Patent Application
20070178460
The present invention is related to the field of methods for detecting the interaction of proteins via the use of fusion proteins, commonly referred to as split-protein sensors or two-hybrid assays.
The introduction of the yeast-two hybrid system by Fields and Song in 1989 was a milestone for the analysis of protein-protein interactions in living cells (cf. U.S. Pat. No. 5,667,973 and Fields, S., and Song, O. (1989), Nature 340, 245-246). However, a major limitation of this classical two-hybrid system lies in its restriction to the detection of those protein-protein interactions that can be reproduced within the nucleus of a yeast cell. To overcome this restriction, an alternative to this two-hybrid method was introduced in 1994 by Johnsson and Varshavsky (cf. WO 95/29195 and Johnsson, N., and Varshavsky, A. (1994), Proc Natl Acad Sci USA 91, 10340-10344). Here, the two interacting proteins are expressed as fusion proteins with an N- and a C-terminal fragment of ubiquitin. Upon interaction of the two proteins a quasi-native ubiquitin is formed and subsequently recognized by ubiquitin-specific proteases, resulting in the cleavage of a reporter protein from the C-terminal fragment of ubiquitin. The split-ubiquitin system allows for the detection of interactions between cytoplasmic as well as membrane proteins. Since the introduction of split-ubiquitin, a variety of other split-protein sensors has been developed, including pairs of fragments of dihydrofolate reductase (DHFR), β-galactosidase, β-lactamase, inteins, green fluorescent protein (GFP), cAMP cyclase, glycinamide ribonucleotide transformylase, aminoglycoside phosphotransferase, hygromycin B phosphotransferase, and luciferase (cf. Remy, I., and Michnick, S. W. (1999), Proc Natl Acad Sci USA 96, 5394-5399; Rossi, F., Charlton, C. A., and Blau, H. M. (1997), Proc Natl Acad Sci USA 94, 8405-8410; Galarneau, A., Primeau, M., Trudeau, L. E., and Michnick, S. W. (2002), Nat Biotechnol 20, 619-622; Wehrman, T., Kleaveland, B., Her, J. H., Balint, R. F., and Blau, H. M. (2002), Proc Natl Acad Sci USA 99, 3469-3474; Ozawa, T., Nogami, S., Sato, M., Ohya, Y., and Umezawa, Y. (2000), Anal Chem 72, 5151-5157; Ozawa, T., Kaihara, A., Sato, M., Tachihara, K., and Umezawa, Y. (2001), Anal Chem 73, 2516-2521; Ghosh, I., Hamilton, A. D., and Regan, L. (2000), Journal of the American Chemical Society 122, 5658-5659). Among these systems only split-ubiquitin was successfully applied to screen for binding partners. Other sensors were used to monitor the interactions between selected pairs of proteins rather than to find new partners by a random library approach. Robust systems that can be used for identifying interaction partners at any location inside the cell and in different hosts are therefore still needed. Ideally the interaction-induced reassociation of such a split-protein sensor would provide the cell with a growth advantage thus allowing a selection for interacting proteins. However, generating new split-protein sensors is technically demanding as it depends critically on identifying suitable fragments that can reconstitute a native-like and active protein. The chosen fragmentation site has to fulfill at least the following criteria: (i) to yield two fragments that efficiently fold into quasi-native protein only when fused to two interacting proteins; (ii) not to significantly impair the activity of the reconstituted protein; (iii) to yield soluble protein fragments that are not readily degraded in vivo. In previous studies, the challenge of rationally finding such sites has been mostly tackled by trial and error.
It is thus an object of the present invention to overcome the above-mentioned drawbacks of the prior art, i.e. to provide a method for identification of suitable fragmentation sites in a reporter protein especially for use as a split-protein sensor, that is not limited by the above-mentioned drawbacks of rational design, and which especially allows for the identification of suitable fragmentation sites in a reporter protein even in the absence of any structural information such as a crystal structure. Further objects of the invention will become apparent to the person of routine skill in the art in view of the following detailed description of the invention.
