methods described herein form a second aspect of the invention.
  • Such arrays comprise the "tagged" protein expression library, immobilised, usually on a solid support.
  • a range of possible solid supports are in common usage in the area of arrays and any of these "substrates” can be utilised in the production of arrays of the present invention.
  • protein array relates to a spatially defined arrangement of one or more protein moieties in a pattern on a surface.
  • the protein moieties will be attached to the surface either directly or indirectly.
  • the attachment can be non-specific (e.g. by physical absorption onto the surface or by formation of a non-specific covalent interaction).
  • the protein moieties will be attached to the surface through the common marker moiety linked to each protein using the methods described herein.
  • the protein moieties may be incorporated into a vesicle or liposome which is tethered to the surface.
  • each position in the pattern may contain one or more copies of:
  • a sample of a single protein type in the form of a monomer, dimer, trimer, tetramer or higher multimer
  • a sample of a single protein type bound to an interacting molecule e.g. DNA, antibody, other protein
  • a sample of a single protein type bound to a synthetic molecule e.g. peptide, chemical compound
  • the surface which supports the array may be coated/derivatised by chemical treatment, for instance.
  • suitable surfaces include glass slides, polypropylene or polystyrene, silica, gold or metal support or membranes made of, for example, nitrocellulose, PVDF, nylon or phosphocellulose.
  • the methods of the present invention allow tagging of all proteins in a given proteome specifically at either the N- or C-terminus. Whilst some proteins may not tolerate N-terminal extensions and others might not tolerate C-terminal extensions, it is likely that the vast majority of proteins will tolerate one or other such extensions.
  • Existing library cloning methods simply cannot address this problem since they clone genes either as full-length, unmodified cDNAs or as random and almost inevitably truncated fusions to some protein partner. Compared to the latter, the present methods allow the position of the tag to be targeted to the sequences at or close to the N- or C-terminal residues of the cDNA products such that fusion to eg.
  • the method of immobilising proteins in an array as described herein is through specific rather than non-specific interactions, and these specific interactions are a function of the tag added to the termini of each cDNA.
  • the methods described herein can be used to screen purified, immobilised proteins which have been expressed in non-bacterial host organisms to aid maintenance of function through correct folding and post-translational modification, whereas existing methods such as phage display or ⁇ -cDNA expression libraries are restricted to bacterial hosts in which the majority of eukaryotic proteins are found to be synthesised in a non-functional form, either due to mis-folding or incorrect post-translational modification.
  • the methods of the present invention have a wide range of potential in vitro applications, which can be broadly divided into three main areas. These are the study of protein-ligand interactions, the study of protein-protein interactions, and the study of protein-DNA interactions.
  • the methods described herein will allow the rapid profiling of the interactions between a given new chemical entity and all proteins in a given proteome. This can be achieved simply through probing the appropriate proteome array with the NCE at varying stringencies in what might be considered a reverse high throughput screen.
  • the methods can be applied directly to the question of species cross-reactivity, allowing a potential antifungal compound, for example, to be quickly assessed in terms of its interactions with, for example, all proteins in a human proteome; this type of information is likely to prove very useful in any subsequent optimisation of lead compounds.
  • the methods of the present invention can also be used to identify families of proteins, such as serine proteases, through screening proteome arrays with generic inhibitors. This would then allow the subsequent development of biochips displaying, for example, all human serine proteases or, alternately, all kinases or all p450 enzymes for more focused screening of lead compounds.
  • a p450 biochip would have utility in assessing whether a given lead compound is likely to be metabolised or not, since p450-mediated hydroxylation is often the first step in this process and is thought to be one of the primary sources of patient-to-patient variability in drug response; indeed one of the goals of drug design now is to generate compounds which are not metabolised in the first place and here again a p450 chip would have significant potential utility.
  • Protein-protein interactions and multiprotein complexes are of critical importance in cellular biology. Signalling pathways, for example, are commonly initiated by an interaction between a cell surface receptor and an external ligand, and this is followed by a cascade of protein- protein interactions which ultimately result in the activation of a specific gene. Individual protein-protein interactions might be dependent on the presence of a specific ligand or alternatively might be blocked by a specific ligand, whilst some multiprotein complexes will only form in a ligand-dependent manner.
  • a drug might block the binding of a protein or small molecule to a cell surface receptor and hence block the signalling cascade at the beginning; a drag might block a protein-protein interaction or inhibit an enzymatic activity within the signalling cascade; or alternatively, a drug might block formation of specific protein-DNA or protein-protem interactions within the enhanceosome complex.
