KR20030043933A - Method - Google Patents

Abstract

The present invention provides a novel method for preparing proteins and arrays containing such proteins, wherein one or more domains are full length, correctly folded, and the N- or C-terminus are each tagged with one or more markers, and arrays of such proteins for high speed screening To use in

Description

Method {METHOD}

Genome mapping projects are revolutionizing the discovery of therapeutic targets and drug discovery. As new therapeutic targets are identified, high throughput screening of existing combinatorial chemical libraries will suggest a number of potential lead compounds that are active against these targets. While tracking all lead compounds through initial clinical trials is clearly uneconomical, there is no rapid way to assess such lead compounds in terms of the activity profiles that they currently think of for all proteins in the organism. . If available, such methods enable the early assessment of the potential toxicological profile of all lead compounds, and this information can significantly improve the process of determining whether to trace or exclude lead compounds.

There is a complementary need in the pharmaceutical industry to identify all targets of existing drugs (drugs already on the market or still in development), thereby identifying their mechanism of action. The availability of this information will greatly facilitate the process of obtaining regulatory approval of the new drug, since it is clear that the competent authority will consider knowledge of the mechanism of action as the most important. In addition, this type of information will enable improved next generation drug design. Since the majority of drugs probably have at least minor side effects that bind the drug or its metabolites to undesirable targets, all of these target proteins need to be identified to establish the requirements necessary for the design of the improved drug. . However, at present there is no simple way to obtain this information, and the development of many millions of dollars of potential drugs simply stops due to the lack of knowledge of the target of action.

There is increasing recognition that protein-protein interactions are of great importance in controlling cellular responses to both internal and external stress. Thus, specific protein-protein interactions are potential targets for drug-mediated intervention in infectious disease states and other disease states. Currently, yeast two-hybrid assays are the only method available for assessing protein-protein interactions, but this type of in vivo assay can be used to identify specific agonists or antagonists of protein-protein interactions even in non-high throughput formats. It will not be readily compatible with identification. Functional proteome expression arrays, or “proteome chips,” will allow for the determination of the specificity of protein-protein interactions and the specificity of any drug-mediated effects in an in vitro format. Therefore, they will have a lot of potential because they will revolutionize the field of research.

One way to generate functional proteome arrays is to individually clone, express, purify and immobilize all proteins expressed in a particular proteome. Nevertheless, an important initial consideration concerns the absolute size of a given genome with consideration of the availability of sequence data for the entire genome.

By way of illustration of this point of view, a typical bacterial genome is ~5 Mbp, double prime, the current sequence is completely determined and (e. G., Helicobacter pylori (Helicobacter pylori), Escherichia coli (Escherichia coli) and Mycobacterium Mycobacterium tuberculosis : Mycobacterium genome is typically -40 Mbp, mammalian genome is -3 Gbp, plant genome is -10 Gbp Currently the human genome sequence is in the domain in which much of this information is published. It is very questionable whether it will exist, but it is expected to end almost in 2003. It is obviously impossible to predict whether a genome other than the representative organism model will be available in real time frames, but from the perspective of functional proteomics models The organism has only limited value, so in principle within the next four years It may be possible to design and synthesize primers for cloning each of the ~ 100,000 genes in the human genome from the cDNA library, which is very expensive (although millions of dollars at the price of the primers), even though the necessary sequence data is available. Name), requires excessive work process.

However, what about the pharmaceutically relevant organisms that make full sequence data unavailable? These cannot simply be ignored by functional proteomics, is there an alternative? Expression cDNA libraries in principle produce arrays of proteins in combination with nonspecific immobilization, but this technique is very limited because non-specific immobilization generally loses function due to the collapse of the folding of the protein. In addition, all host cell proteins will be immobilized at most to reduce the signal-to-noise ratio and, in the worst case, to obscure the desired results. Thus, the ability to generate functional proteome arrays that are specifically immobilized and purified by conventional motifs or tags without requiring individual protein knowledge of common genomic sequences without affecting function. Means a breakthrough in the field of functional proteomics.

The inventors have solved the above problems by providing a method by which each protein in the proteome can be tagged with conventional markers at defined positions in the protein without requiring any prior knowledge of the DNA sequence of the corresponding gene. I developed a method. The 'tag' is used to impart commonality and specificity to downstream immobilization and purification processes, allowing the creation of spatially defined arrays representing thousands of proteins from a given proteome.

An important consideration here concerns the exact positioning of the 'tag'. When the tag is inserted in-frame into any gene at random location that is not defined, the resulting tagged protein will be cleaved in an undefined manner and in many cases correct folding and hence function will be destroyed. Full length proteins are often identified as having short polypeptide extensions at the N-terminus and C-terminus (or both) that can be cleaved without affecting folding or function. However, when the cleavage removes the N-terminal extension and the C-terminal extension and crosses the domain boundary, the folding or function of the protein is generally impaired.

The present invention provides methods for producing proteins and arrays containing such proteins, each having one or more domains of full length and correctly folded and one or more marker moieties tagged at their N-terminus or C-terminus, respectively, as well as in high-speed screening It relates to the use of such an array.

1A is a diagram illustrating the configuration of a vector pMM106H.

1B is a diagram detailing PCR amplification and exonuclease digestion of experimental genes (GST) prior to tagging.

Figure 1c is a diagram detailing the specific ligation and PCR amplification to introduce the tag.

1D is a diagram showing the reaction between glutathione catalyzed by GST and 1-chloro-2,4-dinitrobenzene.

The methods described herein allow for the precise insertion of a tag in the correct translation frame at the N- or C-terminus of each protein or in a region adjacent to either terminus that is not critical for the folding and function of the protein. When specifically immobilized in an array, the individual tagged proteins are correctly folded to retain their function. In the case of multidomain proteins in which individual domains have distinct functions, the methods described herein allow for the insertion of the tag within the entire coding sequence, excluding an external specific domain boundary, thereby allowing specific immobilization within the array. If so, ensure that each tagged domain is correctly folded to retain functionality.

Since each protein in the array will be fully functional, the array can be screened directly to identify drug targets and other biologically relevant molecules. Spatial confinement of the array can directly correlate the phenotype of each protein to its genotype which allows for the identification of 'hits'.

In one aspect, the present invention provides a method of producing one or more proteins wherein the one or more domains are full length and correctly folded and each tagged at the N-terminus or C-terminus by one or more marker moieties.

(a) providing at least one DNA molecule having an open reading frame encoding said protein with a 5 'and / or 3' untranslated region;

(b) amplifying the DNA molecule under conditions that statistically incorporate α-S-dNTP and dNTP into the daughter DNA molecule;

(c) specifically protecting the 5 'end or 3' end of the DNA molecule from nuclease degradation;

(d) the DNA molecule is first treated with 5 '→ 3' nuclease or 3 '→ 5' nuclease to generate a set of nested deletions, and then the start codon of the open translation frame. Or treating with a single chain nuclease under conditions that allow removal of said 5 'or 3' untranslated region comprising a stop codon;

(e) cloning the fragment generated in step (d) into an expression vector containing coding sequences for one or more 5 ′ or 3 ′ marker moieties;

(f) expressing the encoded protein

It includes.

The DNA molecule (s) preferably incorporates a single α-S-dNTP, more preferably α-S-dTTP or α-S-dATP.

The marker moiety can be a peptide sequence, for example a hexa-histidine tag, an antibody epitope or a biotin analogue, or indeed a complete protein, or protein domain, for example a maltose binding protein domain. The marker portion itself may be post-translationally modified, for example biotin or lipid molecules may be added. In a preferred embodiment, the marker moiety will enable purification of "tagged" proteins.