This object and yet further objects are achieved inter alia by a method for the identification of suitable fragmentation sites in a reporter protein, and related thereto, recombinant DNA sequences and, encoded thereby, first and complementary second subdomains of a reporter protein, host cell lines transformed with said recombinant DNA sequences, a kit of parts comprising DNA-based expression vectors, a method for detecting an interaction between proteins, a use of random circular permutation and a use of a host cell line allowing for homologous recombination according to the independent claims.
Most biological processes are controlled by protein-protein interactions and split-protein sensors have become one of the few available tools for the characterization and identification of protein-protein interactions in living cells. Here we introduce a generally applicable combinatorial approach for the generation of new split-protein sensors and apply it to the (β/α)8-barrel enzyme N-(5′-phosphoribosyl)-anthranilate isomerase Trp1p from Saccharomyces cerevisiae (cf. Braus, G. H., Luger, K., Paravicini, G., Schmidheini, T., Kirschner, K., and Hutter, R. (1988), J Biol Chem 263, 7868-7875). These so-called split-Trp protein sensors are capable of monitoring the interactions of pairs of cytosolic and membrane proteins. One of the selected split-Trp pairs (44Ntrp and 44Ctrp) was chosen by means of an example and successfully applied to monitor protein-protein interactions both at the membrane as well as in the cytosol of yeast. Its selected fragmentation site would not have been easily predicted by theoretical considerations, thus underlining the power of the evolutionary approach according to the invention. The direct read-out through complementation of tryptophan auxotrophy qualifies the split-Trp system for high-throughput applications in yeast and bacteria. Of course, appropriately engineered trp1-deficient host strains are required for such assays, which are however either readily available or easily to be made by the person of routine skill in the art. In addition, the introduced combinatorial approach allows for generating split-protein sensors of almost any reporter protein, thereby yielding tailor-made sensors for different applications.
Trp1p is a relatively small (25 kD), monomeric protein that catalyzes the isomerization of N-(5′-phosphoribosyl)-anthranilate in the biosynthesis of tryptophan (cf. Eberhard, M., Tsai-Pflugfelder, M., Bolewska, K., Hommel, U., and Kirschner, K. (1995), Biochemistry 34, 5419-5428). The DNA coding sequence of Saccharomyces cerevisiae is given in SEQ ID NO: 1, the corresponding amino acid sequence is given in SEQ ID NO: 2. Creating a pair of Trp1p fragments (split-Trp) that only reconstitute the enzymatic activity when linked to interacting proteins allows monitoring this protein interaction through a simple growth assay: otherwise trp1 yeast strains expressing such a split-Trp fusion pair would not be able to grow on medium lacking tryptophan. As many different trp1 strains exist, the interaction assay could be applied immediately in different genetic backgrounds, adding a further attractive feature to a split-Trp sensor. Trp1p is a well-studied member of the prominent class of proteins that fold into a (β/α)8-barrel, which is the most commonly occurring fold among enzymes. The herein presented approach of identifying suitable fragmentation sites in a reporter protein is thus very broadly applicable. This folding motive has been previously subjected to circular permutation and has been expressed as two separate fragments that spontaneously associate into a functional enzyme (cf. Luger, K., Hommel, U., Herold, M., Hofsteenge, J., and Kirschner, K. (1989), Science 243, 206-210; Eder, J., and Kirschner, K. (1992), Biochemistry 31, 3617-3625). Furthermore, it has been proposed that the (β/α)8-barrel evolved by tandem duplication from a (β/α)4-domain (cf. Hocker, B., Schmidt, S., and Sterner, R. (2002), FEBS Lett 510, 133-135). In addition to any practical applications it would therefore add to our understanding where the (β/α)8-barrel can be split into two fragments that, in contrast to previously described pairs of fragments, reconstitute quasi-native Trp1p only when fused to interacting proteins.