  • the transcription factor NF- ⁇ B is involved in cellular processes as diverse as immune and inflammation responses, limb development, septic shock, asthma, and HIV propeptide production.
  • the majority of the intracellular signalling cascades in NF- ⁇ B activation are common to all these process so do not represent viable targets for intervention. The differences between the responses therefore lie in either the original ligand-receptor interaction or in the formation of specific enhanceosome complexes.
  • NF- ⁇ B is known to bind to at least 14 different enhancer elements and the enhanceosome complexes therefore represent potential therapeutic targets.
  • delineation of an individual enhanceosome complex requires knowledge of both the number of individual DNA-binding proteins involved and also their protein-protein interactions with each other.
  • a proteome array can be screened with specific DNA probes to identify novel DNA binding proteins, Alternatively, the proteome array can be screened with the transactivation domain of a given transcription factor to identity other proteins with which it interacts. Cross correlation of such screens should allow identification of new components of specific enhanceosome complexes
  • the protein arrays generated by the methods of the present invention will also, allow the selection of molecules which recognise each protein displayed in the arrays.
  • the selected molecules will be antibodies or antibody-like proteins and will be displayed on phage or on ribosomes or will be covalently linked to the encoding mRNA.
  • a phage displayed antibody library can be applied to each immobilised protein in the array and non-binding antibodies removed by washing.
  • the selected phage can then be recovered and used to infect bacteria according to normal procedures.
  • the phage-infected bacteria can then produce either phage particles displaying the selected antibodies for further rounds of selection, or they can produce soluble antibody fragments for direct use.
  • the terms 'antibody' or 'antibody fragments' here refer to single chain Fvs, FAB fragments, individual light or heavy chain fragments, derived from mouse, human, camel or other organisms.
  • the protein array will be in microwell format such that after the selection step, the phage particles can be recovered by addition of appropriate bacterial cells to each well where they will become infected by the selected phage particles. Growth media can then be added to each well and the infected bacteria allowed to grow and express the antibody fragments, whilst maintaining the physical separation of the antibody fragments selected to each immobilised protein in the array. If so desired, new phage particles produced by the infected bacteria can be used in subsequent rounds of selection.
  • Such procedures are now routine for selecting polyclonal or monoclonal antibody fragments to a single purified and immobilised protein. In effect then the original protein arrays here will allow the generation of polyclonal or monoclonal antibody fragments to thousands of correctly folded proteins in a massively parallel manner whilst otherwise using standard in vitro antibody selection methods.
  • the selected, solubly expressed antibody fragments from each well of the original array can themselves be immobilised in to a new spatially defined array such that the antibody fragments in each position of the new array were selected against the proteins immobilised in -a single, defined position in the original array.
  • the antibody arrays so- generated will contain at each position either polyclonal or monoclonal antibody fragments, depending on the number of rounds of selection carried out prior to immobilisation of the soluble antibody fragments.
  • Such antibody arrays will have a number of potential uses including capture of individual proteins from a crude cell or tissue lysate for differential expression monitoring of the relevant proteome.
  • the antibody-captured proteins might be screened directly for ligand-binding function.
  • any one monoclonal antibody might bind to the target protein so as to block its function, but another monoclonal antibody might bind but not block function.
  • a polyclonal set of antibodies to all proteins in a proteome however is likely to contain individual antibodies which have the desired ability to bind but not affect function and will, in addition, contain individual antibodies which recognise all post-translational modifications of a given protein.
  • polyclonal rather than monoclonal antibody arrays generated as described will likely be advantageous for screening captured proteins directly for function.
  • the antibody arrays created by the methods described here will have the advantage that all proteins immobilised on the array will be stable under similar conditions.
  • the proteins captured from the crude cell or tissue lysate will not be recombinant but will have been naturally expressed.
  • the captured proteins can be screened for function or ligand binding etc directly after capture from the crude cell or tissue lysate, which should aid maintenance of function.
  • the present invention provides:
  • a method of screening one or more compounds for biological activity which comprises the step of bringing said one or more compounds into contact with a protein array as defined herein and measuring binding of the one or more compounds to the proteins in the array;
  • a method of generating an antibody array which comprises bringing a protein array, as defined herein, into contact with an antibody library, such that one or more proteins in the protein array bind to at least one antibody in the antibody library, removing any unbound antibodies and immobilisation of those antibodies bound to proteins in the protein array;
  • the methods (i), (ii), (iii) and (vi) may also include the step of first providing the array according to one or more of the methods of the present invention.