Thus, the methods of the present invention allow for specific modifications of each member of the cDNA library in a manner that does not depend on any knowledge of the sequence of individual genes within a single port. Instead, the method relies on the unprocessed cleavage of each cDNA by nucleases to remove the 5'-untranslated or 3'-untranslated regions of each cDNA. Subsequently, additional known DNA sequences encoding known marker moieties are appended to the resulting nested deletion set of each cDNA. If the marker moiety is the same reading frame as the individual cDNA and is not advanced by any intraframe termination codon, each genetically modified cDNA produced according to the method of the present invention may be N-terminus or C-terminus. Will encode individual proteins that carry a common moiety fused to, eg, a polypeptide “tag”. Screening for correctly folded and tagged proteins can then cause all the out-of-frame fusions to be discarded for all cleavage and tags that cross domain boundaries and affect the folding (and thus function) of the individual proteins.

Since all members of the cDNA library will be modified in the same way, the net result will be that all proteins encoded by the cDNA library will be tagged with a common portion at their N-terminus or C-terminus.

In general, a protein expressed from a cDNA library will be “tagged” and can be readily identified and isolated. Once isolated, the protein can be attached to, for example, a microarray. Attachment may be performed by the tag itself, or alternatively by other parts that first attach to the protein.

Arrays formed by the methods described herein form a second aspect of the present invention. Such arrays include a "tagged" protein expression library and are typically immobilized on a solid support. Those skilled in the art will understand that a range of solid supports is commonly available in the field of arrays, and any of these "substrates" may be used to create the arrays of the present invention.

As already described, the term “protein array” as used herein relates to a spatially defined array of one or more protein portions in any pattern on the surface. The protein portion is preferably attached directly or indirectly to the surface. The attachment can be nonspecific (eg, by physical adsorption to the surface or by nonspecific covalent interactions). In a preferred embodiment, the protein moiety will be attached to the surface via conventional marker moieties linked to each protein using the methods described herein.

In other embodiments, the protein portion can be incorporated into vesicles or liposomes bound to the surface.

Thus, for example, each position in the pattern

(a) a sample of a single protein type (monomer, dimer, trimer, tetramer or higher multimer form);

(b) samples of a single protein type (eg, DNA, antibodies, other proteins) bound to the interacting molecule;

(c) a sample of a single protein type (eg, peptide, compound) bound to the synthetic molecule; or

(d) a mixture of 2 to 100 different tagged proteins at each position in the pattern of the array.

It may include one or more copies of either.

The surface supporting the array can be coated or derivatized by, for example, chemical treatment. Examples of suitable surfaces include glass slides, polypropylene or polystyrene, silica, gold or metal supports or membranes made of, for example, nitrocellulose, PVDF, nylon or phosphocellulose.

As discussed herein, the methods of the present invention specifically tag all proteins within a given proteome at the N-terminus or C-terminus. Some proteins do not tolerate N-terminal extension, while others do not tolerate C-terminal extension, while a significant number of proteins are believed to tolerate one of these extensions. However, existing library cloning methods simply cannot address this problem because they clone full-length unmodified cDNAs, or cloned random and almost indispensably truncated fusions for several protein partners. to be. Compared with the latter case, the method of the present invention targets the position of the tag to or at or near the sequence of the N-terminal or C-terminal residue of the cDNA product such that, for example, fusion to a given peptide partner may cause This can be done without affecting folding or functionality. Compared to the former case, the method of immobilizing proteins in the array as described herein is via specific interactions rather than nonspecific interactions, which specific interactions of the tag added to the ends of each cDNA to be. In addition, the methods described herein can be used to screen for purified and immobilized proteins expressed in non-bacterial host organisms that aid in maintaining function through correct folding and post-translational modifications, but phage display or lambda-cDNA expression Existing methods, such as libraries, are limited to bacterial hosts, where the majority of eukaryotic proteins have been found to be nonfunctionally synthesized due to misfolding or incorrect post-translational modifications.

The method of the present invention is applicable to a wide range of in vitro fields which can be classified into three main fields. The three main areas of study are the fields of protein-ligand interactions, the fields of protein-protein interactions, and the fields of protein-DNA interactions.

Protein-ligand interaction

The methods described herein will enable fast profiling of the interaction between any new chemical entity and all proteins in a given proteome. This can be achieved by simply probing an appropriate proteome array using NCE under variable stringency that may allow for reverse high throughput screening. Reading of this screening will be useful directly in some of the situations described below.

High throughput screening programs in which libraries of compounds are tested for cells or whole organisms often result in changes in phenotype without known targets prior to screening. However, subsequent confirmation of the primary target can be a very difficult process. The method of the present invention can be applied directly to this type of problem, because the method of the present invention can produce a functional proteome array for the species contemplated, and then by screening the array with a lead compound It can be identified which protein in the proteome is targeted. This highly parallel method for identifying protein-ligand interactions will greatly speed up, also simplify, and enable identification of weaker secondary interactions that may be important. In addition, the method can be directly applied to species cross-reactivity problems, for example to quickly assess potential antifungal compounds in terms of their interaction with all proteins in the human proteome; This type of information is believed to prove very useful for any subsequent optimization of the lead compound.

Current high throughput screening methods allow for the rapid identification of small molecules that bind to certain proteins that have already been identified as potential therapeutic targets themselves. However, these methods do not address the question of how selective any given interaction is, but knowledge about whether or not to track a given lead compound; Recognized knowledge is potentially important in determining whether a compound targeting a single protein disagrees with the fact that it has fewer side effects than fitting multiple related or unrelated proteins.

There are many examples of compounds successfully developed through the third stage, but clinical trials have not received regulatory approval because their main mechanism of action is unknown. The antidepressants Mianserine, Trazadone and Pfizer's antiarthritis teniab are examples of unregulated approval, and millions of dollars in each of them have become useless. The methods described herein can potentially be used to resurrect these failed drugs because they can detect the primary targets of these drugs, and subsequently demonstrate mechanisms of action, and are very expensive clinical trials. The data already replaces regulatory approval.

All existing drugs have side effects, large or small, and one example is clozapine, an antipsychotic that is of interest. If the molecular origin of these side effects can be determined, this will greatly facilitate the design of next generation drugs with optimized main effects and minimized side effects. In addition, the methods described herein can be applied directly to such problems, because in generating a profile of the interaction between any compound and all the proteins in the proteome, an abnormal second interaction will be identified, which is followed by This is because they can be evaluated in terms of whether or not they are associated with known side effects.

In addition, the methods of the present invention can be used to identify groups of proteins such as serine proteases by screening proteome arrays using common inhibitors. This allows for the subsequent development of biochips representing all kinases or all p450 enzymes for more intensive screening of, for example, all human proteases or alternatively lead compounds. For example, p450 biochips may be useful for evaluating whether certain lead compounds will be metabolized or not, because p450-mediated hydroxylation is often the first step in the process and patients in drug response One of the primary causes of versus patient variability; In fact, one of the goals of current drug design is because it is thought to produce compounds that are not primarily metabolized; The p450 chip has significant potential usefulness.

Protein-protein interactions

Protein-protein interactions and polyprotein complexes are important in cell biology. For example, signaling pathways are usually initiated by interactions between cell surface receptors and exogenous ligands, which involve a cascade of protein-protein interactions that ultimately result in activation of specific genes. Individual protein-protein interactions may depend on the presence of specific ligands, or alternatively may be blocked by specific ligands, while some polyprotein complexes will only be formed in a ligand-dependent manner.

Thousands of new protein-protein interactions have been identified using two-hybrid technology. The methods described herein overcome the limitations of these methods and can be used to screen proteome arrays using individual labeled proteins to determine the relative strength of individual interactions as well as interaction partners. In addition, the method of the present invention can be applied to identify them even when the assembly of the polyprotein complex components is ligand dependent.

An example of using this method in this way in defining new protein-protein interactions is the identification of signaling partners of the cell substrate domain of specific cell surface receptors involved in disease states; The identification of such signaling partners may be directly related to the pharmaceutical prospects, since protein-protein interactions may directly mean possible therapeutic targets.