As used herein, a “reporter protein” is understood as a protein or peptide, which possesses a unique activity in vivo and/or in vitro, and which produces a signal that allows the active protein to be easily discernable even within a complex mixture of other proteins or peptides, especially in vivo. Reporter proteins as understood herein are e.g. (i) proteins which are essentially involved in the biosynthetic pathway of formation of an amino acid or an other essential metabolite that is crucial for the organism to survive on medium lacking the respective amino acid or metabolite; or (ii) proteins which are detectable by a characteristic color assay when, preferably in vivo; etc.
As used herein, a “suitable fragmentation site” is understood as an especially randomly chosen position in the amino acid chain (and/or the corresponding gene sequence, respectively), at which a given reporter protein is fragmented into a first subdomain and a complementary second subdomain (and/or the corresponding first subsequence and the complementary second subsequence, respectively), wherein the fragmentation site is suitable in the sense of the present invention, when it fulfils the following demands: (i) to yield two fragments that efficiently fold into quasi-native protein only when fused to two interacting proteins; (ii) not to significantly impair the activity of a reconstituted protein by bringing the two fragments into close proximity especially in vivo; (iii) to yield soluble protein fragments that are not readily degraded in vivo.
As used herein, the term “detectable”, especially “detectable when active” is understood as follows. Detection in the sense of the present invention includes any direct or indirect method of testing for the presence of a reporter protein, especially when reconstituted by fragments thereof, e.g. by chemical, physical, or visual means. Most preferably, detection is performed by a color assay, e.g. fluorescence, chemiluminescence or the like, (in vivo and/or in vitro) and/or a growth assay (in vivo)
As used herein, a “first subdomain” and a “complementary second subdomain” of a reporter protein are understood as follows. A first subdomain represents a first successional part (either an N-terminal-, C-terminal-, integral part or even a part involving both the N-terminal- and the C-terminal part) of a native reporter protein. A complementary second subdomain represents a complementary second part (either an N-terminal, C-terminal, integral part or even a part involving both the N-terminal- and the C-terminal part). The first subdomain and the complementary second subdomain essentially resemble the wild-type sequence, when viewed together, wherein overlapping sequences between both subdomains, that are present in both the first subdomain and the complementary second subdomain can be tolerated as long as the activity of the enzyme is not significantly negatively affected. Moreover, minor deletions, additions or other alterations to the overall sequence can be tolerated, especially at the N-terminus or the C-terminus, as long as the activity of the reporter protein, either as a whole or when reconstituted by its fragments, is not significantly negatively affected.
As used herein, a “first subsequence” and a “complementary second subsequence” are understood as gene sequences encoding for the above-mentioned first subdomain and complementary second subdomain.
As used herein, a “color assay” is understood as a manually or device-supported detection of a change in optical appearance of a sample comprising the reporter protein, or a reporter protein reconstituted by its fragments, inc1. color developments as well in the visible as in the invisible spectrum. Color assays are especially preferred, that can be qualitatively detected by the unaided eye e.g. by coloration of living cells in vivo (colonies on a plate or the like), and that can be additionally quantified in an in vitro assay, e.g. for determining the intensity of an interaction between two proteins.
As used herein, a “growth assay” is understood as an assay, that allows for the growth of a cell, e.g. a colony on a plate, when the reporter protein is present or actively resembled by its fragments, and wherein cells fail to grow, when the reporter protein is not present or actively resembled by its fragments. Most preferably, the growth assay suchlike allows for a simple visual selection of positives.
As used herein, “stringent conditions” for hybridization of DNA are understood as follows. Given a specific DNA sequence, a person of skill in the art would not expect substantial variation among species within the claimed genus due to hybridization under such conditions, thus expecting structurally similar DNA.