  • the present invention provides:
  • FIGURE la shows the construction of the vector pMM106H
  • FIGURE lb shows details of the PCR amplification and exonuclease digestion of an example gene (GST) prior to tagging;
  • FIGURE lc shows details of the specific ligation and PCR amplification to introduce the tag
  • FIGURE Id shows the reaction between Glutathione and l-chloro-2,4- dinitrobenzene catalysed by GST.
  • the Inventors constructed a vector pMM106H derived from pUC19 which contains a strong hybrid promoter (Ptrc) to drive the expression of genes cloned into an Nco I site immediately downstream of the promoter sequence.
  • the Inventors inserted a 676 bp nonsense DNA sequence as a sniffer fragment between the Nco I site and a downstream Hpa I site.
  • Hpa I is a blunt-end cutter and is positioned to cleave the vector such that the downstream DNA encodes a polyasparagine, hexahistidine peptide if the reading frame is on the first base of the blunt-end.
  • GFP green fluorescent protein
  • amber stop codon will result in a small amount of the full length fusion protein for visualisation of green colonies, while most of the fusion protein will terminate , immediately after the His tag and can be used for subsequent immobilisation and enzyme assays.
  • a number of different peptides of proteins whose soluble expression confers some observable phenotype on the cells, could be used in place of GFP as markers for expression and folding of the tagged proteins. These include, but are not limited to, chloramphenicol acetyl transferase, ⁇ -galactosidase, the lacZ fragment of ⁇ -galactosidase, and proteins capable of repressing transcription, such as the ⁇ -CI repressor.
  • the template used in the procedure outlined below was pGSTN.
  • This plasmid was constructed by first PCR-amplifying the Schistosoma japonicum glutathione S transferase (GST) gene from pGEX-2T (Pharmacia) under standard conditions using primers 'GSTfwd2' (5 ' -ATG CTG CAG ACG TCA ACA GTA TCC ATG GCC CCT ATA CTA GG-3 ' ) and 'GSTHindllF (5 ' -GCG AGG AAG CTT GTC AAT CAG TCA CGA TGA ATT ccc G- 3 ' ).
  • the Inventors amplified the GST gene from the construct pGSTN using the polymerase chain reaction with custom-designed vector-specific primers 'STforward'
  • Each PCR reaction was carried out in a standard buffer (lOmM Tris.HCl pH8.8, 25mM KC1, 5mM (NH 4 ) 2 SO 4 , 2mM MgSO 4 , 10% DMSO).
  • a standard buffer lOmM Tris.HCl pH8.8, 25mM KC1, 5mM (NH 4 ) 2 SO 4 , 2mM MgSO 4 , 10% DMSO.
  • Each of the four PCR reactions then also contained a non-standard deoxynucleotide triphosphate mix, as follows:
  • Reaction 1 200 ⁇ M dATP, 200 ⁇ M dTTP, 200 ⁇ M dCTP, 150 ⁇ M dGTP, 50 ⁇ M ⁇ -S- dGTP;
  • Reaction 2 200 ⁇ M dATP, 200 ⁇ M dTTP, 200 ⁇ M dGTP, 150 ⁇ M dCTP, 50 ⁇ M ⁇ -S- dCTP;
  • Reaction 3 200 ⁇ M dATP, 200 ⁇ M dGTP, 200 ⁇ M dCTP, 150 ⁇ M dTTP, 50 ⁇ M -S- dTTP;
  • Reaction 4 200 ⁇ M dGTP, 200 ⁇ M dTTP, 200 ⁇ M dCTP, 150 ⁇ M dATP, 50 ⁇ M ⁇ -S- dATP.
  • amplification of the template DNA in the presence of ⁇ -S-dNTPs can of course be carried out by primer extension reactions using many different DNA polymerase, including thermostable polymerases which lack a 3' to 5' exonuclease activity, such as
  • Tap polymerase and non-thermostable polmerases, such as T4 DNA polymerase or the Klenow fragment of DNA polymerase I.
  • Any restriction enzyme which generates a 5 '-overhang could also be used in place of Aat II providing the requisite site is incorporated into the design of the PCR primers; in this case, generation of the 5'-overhang would be followed by a DNA-polymerase-mediated fill-in reaction in which the relevant ⁇ -thio dNTPs were used in place of the dNTPs such that the new 3 '-end of the PCR product is now protected from exonuclease digestion.