Protein-DNA Interaction

About 10% of all genes in the human genome are expected to encode transcription factors, but only a few of these have now been identified. The binding of specific transcription factors to DNA enhancer elements, often in response to external stimuli, is a requirement for the formation of an enhancementosome complex that switches on gene expression. There are several points where gene expression can in principle be affected by drug administration: drugs block the signaling cascade initially by blocking the binding of proteins or small molecules to cell surface receptors; The drug blocks protein-protein interactions or inhibits enzymatic activity in the signaling cascade; Or in the alternative, the drug blocks specific protein-DNA or protein-protein interactions in the enhancersome complex. As an example, the transcription factor NF-κB is involved in as many cellular processes as immune and inflammatory responses, limb development, septic shock, asthma and HIV propeptide production. The majority of intracellular signaling cascades in NF-κB activation are common to all of these processes and therefore do not represent living targets for mediation. Thus, the differences between the reactions are due to the underlying ligand-receptor interactions or the formation of specific enhancesome complexes. NF-κB is known to bind to at least 14 different enhancer elements, and thus enhancesomes represent potential therapeutic targets. However, the design of individual enhancersome complexes requires knowledge of a number of individual DNA-binding proteins involved and also knowledge of their protein-protein interaction interactions. Both problems can be handled directly. Proteome arrays can be screened with specific DNA probes to identify novel DNA binding proteins. Alternatively, the transactivation domain of a given transcription factor can be used to screen the proteome array to identify other proteins with which it interacts. The cross correlation of these screenings allows the identification of new components of specific enhanceosomal complexes.

In addition, the protein arrays produced by the methods of the present invention allow the selection of molecules that recognize each protein represented in the array. In a preferred embodiment, the selected molecule will be an antibody or antibody like protein and will be expressed on phage or ribosomes or covalently linked to the coding mRNA.

Thus, phage manifest antibody libraries can be used for each immobilized protein in the array and for unbound antibodies removed by washing. The selected phage can then be recovered and used to infect the bacteria according to the general procedure. Phage infected bacteria can then produce phage particles representing antibodies selected for further rounds of selection, or generate soluble antibody fragments for direct use. As used herein, the term 'antibody' or 'antibody fragment' refers to short chain Fvs, FAB fragments, individual light chain fragments or heavy chain fragments derived from mice, humans, camels or other organisms.

In a preferred embodiment, the protein array is in a microwell format such that after the selection step, phage particles can be recovered by adding the appropriate bacterial cells to each well where the bacterial cells will be infected by the selected phage particles. Growth medium is then added to each well and the infected bacteria are grown and the antibody fragments are expressed while maintaining the physical isolation of the antibody fragments selected for each immobilized protein in the array. If desired, new phage particles produced by infected bacteria can be used in subsequent selection rounds. This procedure is a general method for selecting polyclonal or monoclonal antibody fragments for a single purified and immobilized protein. Indeed, the first protein arrays will produce polyclonal antibody fragments or monoclonal antibody fragments for thousands of correctly folded proteins using a highly parallel approach, or in vitro antibody selection methods.

The antibody fragments selected from each well of the original array, and the freely expressed antibody fragments themselves are immobilized into a newly spatially defined array so that the antibody fragments at each position in the new array are immobilized at a defined single position in the original array. Is selected. The antibody array so produced will contain polyclonal antibody fragments or monoclonal antibody fragments depending on the number of choices made prior to immobilization of the soluble antibody fragment.

Such antibody arrays will have a number of potential uses, including the capture of individual proteins from natural cell or tissue lysates for differential expression monitoring of related proteome. Alternatively, the antibody-captured protein can be screened directly for ligand-binding function. In general, any one monoclonal antibody may bind to and block its function while the other monoclonal antibody may bind but not block the function. In the highly parallel method, it is practically impossible to individually assess all monoclonal antibodies against all proteins in the proteome for their ability to bind but not affect function. However, polyclonal antibody sets for all proteins in the proteome are believed to have binding, individual antibodies that possess certain abilities that do not affect one function and individual antibodies that recognize all post-translational modifications of a given protein. do. Thus, it is generally believed that polyclonal antibody arrays will be advantageous for direct screening of the function of the captured protein, rather than the monoclonal antibody arrays produced as described above.

Compared with the original protein array, antibody arrays produced according to the methods described herein have the advantage that all proteins immobilized on the array will be stable under similar conditions. Proteins captured from natural cell or tissue lysates will be naturally expressed, not recombinant. In addition, the captured protein can be screened for function or ligand binding and the like immediately after being captured from natural cell or tissue lysate, which should aid in maintaining function.

In a further aspect, the present invention

(i) screening at least one compound for biological activity, comprising contacting the at least one compound with a protein array as defined herein and measuring the binding of the at least one compound to the proteins in the array. How to;

(ii) contacting a protein array as defined herein with at least one protein, such as a cell surface receptor, and measuring the binding of the protein in the array with at least one specific protein. -Screening one or more proteins for protein interactions;

(iii) contacting the at least one nucleic acid probe with a protein array as defined herein and measuring the binding of the probe to the proteins in the array, at least one for a particular protein-nucleic acid interaction. Methods of screening proteins;

(iv) use in high speed screening of compounds, proteins or nucleic acids of a protein array as defined herein;

(v) use of said array in screening for molecules that recognize each protein in a protein array as defined herein, wherein the molecule is preferably an antibody;

(vi) contacting the antibody library with a protein array as defined herein to cause the at least one protein in the protein array to bind to at least one antibody in the antibody library, removing any unbound antibody, and A method of producing an antibody array, comprising immobilizing these antibodies bound to proteins in the protein array; And

(vii) a method for screening for protein function or abundance comprising contacting the antibody array as defined herein with a mixture of one or more proteins.

To provide.

The methods (i), (ii), (iii) and (vi) may first comprise providing an array according to one or more of the methods of the invention.

The use of proteins derived from the methods described herein forms a further aspect of the invention. Those skilled in the art will appreciate that applications are known in the art for which modified proteins can be used.

In a further aspect, the present invention

(i) Expression of tagged proteins produced by the methods of the invention in a number of expression hosts, ie bacteria, yeasts, mammalian cells (e.g. Walker EA, Clark AM, Hewison M, Ride JP, Stewart PM) Functional Expression, Characterization and Purification of Type 1 Human 11-Beta-hydroxysteroid Dehydrogenase, J Biol Chem 2001 Jun 15; 276 (24): 21343-50; Cai J, Daoud R, Georges E , Functional Expression of Multidrug Resistant Protein 1 in Gros P. Pichia pastoris , Biochemistry 17 July 2001; 40 (28): 8307-16, and Hara H, Yoshimura H, Uchida S, Toyoda Y, Aoki M, Sakai Y, Morimoto S, Shiokawa K, Molecular Cloning and Functional Expression Analysis of cDNA for Human Hepasocin with Hepatocellular Promoting Activity (1), Biochim Biophys Acta July 30, 2001; 1520 ( 1): 45-53);

(ii) use of tagged proteins produced by the method as defined herein;

(iii) generated by a method as defined herein for analysis of the interaction between a protein expressed in a yeast two-hybrid system and another protein by cloning of the modified DNA molecule into a yeast two-hybrid expression vector. Of tagged tagged proteins (see, eg, Staudinger J, Perry M, Elledge SJ, Olson EN.2-Hybrid Systems for Vertebrate Helix-Loop-Helix Protein Interactions in Yeast, J Biol Chem Mar 5, 1993; 268 (7): 4608-11 and Vojtek AB, Hollenberg SM, Cooper JA.Direct Interaction of Mammalian Ras with Serine / Threonine Kinase Raf, Cell 16 July 1993; 74 (1 ): 205-14).