The method according to the invention for the identification of suitable fragmentation sites in a reporter protein, wherein the reporter protein is detectable when active, comprises the steps of:
By using a combinatorial library approach, comprising randomly created first subsequences and randomly created complementary second subsequences, the drawbacks of rational design of split-protein sensors are overcome. Most advantageously, even fragmentation sites of proteins encoded by said subsequences may thereby be identified, which would have never been readily predicted by any rational approach. First subsequences and complementary subsequences are ideally suitable in the context of the present invention, when reconstitution of activity of the corresponding reporter protein only occurs to a significant extent at all, when both corresponding subdomains are forced into close spatial proximity, but do not self-assemble in order to reconstitute a detectable amount of an active reporter protein.
DNA sequences of suitable reporter proteins are readily available to the person of routine skill in the art (step (a)), e.g. from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Bethesda, Md. 20894. Genes encoding for reporter proteins may then be amplified e.g. from a suitable host cell by PCR using standard techniques and primers suitably designed based on the known DNA sequence (vide supra), or the gene encoding for a reporter protein may be completely built up from suitably designed oligonucleotides de novo.
DNA manipulating techniques that may be used in step (b) for the creation of a library based on said DNA sequence are readily apparent to the person of routine skill in the art, either. In short, N- and C-terminal domains of the wild-type reporter protein are amplified separately from a suitable source of DNA by standard PCR techniques, and are subsequently recombined using standard overlap extension PCR techniques in order to recombine and thereby re-arrange the wild-type gene, preferably now containing the N- and C-termini of the wild-type gene connected with each other and as an internal part of the sequence, and preferably comprising a unique restriction site at the wild-type N- and C-termini. At the same time, suitable restriction sites may be designed at the newly created N- and C-termini in order to allow for efficient subsequent cloning steps; most preferably, the restriction site is designed for the same restriction enzyme at both the N- and C-terminus. Most preferably, the rearranged DNA construct is inserted into a high-copy plasmid, the plasmid amplified by standard techniques, and the re-arranged DNA of interest is thereafter cut out of the high-copy plasmid using the restriction sites at the newly created N- and C-termini. The rearranged gene is then incubated with a ligase to yield dimerized, oligomerized and circularized DNA construct. Afterwards, these constructs are digested e.g. with a suitable, random-cut DNAse, and fragments corresponding to the wild-type length are preferably thereafter treated with ligase and polymerase to repair nicks, gaps and to flush the ends of the fragments of the reporter protein. Afterwards, the DNA fragments corresponding to the wild-type length of the reporter protein's gene are isolated e.g. by standard agarose gel electrophoresis procedures. The resulting fragments are preferably blunt-end cloned into a suitable expression vector, which was cleaved at a unique restriction site (preferably blunt-end). The expression vector is especially designed by standard DNA manipulation techniques to provide a construct after blunt-end cloning, in which one of the artificially generated new N- and C-termini is under the control of a promoter sequence and especially fused to a gene encoding for a tag sequence and a gene encoding for first peptide or protein C1, each preferably via a linker sequence. Moreover, the other terminus, respectively, is especially fused to a gene encoding for a preferably different tag sequence and gene encoding for a second peptide or protein C2. Peptides or proteins C1 and C2 are thereby known to interact with each other in vivo, and may e.g. be leucine zippers. The tag sequences may afterwards advantageously be used for the control of correct expression and stability of fusion proteins. After transformation and amplification in a suitable host such as e.g. E. coli XL1Blue to a typical library size of about 104 to 105 independent clones, the vector is linearized at a restriction site at the wild-type N- and C-termini, and an oligonucleotide is inserted into the resulting gap, which is specifically designed to integrate a terminator for the first domain of said reporter protein and a promoter sequence for the second domain of said reporter protein, by homologous recombination in a suitable host such as yeast according to standard procedures. The oligonucleotide is designed and constructed by standard PCR techniques to provide flanking regions both at the 5′ and 3′ ends of e.g. about 50 bp with the gene of the reporter protein in order to allow for successful homologous recombination. Suchlike, the selection of clones possessing fragmentation sites at or nearby the wild-type N- and C-termini can be suppressed. For selecting thereafter, a marker gene is also provided by the oligonucleotide, e.g. encoding for a protein involved in antibiotic resistance. Successful homologous recombination may thus be easily observed by growth in the presence of the respective antibiotic.