  • Exonuclease III is a non-processive 3'- to 5'- exonuclease which is unable to hydrolyse ⁇ -thio-containing nucleotides so, in the present protocol, every time Exo III reaches an ⁇ -thio-deoxynucleotide base, the progressive truncation of the recessed 3 '- end of the PCR product is halted. The net result is thus a nested set of deletions as a consequence of the random incorporation of each ⁇ -S-dNTP at the earlier stage.
  • the ratio of ⁇ -S-dNTP to dNTP used in the original PCR amplifications was determined empirically such that the envelope of nested deletions spanned a 400bp window of sizes centred approximately lOObp shorter than the original full length PCR product.
  • the size range of the truncations obtained can be controlled by altering the ratio of ⁇ - S-dNTP to normal dNTP. This is important when the method is applied to eukaryotic cDNAs because such cDNAs have variable length 3' untranslated regions, with the most common 3'-UTR length falling in the range of 200-300 bp. Since the relative efficiency of incorporation of each of the four ⁇ -S-dNTPs varies according to the identity of the polymerase, it is desirable to use ⁇ -S-dNTP to normal dNTP ratios which are optimised for each of the four bases and for the particular polymerase.
  • the molar ratio of racemic ⁇ -S-dNTP to normal dNTP used will lie in the range 1:1 to 1:3.
  • the nested set of deletions generated by Exonuclease III digestion in the previous step was purified by ethanol precipitation and resuspended in lx mung bean nuclease buffer (50mM sodium acetate pH5.0, 30mM NaCl, lmM ZnSO 4 ).
  • the digested DNA was treated with (2units/ ⁇ g) 30 Units of mung bean nuclease in a lOO ⁇ l reaction at 30°C for 30 minutes. This process removed the 5'- and 3 '-overhangs to yield blunt- end products.
  • the reaction was stopped by the addition of EDTA to a final concentration of 5mM.
  • the digested products were purified using a QIAquick PCR purification kit (Qiagen), digested with Nco I, and separated on a 1% agarose/TBE gel, using a lOObp DNA ladder as a standard. Products ranging in size from 800 to lOOObp were extracted from the agarose using a QIAquick gel extraction kit (Qiagen).
  • Qiagen QIAquick PCR purification kit
  • SI nuclease can also be used to remove the 5 '-overhang from the nested set of 3 '-deletions generated by a 3'- 5'- exonuclease.
  • a number of other standard molecular biology methods for generating a nested.set of deletions represent obvious variations on the original procedure. These include, but are not limited to, the use of any 3'- to 5'- exonuclease, any 5'- to 3'- exonuclease, or any endonuclease which truncates progressively from the termini of a linear DNA fragment.
  • the initial PCR amplification can be performed using the same reverse primer as above but with a forward primer which binds approximately 2 kb upstream of the start of the GST gene. This will generate a fragment in which the GST gene is flanked by >2 kb on the 5' end and only 84 bp on the 3' end.
  • the purified PCR fragment can then be treated with Bal 31 nuclease, which progressively degrades linear duplex DNA from both the 5'- and 3'-ends.
  • the enzyme is non- processive and the rate of degradation of the DNA depends on the time and temperature of the reaction, as well as the base composition of the DNA. Since the flanking region on the 3 '-end of the GST gene in the PCR product is significantly shorter than that on the 5 'end, degradation up to and beyond the stop codon will occur long before the start codon is reached from the other end. Time course experiments allow the optimum reaction conditions for removal of up to 400bp from the 3' end of the PCR product to be determined.
  • the resulting nested set of deletions can then be blunt-ended to remove any remaining single-stranded regions, digested with a unique restriction enzyme encoded at the 5 '-end of the gene by the original vector, and directionally cloned into the tag vector.
  • Lambda exonuclease can be used to generate a nested set of 5 '-deletions.
  • the preferred substrate for this enzyme is 5' phosphorylated double stranded DNA so one end of the DNA substrate can be easily protected by having a 5' hydroxyl terminus.
  • 3- exonuclease can be removed by a number of different enzymes, including T4 DNA polymerase or a single-stranded DNA specific nucleases such as R Ase T or Exonuclease T or mung bean nuclease.