(iv) as defined herein for immobilization on an affinity column / substrate, for example to enable purification by affinity chromatography of a) interacting proteins, b) DNA or c) chemical compounds. Use of tagged proteins produced by the method (eg, Rhodes N, Gilmer ™, Lansing TJ. Expression and Purification of Active Recombinant atm Proteins from Transiently Transfected Mammalian Cells, Protein Expr Purif May 2001; 22 (3): 462-6: Zwicker N, Adelhelm K, Thiericke R, Grabley S, Hanel F. Strep-Tag II, Biotechniques for one-step affinity purification of the active bHLHzip domain of human c-Myc, August 1999; 27 (2): 368-75, and Giuliani CD, Iemma MR, Bondioli AC, Souza DH, Ferreira LL, Amaral AC, Salvini TF, Selistre-de-Araujo HS, active recombinant lysine 49 phosphor as a fusion protein in bacteria Expression of Lipase A (2) Myotoxin, Toxicon October 2001; 39 (10): 1595-600).

(v) Use of a tagged protein produced by a method as defined herein for immobilization by affinity purification for detection by antibodies (ELISA assay) as a diagnostic tool (see, eg, Doellgast GJ, Triscott MX). , Beard GA, Bottoms JD, Cheng T, Roh BH, Roman MG, Hall PA, Brown JE.Sensibility for the detection of Clostridium botulinum neurotoxins A, B and E using signal amplification by enzyme binding aggregation assays Enzyme Linked Immunosorbent Assay, J Clin Microbiol September 1993; 31 (9): 2402-9).

(vi) Use of a tagged protein produced by a method as defined herein as a probe for cDNA microarrays to identify DNA binding proteins (e.g., DeRisi J, Penland L, Brown PO, Bittner ML, Meltzer PS, Ray M, Chen Y, Su YA, Trent JM, Use of cDNA microarrays to analyze gene expression patterns in human cancer, Nat Genet Dec. 1996; 14 (4): 457-60).

(vii) Tagging produced by the method as defined herein to identify the identity of the protein in the 'proteome' by mass spectrometry of the expressed protein component of the starting material or source library modified by the method of the present invention. Of modified proteins (see, for example, Bordini E, Hamdan M. Matrix supported laser desorption / ionization flight times and studies of several covalent and noncovalent complexes by electrospray mass spectroscopy, Rapid Commun Mass Spectrom) 1999; 13 (12): 1143-51)

To provide.

Preferred features of each aspect of the invention may be mutually applicable to other aspects.

The invention will be further described by the following non-limiting examples which are not intended to limit the scope of the invention in any way.

(a) vector construction (see FIG. 1A)

We constructed a vector pMM106H derived from pUC19 containing a strong hybrid promoter (P trc ) to drive the expression of the cloned gene into the Nco I position immediately downstream of the promoter sequence. We inserted a 676 bp nonsense DNA sequence as a stuffer fragment between the Nco I position and the downstream Hpa I position. Hpa I is a blunt- ended cutter and is positioned to cleave the vector so that when there is a reading frame on the first base of the blunt end, the downstream DNA encodes the hexahistidine peptide, polyasparagine. Behind the hexahistidine tag is an amber stop codon (TAG) followed by a gene encoding the green fluorescent protein (GFP) of the jellyfish Aequorea victoria . The configuration of pMM106H was confirmed by sequencing.

Genes cloned into pMM106H as Nco I / blunt end fragments produce fusions to His-tag and GFP only if the correct translation frame is generated at the Hap I position during cloning. As used herein, GFP was used as a reporter gene that facilitates visual screening of clones expressing His-tags, and was also used to provide signs of correct folding of the fusion protein, because GFP was in the correct form. This is because it only shows activity when it is folded.

The amber stop codon produces a small amount of full length fusion protein for visualization of green colonies, while most of the fusion protein will be terminated immediately after His-tag and can be used for subsequent immobilization and enzymatic analysis. Many different peptides of proteins in which soluble expression confers some observable phenotype on cells can be used in place of GFP as markers for expression and folding of tagged proteins. Examples thereof include chloramphenicol acetyl transferase, β-galactosidase, lacZ fragments of β-galactosidase, and proteins capable of inhibiting transcription, such as, for example, λ-CI refreshers, but are not limited thereto. no.

PGSTN was used as a template in the procedure described below. This plasmid was first synthesized with Schistosoma japonicum glutathione S transferase (GST) gene derived from pGEX-2T (Pharmacia) under standard conditions, primer 'GSTfwd2'(5'-ATG CTG CAG ACG TCA ACA GTA). TCC ATG GCC CCT ATA CTA GG-3 ') and' GSTHindIII '(5'-GCG AGG AAG CTT GTC AAT CAG TCA CGA TGA ATT CCC-3') was constructed by PCR amplification. These primers introduced an Nco I restriction site at the initiation codon of GST, mutated the second residue of GST from serine to alanine, followed by a termination todon at the 3'-terminal multiple cloning site of the GST gene, followed by a HindIII restriction site. Was introduced. The PCR product was then cloned into pTrcHis (Invitrogen) previously digested with Nco I / Hin dIII as Nco I / HindIII fragments under standard conditions to generate pGSTN.

(b) PCR amplification and exonuclease digestion of genes prior to tagging (see FIG. 1B)

We have designed a vector-specific primer 'ST forward'(5'-ATG CTG ACG TCA TGA GGC CCA TGG GGC that binds to the vector at 156 bp upstream of the start codon and 84 bp downstream of the stop codon, respectively. GST gene from pGSTN construct by PCR using CCG GAT AAC AAT TTC ACA CAG G-3 ') and' ST reverse '(5'-GCG GAT CCT TGC GGC CGC CAG GCA AAT TCT GTT T-3') Was amplified. Separately 30 cycles of PCR (94 ° C. 1 min; 57 ° C. 1 min; 72 ° C. 2 min) were performed in four 100 μl reactions. Each PCR reaction contained about 20 ng of template DNA, 50 pmol of each primer and 2 units of Pwo polymerase. Each PCR reaction was performed in standard buffer (10 mM Tris-HCl pH 8.8, 25 mM KCl, 5 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ , 10% DMSO). Each of the four PCR reactions then also contained the following non-standard deoxynucleotide triphosphate mixtures:

Reaction 1) 200 μM dATP, 200 μM dTTP, 200 μM dCTP, 150 μM dGTP, 50 μM α-S-dGTP;

Reaction 2) 200 μM dATP, 200 μM dTTP, 200 μM dGTP, 150 μM dCTP, 50 μM α-S-dCTP;

Reaction 3) 200 μM dATP, 200 μM dGTP, 200 μM dCTP, 150 μM dTTP, 50 μM α-S-dTTP;

Reaction 4) 200 μΜ dGTP, 200 μΜ dTTP, 200 μΜ dCTP, 150 μΜ dATP, 50 μΜ α-S-dATP.

Many different DNA polymerases, including Tap polymerase and non-thermal stability, including thermostable polymerases lacking 3 '→ 5' exonuclease activity as well as amplification of template DNA in the presence of α-S-dNTP. It can be carried out by primer extension reaction using polymerases such as T4 DNA polymerase or Klenow fragment of DNA polymerase I.

The inclusion of a single α-thio deoxynucleotide triphosphate in each particular PCR mixture results in random, statistical incorporation of the relevant α-S-dNTPs into the specific final PCR product. These modified nucleotides are not substrates of exonuclease III and are used to stop the progressive removal of nucleotides by the enzyme. The four individual PCR mixtures were pooled, purified using QIAquick PCR cleanup kit (Giagen) under standard conditions and completely digested with restriction enzyme Aat II. The resulting about 1000 bp PCR product was then gel purified. Degradation with Aat II produced 3'-protrusions resistant to exonuclease III activity, thereby protecting the 5'-terminus of the PCR product from degradation.

Alternative methods for specifically protecting one end of a PCR product from exonuclease cleavage can be readily implemented to include, but are not limited to: Any restriction enzyme that produces a 3'-overhang of 4 or more bases can be used in place of AatII when the required position is incorporated in the PCR primer design. Any restriction enzyme that produces a 5′-protrusion can also be used in place of AatII when the necessary site is incorporated in the PCR primer design; In this case, after the 5'-protrusion is generated, if a DNA-polymerase-mediated fill-in reaction in which the relevant α-thio dNTP is used instead of dNTP is performed, a novel 3'- The terminus is protected from exonuclease cleavage.

10-15 μg of the cleaved PCR product is then incubated with 75 units of exonuclease III 75 μg of DNA for 30 minutes at 37 ° C. in 150 μl reaction. The exo III cleavage is performed in standard reaction buffer (66 mM Tris-HCl, pH 8.0, 6.6 mM MgCl ₂ , 5 mM DTT, 50 μg / ml bovine serum albumin). This condition ensures completion of cleavage by exo III. The enzyme was then inactivated by heating to 75 ° C. for 15 minutes. The product of the exo III cleavage is a nested set of deletions obtained from the 3'-end of the PCR product.

Exonucleases III are non-processing 3 ′ → 5′-exonucleases, and because exo-nucleotides cannot hydrolyze α-thio-containing nucleotides, exo III is α-thio-de Each time an oxynucleotide base is reached, forward cleavage of the degenerative 3 'end of the PCR product is stopped. Therefore all products are nested sets of deletions that are the result of random incorporation of each α-S-dNTP at an early stage. The ratio of α-S-dNTP to dNTP used in the original PCR amplification is determined experimentally so that the envelope of the nested deletion within the 400 bp size is drawn about 100 bp shorter than the original full length PCR product.

The size range of the cleavage obtained as above can be adjusted by changing the ratio of α-S-dNTP to normal dNTP. This fact is important for the application of the method of the present invention to eukaryotic cDNAs, since the cDNAs have various lengths of 3'- having the most common 3'-UTRs in the range of 200-300 bp in length. This is because it has a non-translated domain. Since the relative efficiency of the incorporation of four α-S-dNTPs each depends on the identity of the polymerase, the ratio of α-S-dNTP to normal dNTPs optimized for each of the four bases and for the particular polymerase is applied. It is preferable. The molar ratio of racemic α-S-dNTP to normal dNTPs commonly used will range from 1: 1 to 1: 3.

(c) preparation for removal and cloning of single chain regions (see FIG. 1C)

The nested set of deletions generated by exonuclease III cleavage in the previous step was purified by ethanol precipitation followed by one-fold mung bean nuclease buffer (50 mM sodium acetate pH 5.0, 30 mM NaCl, 1 mM ZnSO ₄ Resuspended). The cleaved DNA was treated with 30 units (2 units / μg) of mung bean nuclease in 100 μl of reaction solution at 30 ° C. for 30 minutes. This process removes the 5'- and 3'-protrusions to obtain blunt-end products. The reaction was stopped by adding EDTA to a final concentration of 5 mM. The cleaved product was purified using a QIAquick PCR Purification Kit (Qiagen), which was digested with NcoI and then separated on a 1% agarose / TBE gel (in this case using 100 bp DNA ladder as standard). Products ranging in size from 800 to 1000 bp were extracted from agarose using a QIAquick gel extraction kit (Qiagen). It is certain that other single chain nucleases such as S1 nucleases may be used to remove 5'-protrusions from nested sets of 3'-deletions produced by 3 '→ 5'-exonucleases. .

(d) Modifications in the manufacture of nested deletions

Many of the other standard molecular biological methods for generating nested sets of deletions are obvious variations of the original method. This method includes the use of any 3 ′ → 5 ′ exonuclease, the use of any 5 ′ → 3 ′ exonuclease, or any endonuclease that cleaves progressively from the end of a linear DNA fragment. Including but not limited to use. For example, initial PCR amplification can be performed using the same reverse primer as described above, but with forward primers that bind about 2 kb upstream of the GST gene initiation. This will produce a fragment where the GST gene is> 2 kb at the 5 'end and occupies only 84 bp at the 3' end. The purified PCR fragment can then be treated with Bal 31 nuclease (an enzyme that progressively cleaves linear double stranded DNA simultaneously from its 5 'and 3' ends). The enzyme is non-processed and the rate of DNA degradation depends on the reaction time and temperature and the base composition of the DNA. The flanking region present at the 3 'end of the GST gene in the PCR product is considerably shorter than the flanking region at the 5' end, so that degradation before or after the stop codon occurs long before the enzyme reaches the initiation codon from the other end. Will occur. Over time experiments can determine the optimal reaction conditions to remove up to 400 bp from the 3 'end of the PCR product. The resulting nested set of deletions can then be blunt-ended to remove any remaining single chain region, cleaved with a unique restriction enzyme encoded at the 5 'end of the gene by the original vector, and oriented into the tag vector. Can be cloned. Alternatively, lambda exonucleases can be used to generate nested sets of 5'-deletions. Preferred substrates of this enzyme are 5'-phosphorylated double chain DNAs, one end of which is easily protected by having a 5'-hydroxyl terminus.

The single chain 3 'overhang of the nested set of 5'-deletions produced by 5' → 3 'exonuclease is a T4 DNA polymerase or a single chain DNA specific nuclease such as RNAse T or exonuclease. It can be removed by a number of different enzymes, including the T or mung bean nuclease.

(e) Cloning and analysis of the modified product (see FIG. 1C)

Restriction enzymes NcoI and HpaI were used to complete the cleavage of the vector pMM106H (3 μg) and gel purified the 2870 bp backbone fragment. After ligating the vector DNA prepared as described above and the restricted product under standard conditions and using this ligation mixture, an E. coli DH5α cell (then recovered and containing 100 μg / ml carbenicillin) Smeared on the stomach).

This cloning method is performed on the entire set of deletions obtained in the previous step. However, only such deletions that excise the end codon and the end just behind the in-frame codon of the GST gene should be able to fuse in-frame to the hexahistidine tag and GFP after the cloning step through this method; Due to the translation termination at the GST termination codon, all other deletion products cloned in this manner should induce out-of-frame fusion to hexahistidine tag and GFP, or unfused GST protein. This is because the blunt end of the deletion product is ligated to the blunt end of the vector, resulting in a gene fusion in which the translation translation frame of the downstream vector DNA is indicated by the original translation frame of the GST coding region. If the deletion product ends in an incomplete codon, the newly attached hexahistidine coding sequence will be out of frame for the GST gene, whereas if the deletion product carries a GST termination codon, the translationality of GST to the hexahistidine tag Fusion will not occur. Thus, hexahistidine- (and GFP-) tagged proteins generated from the general methods described above will need to be GST fused to polyasparagine, hexahistidine tags. They do not need to be clones that are absolutely full length, but the ability to correctly fold and the retention of enzyme activity will be screened in an additional step.

Transformed colonies were visualized under UV light (365 nm) and 30 colonies (about 10% of the total) emitting green fluorescence were visually selected for further analysis. These colonies were replicated plated and analyzed by colony western blot under standard conditions using anti-His-tag and anti-GST antibodies. Since the anti-His-tag antibody binds only to colonies expressing hexahistidine-tagged protein, direct information on the number of colonies expressing in-frame fusion to hexahistidine-tag can be obtained via western blot. On the other hand, since the anti-GST antibody binds close to the C-terminus of the GST protein, it recognizes only colonies expressing the GST protein which is full length or almost full length. We identified 19 colonies (63% of green fluorescent colonies) containing proteins recognized positively by both anti-His-tag and anti-GST antibodies. DNA obtained from 12 of these colonies was amplified, purified and sequenced. Sequencing data confirmed the presence of 10 clones with two full in-frame fusions for the full length GST and short cuts of the GST gene, in which case they were still in the frame with hexahistidine tags. Therefore, the isolation frequency of the full length GST clones obtained by the inventors through this general method is about 17% (of the total number of green fluorescent colonies), while the full or nearly full length GST predicted to retain activity. The isolation frequency of the clones is about 63% (of the total number of green fluorescent colonies).