  • E. coli DH5 ⁇ cells were transformed with one of the full-length, hexahistidine-tagged
  • the crude lysate (500 ⁇ l; lOO ⁇ g) was then mixed with Nickel-NTA magnetic beads (50 ⁇ l; binding capacity 15 ⁇ g hexahistidine-tagged protein) and the beads recovered by sedimentation under a magnetic field. The supernatant was discarded and the beads were washed and then resuspended in a glutathione S transferase assay buffer containing ImM each of glutathione and l-chloro-2,4- dinitrobenzene. End point assay data was collected after 30 minutes at room temperature by measuring the absorbance at 340nm; this wavelength corresponds to the ⁇ max of the product of the GST-catalysed reaction.
  • (a) Vector construction The Inventors constructed a second vector, pMMl 11 , which is essentially the same as pMM106H (see Example 1), except that the 676bp Nco II Hpa I nonsense DNA stuffer fragment is replaced with a 300bp Nco Hpa I fragment derived from the Escherichia coli gdhA gene; the Hpa I cloning site is replaced with a Sma I site, positioned such that the downstream hexahistidine tag is out of frame with the gdhA gene by 2 nucleotides; and the ATG start codon of the GFP gene is replaced with the codon for alanine (GCG).
  • pMMl 11 which is essentially the same as pMM106H (see Example 1), except that the 676bp Nco II Hpa I nonsense DNA stuffer fragment is replaced with a 300bp Nco Hpa I fragment derived from the Escherichia coli gdhA gene; the H
  • the vector has been designed such that an insert cloned into the Sma I site must contain the first nucleotide of a codon at its 3' end to put it in frame with the hexahistidine tag and GFP.
  • the construction of pMMl 11 was confirmed by sequencing.
  • a further advantage of this modified procedure is that a polyA tail can be incorporated into the 5 '-end of the forward primer used in the initial PCR amplification (e.g. Forward-A 5 ' -AAA AAA AAA AAA GAT CGA TCT C AT GAC GGA TAA CAA TTT CAC
  • Example 1 Following the procedure as described in Example 1 for glutathione-S-transferase, the Inventors have demonstrated that the procedure is independent of the sequence of the gene being manipulated. Thus starting with a plasmid encoding human transcription factor NF- ⁇ B p50 and following exactly the procedure described in Example 1 unless otherwise specified, the Inventors have been able to demonstrate the modification of NF- ⁇ B p50 such that the first in-frame stop codon has been excised and replaced by an in-frame fusion to DNA encoding a polyasparagine, hexahistidine tag and GFP (when the amber stop codon is suppressed).
  • the clones that fluoresced green, when excited with far uv light (365 nm) were further characterised.
  • Colony Western blots using an anti-His-tag antibody allowed identification of clones expressing hexahistidine-tagged protein.
  • the soluble protein lysates of these clones were resolved by SDS-polyacrylamide gel electrophoresis and probed with anti-His tag antibody.
  • Immunoreactive signals were observed at approx. 65 kDa M r (corresponding to translationally fused NF- ⁇ B p50 to the hexahistidine tag and GFP) and at approx. 38 kDa M ⁇ (NF- ⁇ B p50-histag).
  • NF- ⁇ B plasmids created via the above methodology.
  • a single carbenicillin-resistant colony was grown to mid-log phase in 10ml liquid culture and then supplemented with lOO ⁇ M IPTG to induce expression of the hexahistidine-tagged NF- ⁇ B p50. After growth for a further 4 hours, cells were harvested and lysed by sonication. SDS- PAGE of the crude lysate showed an overexpressed protein at the expected size (38kDa) which represented roughly 5% of total soluble protein.
  • the protein lysates were prepared using the lysozyme/freeze-thaw method in PBS (phosphate buffered saline pH 7.5) containing 5 mM ⁇ -mercaptoethanol. 200 ⁇ l of the soluble protein lysate from each clone, was applied to the Ni-NTA coated microwell and incubated at room temperature for 45 minutes. At the end of the incubation period, the wells were washed three times with PBST (PBS containing 0.02 % Triton X-100) to remove all the unbound proteins.
  • PBST PBS containing 0.02 % Triton X-100
  • the wells were washed three times with DNA binding buffer ( 10 mM Tris.HCl pH 7.4, 75 mM KC1 containing 5 mM ⁇ - mercaptoethanol with a soak time of 1 minute.
  • DNA binding buffer 10 mM Tris.HCl pH 7.4, 75 mM KC1 containing 5 mM ⁇ - mercaptoethanol with a soak time of 1 minute.