(f) Immobilization and functional analysis of tagged proteins (see FIG. 1D)

E. coli DH5α cells were transformed with one of the full length hexahistidine tagged GST plasmids prepared by the method described above. Single carbenicillin-resistant colonies were grown in medium log phase in 10 ml culture and then fed with 10 μM IPTG to induce the expression of hexahistidine tagged GST. After further growing for 4 hours, cells were collected and lysed by freeze-thaw / lysozyme. SDS-PAGE of crude lysate confirmed overexpressed protein (approximately 20% of total soluble protein) with expected size (27 kDa) and small amount of 54 kDa GST-hexahistidine-GFP fusion generated through amber suppression It was. The crude lysate (500 μl; 100 μg) was then mixed with nickel-NTA magnetic beads (50 μl; binding force 15 μg hexahistidine-tagged protein) and recovered by sedimentation under magnetic field. The supernatant was then discarded and the beads washed, and then resuspended in glutathione S transferase assay buffer containing 1 mM each of glutathione and 1-chloro-2,4-dinitrobenzene. The absorbance at 340 nm was measured to collect endpoint analysis data after 30 minutes at room temperature; The wavelength then corresponds to λ _max of the GST-catalyzed reaction product.

As a control, DH5α cultures containing a plasmid encoding a parent vector (pMM106H) or an unrelated His-tagged protein (alanine racemase) were grown, followed by induction, collection, lysis and analysis. . GST activity was detected only on beads mixed with crude lysate containing His-tagged GST, which was due to His-tagged GST where the observed GST activity was specifically immobilized and also the protein was subjected to specific immobilization. It is to clarify that it retains the activity against.

After completing the enzyme assay, the protein was eluted from the magnetic beads by addition of a buffer containing 100 mM imidazole and analyzed by SDS-PAGE. This indicates that the sample representing the positive activity assay contains a single immobilized protein with the exact size (27 kDa) predicted for glutathione S transferase, whereby the activity observed on the beads is recombinant His-tagged. It was confirmed that it is due to only protein.

Example 2

(a) vector composition

The inventors found that pMM111 (676 bp NcoI / HpaI nonsense DNA stuffer fragment, the second vector, was substantially substituted with pMM106H of Example 1 except that the 300 bp NcoI / HpaI fragment derived from Escherichia coli gdhA gene was substituted. Same as); In this case, the HpaI cloning position was replaced with the SmaI position such that the downstream hexahistidine tag was located 2 nucleotides outside from the frame carrying the gdhA gene; The ATG initiation codon of the GFP gene was substituted with alanine codon (GCG). The vector was designed such that the insert cloned at the SmaI position contained the first nucleotide of the codon at the 3 'end to introduce this insert into the frame containing the hexahistidine tag and the GFP. The configuration of pMM111 was confirmed by sequencing.

(b) Modifications to introduce tags

The inventors then carried out the same method as described in Example 1, except that it was modified as follows. First, only α-S-dTTP was introduced into the original PCR amplification process, ie, reaction 3 in section (b). Second, the final product was cloned into the NcoI and SmaI positions of vector pMM111.

This method has several theoretical advantages over Example 1 described above. These advantages can be seen mainly through statistics on the incorporation of a single α-thio dNTP into the original PCR product. Therefore, when consumable exonuclease III digestion is performed, the nested set of 3'-degenerative deletions will terminate with all 3'-thymidine bases, but not all of the 4 nucleotides. The SmaI position in pMM111 of these fragments when 3'-T is that of the first co-termination stop codon of the GST gene or is of the preceding sequence and is in the same translation frame as 'T' of the first co-termination codon Cloning of the will result in in-frame fusion to the hexahistidine tag and GFP (since degradation with SmaI creates a gap of two nucleotides before the coding sequence of the tag). Statistically four times less nested deletions would be produced by exonuclease hydrolysis in this modification method than in the method of Example 1. However, all three possible stop codons contain 'T' as their first base, so they will all be present in the set of deletions making up a ratio four times higher than that of the entire set of deletions. For the probability of any 'T' present in the same decryption frame as the first 'T' of the stop codon, 33% of all codons obtained with this variant must be fused in-frame for the His tag encoded by the vector, Defects that affect folding (and thus function) produce 'white' colonies (for this reason, presented in Example 1). The inventors found that following this modification method, the ratio of clear, full-length clones in the 'green' population was higher than when the method of Example 1 was performed. Of course, all known initiation codons (ATG, GTG, TTG, ATT, CTG) include 'T' in the second position, so the same applies to the 'starting' codons.

A further advantage of this modification method is that the polyA tail of the forward primer (eg, forward A; 5'-AAA AAA AAA AAA GAT CGA TCT CAT GAC GGA TAA CAA TTT CAC ACA GG-3 ') was used for initial PCR amplification. Can be incorporated at the 5'-end. During an amplification process where the ratio of dTTP: α-S-dTTP is 3: 1, it is likely that one or more α-S-dTTP residues will be incorporated at the end of the complementary chain (5 'end of the PCR product). Such incorporated nucleotides will be resistant to exonuclease III degradation, thus eliminating the requirement for an enzymatic step to specifically protect the ends of the PCR product from degradation.

Example 3

(a) modification of a second protein using a hexahistidine tag

In carrying out the method described in Example 1 for glutathione-S-transferase, the present inventors demonstrated that the method is independent of the sequence of the gene being manipulated.

Therefore, unless otherwise indicated, the present invention was initiated with a plasmid encoding the human transcription factor NF-κB p50 and followed by the method described in Example 1, and the inventors followed the first intraframe stop codon (when the amber stop codon was inhibited). A method was described for modifying NF-κB p50 to be cleaved and replaced with an in-frame fusion to DNA encoding the polyasparagine, hexahistidine tag and GFP. Clones that emit green fluorescence when excited with far ultraviolet light (365 nm) were further characterized. Clones expressing hexahistidine tagged proteins with colony western blot using anti-His-tag antibodies were identified. Soluble protein lysates of the clones were analyzed by SDS-polyacrylamide gel electrophoresis and probed with anti His tag antibody. Immunoreactive signals were observed at about 65 kDa M _r (corresponding to hexahistidine tag and GFP translatable to NF-κB p50) and about 38 kDa M _r (NF-κB p50-His tag). In addition, a signal of what might be a degradation product corresponding to his tagged GFP protein was observed at about 27 kDa M _r . Sequencing data confirmed that some clones encode a complete in-frame fusion of NF-κB, either full length or nearly full length, for the hexahistidine tag. In one experiment, 190 colonies were screened for green fluorescence. A total of 38 clones (20% of the total number of screened clones) emitted green fluorescence when excited with ultraviolet light (365 nm). Colony Western blot was performed using anti-His tag antibody, indicating that 29 out of 38 clones expressed hexahistidine tag. From the sequencing data, 18 of these clones were in-frame fusions of NF-κB p50 to hexahistidine tags, either full length or nearly full length; Seven of these clones were His full-tagged NF-κB p50 gene, and the remaining 11 His-tagged clones were short fragments consisting of 1-4 amino acid residues. This experiment clearly demonstrates the advantages of having a reporter system that indicates expression and accuracy of protein folding. About 50% of the clones that emit green fluorescence were full length intraframe fusions to His tags, or had His-tags with minor truncation fused in frame without crossing domain boundaries.

(b) Immobilization and functional analysis of hexahistidine tagged NF-κB p50

E. coli DH5α cells were transformed with one of the full length hexahistidine tagged NF-κB plasmids prepared by the method described above. Single carbenicillin-resistant colonies were grown in medium log phase in 10 ml culture and supplemented with 100 μM IPTG to induce the expression of hexahistidine tagged NF-κB p50. After further growing for 4 hours, cells were collected and solubilized by ultrasound. SDS-PAGE of the crude lysate revealed that the protein of expected size (38 kDa), which accounts for about 5% of the total soluble protein, was overexpressed.

The double stranded oligonucleotide, 'κB motif', contains a palindromic binding site for NF-κB p50 and was labeled with digoxigenin at the 3'-base using a 3'-terminal transferase under standard conditions.

Protein lysates were prepared using the lysozyme / freeze-thaw method in PBS (phosphate buffered saline, pH7.5) containing 5 mM β-mercaptoethanol. 200 μl of soluble protein lysate from each clone was injected into Ni-NTA coated microwells and then incubated for 45 minutes at room temperature. In the last step of incubation, the wells were washed three times with PBST (PBS containing 0.02% Triton X-100) to remove all unbound proteins. The wells were washed three times by immersion in DNA binding buffer (75 mM KCl containing 10 mM Tris HCl, pH7.4, 5 mM β-mercaptoethanol) for 1 minute. A 3′-digoxigenin labeled κB motif (2 pmol) was added to the wells in 200 μl of DNA binding buffer containing 1 μg poly (dI-dC) nonspecific DNA. After further incubation for 30 minutes, unbound DNA was removed by washing three times with 25 mM KCl containing 10 mM Tris-HCl pH 7.4, 0.02% Triton X-100. Antidigoxigenin antibody-alkaline phosphate conjugates were diluted to a concentration of 150 mU / ml in 'antibody dilution buffer' (10 mM Tris-HCl, pH 7.4, 25 mM potassium chloride) supplemented with 0.2% bovine serum albumin. Diluted antibody (200 μl) was then added to the microwells. After 30 min at room temperature, microwells were washed with 'antibody dilution buffer' (3 x 350 μl) supplemented with 0.02% Triton X-100 to remove unbound antibodies. Then 200 μl of 250 μM p-nitrophenyl phosphate (pNPP), alkaline phosphate substrate (100 mM Tris-HCl, pH9.5, 100 mM NaCl, 50 mM MgCl ₂ ) was added to the wells and overnight at room temperature The reaction was allowed to proceed, and each well was quantitatively analyzed at 405 nm when yellow colored (product, indicating that p-nitrophenyl was formed). The hydrolysis background ratio of the substrate, pNPP, was low, confirming that the wells developed yellow color, and it was immediately confirmed that the analytical result was positive. We do not add any of the crude lysates, labeled oligonucleotides, or antibodies as a control in this assay, or add a 20-fold excess of unlabeled double stranded oligo, or hexahistidine tagged NF-κB Crude lysate containing p50 was replaced with the same amount of crude lysate obtained from DH5α cells expressing hexahistidine tagged GST in the same vector background.

In this assay, NF-κB p50 initially binds to labeled oligonucleotides via specific binding sites. The protein-DNA complex is then transferred in microwells with any unbound, labeled oligos via hexahistidine tags and all other proteins (including complexes between labeled oligos present in crude lysate and other DNA binding proteins). Immobilized and washed it. Because the antibody-conjugate recognizes the label on the oligo rather than the hexahistidine tagged protein, the positive signal in this assay is only when the NF-κB p50-DNA interaction is maintained when NF-κB p50 is immobilized via the tag. Could be observed; If this interaction is not maintained, the oligo will be lost during the cleaning step and no discoloration will be observed.

The inventors found that the yellow product contained hexahistidine tagged NF-κB p50 crude lysate and digoxigenin labeled oligonucleotides and was detected only in microwells to which an antidigoxigenin antibody-alkaline phosphate conjugate was added. It was. This is due to the fact that the discoloration observed is due in particular to the immobilized NF-κB p50-oligonucleotide complex and that NF-κB p50 retains activity against specific immobilization.

Example 4

(a) Identification of 1 protein from 10 gene pools

We performed this method in the same manner as described in Example 1, except that it was specified with 10 different gene pools listed in the table below. We have produced an array of product proteins that have been specifically modified so that each position in the array corresponds to a single recombinant protein immobilized via an attached tag as a result of the method. The inventors then screened this array by functional assay to successfully identify each component of the protein of the pool.

Size and function of 10 genes in the pool gene Size (bp) Source and function Glutathione S Transferase 950 Germ ; Detoxification NF-κB p50 1165 human ; Transcription factor Maltose binding protein 1325 Germ ; Carbohydrate Transport Alanine racemase 1342 Germ ; Cell wall biosynthesis Nuclear Factor (NFAT) in Activated T Cells 1087 Rat animal; Transcription factor Indoleglycerol phosphate synthase 1528 Germ ; Amino acid biosynthesis Phosphoribosylanthranilate isomerase 920 Germ ; Amino acid biosynthesis Tryptophan synthase (α-subunit) 1122 Germ ; Amino acid biosynthesis Chymotrypsin inhibitor 2 389 barley ; Serine Protease Inhibitors β-lactamase 1040 Germ ; Antibiotic resistance

Initially, all 10 genes were subcloned into the framework of the same pTrcHisA vector because, above all, to simulate the situation faced by the cDNA library. In order to amplify the genes encoded in the framework of the pTrcHisA vector, the primers 'ST forward' and 'ST reverse' described in Example 1 were designed to make universal primers.

Primer 'ST forward' is designed to encode multiple restriction positions as follows.

Therefore, either restriction enzyme AatII or SfiI produced 3′-protrusions for exonuclease protection purposes. To orientally clone in the last step of the modification method, in the present example the inventors produced sticky ends that matched the NcoI cloning position of the tag vector pMM106H used in this experiment, despite statistically more frequent cleavage in the library. And none of the 11 genes in the pool of this example were cleaved, the method using Bsp HI was chosen. Essentially any of the primers encoding restriction positions could be used to provide a tag vector containing the same cloning position downstream of the promoter; In this case, SfiI has a recognition sequence of 8 bp so that the frequency of randomly generating SfiI positions in a given gene is lower than that of 6 bp (1 / 4,096), which is the same as that of BspHI (6.5). In this respect, SfiI has a significant advantage when used in a larger library format, since it will be 10 × ^1/4 ). The tag vector pMM106H is a 'ATG' vector, i.e., a vector that overlaps the 5'-cloning position (NcoI) with an ATG start codon located downstream of the ribosomal binding site (RBS) to express a native protein. However, in the methods described herein, the inventors do not use cloned genes that carry restriction sites common to the start codons. Instead, we use a vector coding promoter that initiates transcription to produce an mRNA that carries the signal necessary for initiation of translation provided by the cloned genes themselves. Therefore, in this example, all genes present in the original pool retain the initiation codon immediately preceded by the RBS, whether or not there is a cloning position in the ATG. Since the primer 'ST forward' binds upstream of RBS in all 11 initial clones, subsequent post-cloning cloning using any of the restriction sites that the primer encodes will result in the newly modified genes being combined with these original RBS and ATG. Together they will be introduced into the tag vector to ensure translation. In the cDNA library format, similar conditions apply in that every full length cDNA will have its own 5'-untranslated region (UTR) containing the eukaryotic translation initiation signal. All conditions necessary for proper translation to be initiated are such that each modified cDNA along with its 5'-UTR provides a transcription initiation signal to a single port in a sequence dependent manner for a broad set of PCR primers equivalent to those used in this example. Cloning into eukaryotic vectors that can be used to modify each cDNA library member.

The experimental method was performed as described in Example 1 with the following modifications. As a template for initial PCR amplification using primers' ST forward 'and' ST reverse ', a molar pool equivalent to all 10 genes was used and these fragments were cut with AatII to protect the 5' terminus. Exonuclease Ⅲ and mung bean nuclease were cut to claim was performed identically produced as in Example 1, the processed short after BspHI, the BspHI by restriction fragment present in a specific only in the forward PCR primer binding site in vector pMM106H To produce sticky ends for cloning. The resulting fragments were gel purified and ligated to the vector. Transformed cells were visualized under UV light (365 nm) and colonies emitting green fluorescence were visually selected for analysis by Western blot. About 2% of the total number of transformed colonies emitted green fluorescence. Of these, 103 (42%) colonies expressed proteins recognized by anti His tag antibodies. The colonies were inoculated in 1.5 ml of culture solution contained in blocks containing 96 deep wells each and then grown overnight. Cells were collected by centrifugation and then lysed by freeze-thaw / lysozyme. Each crude lysate was then placed in each well of a nickel-NTA-coated 96-well plate and washed to remove unbound proteins, leaving each His tagged recombinant protein immobilized in each well. It became. The immobilized proteins were then analyzed for GST or NF-κB activity using the assays described in Examples 1 and 3, containing positive 'hits' in each case, whether green or yellow. Wells were confirmed.