  • the 3' digoxigenin labelled KB motif (2 pmol) was added to the wells in 200 ⁇ l of the DNA binding buffer containing 1 ⁇ g of poly (dl-dC) non-specific DNA. After another 30 minutes incubation the unbound DNA was removed by washing the wells three times with 10 mM Tris.HCl pH 7.4, 25 mM KC1 containing 0.02% Triton X-100.
  • An anti-digoxigenin antibody-alkaline phosphatase conjugate was diluted to 150mU/ml in 'antibody dilution buffer' (lOmM Tris.HCl pH7.4, 25mM potassium chloride) supplemented with 0.2% bovine serum albumin.
  • the diluted antibody (200 ⁇ l) was then applied to the microwells. After 30 minutes at room temperature, unbound antibody was removed by washing the microwells with 'antibody dilution buffer' (3x350 ⁇ l) supplemented with 0.02% Triton X-100.
  • the Inventors omitted either the crude lysate, or the labelled oligonucleotide, or the antibody, or added a 20-fold excess of unlabelled duplex oligo or replaced the hexahistidine-tagged NF- ⁇ B p50 containing crude lysates with equivalent amounts of a crude cell lysate from DH5 ⁇ cells expressing hexahistidine- tagged GST in the same vector background.
  • NF- ⁇ B p50 first binds to the labelled oligonucleotide via the specific binding site.
  • the protein-DNA complex is then immobilised in the microwells via the hexahistidine tag and all other proteins (including complexes between the labelled oligo and other DNA binding proteins present in the crude lysate) together with any unbound, labelled oligo, are then washed away.
  • the antibody-conjugate recognises the label on the oligo, not the hexahistidine-tagged protein, a positive signal in the assay can only be observed if the NF- ⁇ B p50-DNA interaction is maintained on immobilisation of NF- ⁇ B p50 via the tag; if this interaction is not maintained, the oligo will be lost during the washing steps so no colour change will be observed.
  • the Inventors have applied the procedure exactly as described in Example 1 except where specified to the pool of 10 different genes listed in the table below.
  • the Inventors have generated arrays of the resultant specifically modified proteins such that each position in the array corresponds to a single recombinant protein immobilised through the tag appended as a result of this procedure.
  • the Inventors have then screened the array by functional assay and have successfully identified individual protein components of the pool.
  • the primers 'STforward' and 'STreverse' described in Example 1 were designed to be universal primers for the amplification of genes encoded within a pTrcHisA vector backbone.
  • the primer 'STforward' was designed such that it encodes a number of restriction sites as follows:
  • any of the primer encoded restriction sites could be used providing that the tag vector contains an equivalent cloning site downstream of the promoter; Sfi I would have significant advantages in this regard in a larger library format because it has an 8bp recognition sequence so the frequency of random occurrence of an Sfi I site within a given gene will be much lower (1 in 6.5xl0 4 ) than that for a 6bp recognition sequence such as that of Bsp HI (1 in 4,096).
  • the tag vector pMM106H is an 'ATG' vector, i.e. the 5 '-cloning site (Nco I) overlaps the ATG start codon positioned downstream of a ribosome binding site (RBS) for expression of native proteins.
  • the Inventors are not reliant on the cloned genes having a common restriction site at their start codons. Instead, the Inventors simply rely on the vector-encoded promoter initiating transcription to produce mRNA, with the requisite signals for translational initiation being provided by the cloned genes themselves.
  • Example.1 The experimental procedure was carried out as described in Example.1 with the following modifications.
  • An equimolar pool of all ten genes was used as the template for initial PCR amplification using primers 'STforward' and 'STreverse', after which fragments were digested wit Aat II to protect the 5' end.
  • Exonuclease HI and mung bean nuclease-treated fragments were generated exactly as in Example 1 and were then digested with Bsp HI, which restricts the fragments uniquely within the forward PCR primer binding site and generates a cohesive end for cloning into the vector pMM106H.
  • the resulting fragments were gel purified and ligated in to the vector.
  • Transformed cells were visualised under UN light (365nm) and colonies which fluoresced green were selected by eye for analysis by Western blot. Approximately
  • the Inventors have used the procedures described in these Examples to create arrays of functional proteins in a microwell format and using these arrays the Inventors have successfully identified three different proteins from a pool based on either specific protein-ligand interactions (GST activity assay) or specific protein-DNA interactions (NF- ⁇ B binding assay).
  • GST activity assay specific protein-ligand interactions
  • NF- ⁇ B binding assay specific protein-DNA interactions

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