In the first assay, three proteins in the array were found to exhibit GST activity (about 3% hit ratio). Sequencing of the plasmids revealed that the three genes were in-frame fusions between the GST gene, the hexahistidine tag, and the GFP gene (two of these three fusions were full-length GST and the other had little effect on activity) You can see that it encrypts the chain).

In the second assay, three proteins in the array showed positive 'κB -motif' DNA binding activity. Further characterization of the positive clones revealed that two of the clones were in-frame fusions of the NF-κB p50 gene to the hexahistidine tag, one of which was nearly full length (one amino acid cleaved). On the other hand, it was found that the other was more cleaved but contained the entire DNA binding domain of NF-κB p50. Interestingly, since the assay is designed to bind to cognate DNA sequences, the cleavage of the still, intact, folded and functional DNA binding domain will appear positive in this assay. The third clone was found to fuse in frame the DNA binding domain of murine animal transcription factor NFAT to His tag. The 3'-digoxigenin labeled 'κB motif' used in this assay contains a specific, high affinity (Kd is nearly pM) binding site for NF-κB p50, but this same binding site is also nearly It is specifically recognized by the DNA binding domain of NFAT with an affinity that is nM. The results thus explain that functional interrogation of the arrays produced by the present method can identify specific interactions and weaker interactions that are specific but biologically relevant. In subsequent experiments, an array containing about 340 His-tagged proteins was prepared according to the method of this example. Analysis of the array by GST activity analysis revealed that 8% of all proteins in the array possess strong GST activity. In addition, PCR analysis was performed on a pool of 340 coding plasmid DNA using gene specific primers, and the results showed that each gene appeared in His-tagged collections. Thus, the data further confirmed that the method of this example is sequence dependent and can be used to collect different genes.

Therefore, in summary, we can prepare an array of functional proteins in a microwell format using the methods described in the above examples, and we use this array to identify specific protein-ligand interactions (GST activity assays). ) Or three different proteins from the pool can be successfully identified based on specific protein-DNA interaction (NF-κB binding assay).

[Sequence list]

Claims (41)

A method of making one or more proteins in which one or more domains are full length, correctly folded and each tagged with one or more marker moieties at either the N-terminus or the C-terminus, (a) providing one or more DNA molecules having an open translation frame encoding said protein and a 5 'and / or 3' untranslated region; (b) amplifying the DNA molecule under conditions that statistically incorporate α-S-dNTP and dNTP into the daughter DNA molecule; (c) specifically protecting the 5 'or 3' end of the DNA molecule from nuclease degradation; (d) treating the DNA molecule with 5 '→ 3' nuclease or 3 '→ 5' nuclease to form a set of nested deletions, followed by initiation of the open reading frame Treating with a single chain nuclease under conditions that remove the 5 'or 3' untranslated region comprising the codon or the stop codon; (e) cloning the fragment produced by step (d) into an expression vector containing coding sequences for at least one 5 'or 3' marker moiety; And (f) expressing the encoded proteins How to include. The method of claim 1, wherein a single α-S-dNTP is statistically incorporated by amplifying the DNA molecule. The method of claim 2, wherein the single α-S-dNTP is α-S-dTTP or α-S-dATP. 4. The method of claim 1, wherein the nuclease is an exonuclease III or a λ exonuclease. 5. The method of claim 1, wherein the single chain nuclease is mung bean nuclease or T4 DNA polymerase. The method of any one of claims 1 to 5, wherein the marker portion confirms the expression of the open reading frame. 6. The method of any one of claims 1 to 5, wherein the marker portion confirms folding of the open reading frame. 8. The method of any one of claims 1 to 7, wherein the marker portion encodes a green fluorescent protein. 8. The method of claim 1, wherein the marker moiety is a peptide sequence (eg, hexa-histidine tag), a complete protein or a protein domain (eg, maltose binding protein domain). 9. The method of claim 9, wherein the tag purifies each protein in the array. The method of claim 1, wherein the tag is inserted to replace the start codon or stop codon for each protein. The method of claim 1, wherein the tag is inserted in-frame in a region adjacent to each distal end of the protein, which is not critical for folding and function. 11. The method of any one of claims 1 to 10, wherein the tag is inserted into a frame within an open decrypted frame, but outside of a particular domain boundary that is not critical for folding and functioning. The method according to any one of claims 1 to 13, wherein the amplification of the DNA molecule obtained in step (a) is performed by a non-proof-reading polymerase (eg, Taq polymerase) or DNA polymerase I. The method performed by the Klenow fragment. The method of claim 1, wherein the ratio of α-S-dNTP to dNTP is 1: 1 to 1: 3. The method of claim 1, wherein the 5 ′ → 3 ′ or 3 ′ → 5 ′ nuclease is unable to hydrolyze α-S-phosphodiester bonds. The method of any one of claims 1 to 16, wherein the DNA molecule is cDNA synthesized by reverse transcription from an mRNA sequence. 18. The method of any one of claims 1 to 17, wherein the method is performed simultaneously on a plurality of DNA molecules. 19. The method of any one of the preceding claims, wherein the method is performed on a population of DNA molecules in a single pot. 20. A library of tagged proteins synthesized by the method of any one of claims 1-19. (a) separating each member of the library of claim 20 by a clone; (b) expressing each tagged protein in a spatially isolated format; (c) purifying each tagged protein by a marker portion; And (d) placing each protein in a spatially limited array Method for producing a protein array comprising a. An array comprising a protein prepared by the method as defined in claim 1 or prepared by the method as defined in claim 21. The array of claim 22, wherein the components of the array are immobilized, for example, on a solid surface. The array of claim 22 or 23, wherein each protein is immobilized by a tag portion. At least one compound for biological activity, comprising contacting the at least one compound with a protein array as defined in any one of claims 22 to 24 and determining whether one or more compounds are bound to the proteins in the array. How to screen. 25. A method comprising: contacting one or more proteins, eg, cell surface receptors, with an array as defined in any one of claims 22-24, and determining the binding of one or more specific proteins to the proteins in the array. , A method of screening one or more proteins for specific protein-protein interactions. A method for a specific protein-nucleic acid interaction comprising contacting at least one nucleic acid probe with an array as defined in any one of claims 22 to 24 and determining whether the probe binds to a protein in the array. Method of screening one or more proteins. Use of an array as defined in any one of claims 22 to 24 in high speed screening of compounds, proteins or nucleic acids. Use of an array as defined in any one of claims 22 to 24 in the screening of molecules that recognize each protein in the array, preferably antibodies. Contacting an antibody library with a protein array as defined in any one of claims 22 to 24 so that at least one protein in the protein array is bound with at least one antibody in the antibody library, removing any unbound antibodies, and A method of making an antibody array, comprising immobilizing antibodies bound to a protein in the protein array. A method for screening protein function or abundance comprising contacting an antibody array as defined in claim 30 with a mixture of one or more proteins. 31. The method of any one of claims 25 to 27 or 30, comprising first providing a protein array as defined in any one of claims 22 to 24. The method of claim 21, wherein the proteins in the array are purified and immobilized in one step. Use of tagged proteins prepared by the methods defined herein. Use of a tagged protein prepared by the method defined herein in analyzing the interaction between the expressed protein and other proteins. Tagging prepared by the method as defined herein in immobilization on an affinity column / substrate, e.g. to purify a) interactive protein, b) DNA or c) chemical compound by affinity chromatography. For using purified protein. Use of a tagged protein produced by the method defined herein in immobilization by affinity purification (ELISA assay) for interrogation by antibody as diagnostic means. Use of tagged proteins prepared by the methods defined herein as probes for cDNA microarrays. Use as defined in claim 38 in the identification of DNA binding proteins. Use of tagged proteins prepared by the methods defined herein in identifying proteins in the 'proteome'. 41. The use of claim 40 in performing mass spectrometric analysis of expressed protein components of a source library or starting materials modified by the methods of the invention.