Step (c) is preferably carried out by growing the respective transformants of the library on medium which e.g. lacks a nutrient, e.g. an amino acid, or which provides a substrate for a color reaction. Thus, preferably a growth assay or a color assay is performed, thereby allowing for easy selection of those transformants which lead to a restoration of activity of the reporter protein, which is e.g. essentially involved in the synthesis of said nutrient, e.g. said amino acid, or in said color reaction. Step (c) especially involves the elimination of false positives, i.e. first subdomains and complementary second subdomains, that reconstitute an active reporter enzyme by self-reassembling, i.e. without the need of an outer influence forcing the two domains into close spatial proximity. This can be done e.g. by fusing the respective first and second subdomains of the reporter protein to first and second peptides or proteins, that do not interact with each other, and/or by testing the respective first and second subdomains without any first and second peptides fused thereto at all, and/or by testing constructs lacking the first or the second subdomain, respectively. These assays can be performed by techniques commonly known in the art of e.g. two-hybrid assays.
Identification of suitable subdomains and subsequences, i.e. suitable fragementation sites, can be performed by common DNA-and/or protein sequencing techniques.
According to a preferred embodiment, the reporter protein is detectable in vivo and/or in vitro, both as full length protein and when actively resembled by a first subdomain and a complementary second subdomain, by a means chosen from the group consisting of color assays and growth assays.
Growth assays provide the advantage of a selection step, i.e. only positives grow under the chosen conditions, thus eliminating the need of further screening all individuals of the library. Exemplarily, only positives that comprise a suitable combination of first subdomain and complementary second subdomain grow as colonies on nutrition-specific plates. Color assays, moreover, can be individually designed depending on the specific reporter protein, when this reporter protein is involved naturally in or artificially usable for a color-developing reaction. In some cases, a substrate for such a reporter protein may be incorporated into the growth medium, e.g. the plate, whereupon colored colonies appear due to reconstitution of an active reporter protein by a first subdomain and a complementary second subdomain in vivo. Quanification of such an in vivo color assay may be optionally performed with samples obtained from such colonies. The general procedure of growth assays, color assays and subsequent quantification of the color assay are known in principle from the classical two-hybrid system, cf. eg. U.S. Pat. No. 5,667,973, incorporated herein by reference.
In an especially preferred embodiment, individuals of the library as defined in (b) are either prokaryotic or eukaryotic host cells, comprising:
In vivo assays are at least in the first step preferred, e.g. as a growth assay as outlined above. Thus, prokaryotic or eukaryotic host cells are provided, that are manipulated suchlike to allow for the (co-)expression of both the first and the complementary second subdomain of the reporter protein. Depending on the specific application, both subdomains may of course be encoded by one and the same, or by separate vectors. In most cases, encoding by one and the same vector will be favourable. A vast amount of suitable expression vectors for use as a basis in this respect are available to the person of routine skill in the art, e.g. the pRS316-based yeast expression vector (cf. Sikorski, R. S., and Hieter, P. (1989), Genetics 122, 19-27, incorporated herein by reference).
It is especially preferred that the screening for restoration of detectable activity of said reporter protein, when said first subdomain and said complementary second subdomain are brought into close proximity as defined in (c), comprises the following steps:
By creating said first fusion sequence and said second fusion subsequence, the first subdomain and the complementary second subdomain are forced into close spatial proximity, thus allowing for a screening for restoration of activity of the reporter protein, when the subdomains are forced into close proximity. Preferably, said first protein or peptide and said second protein or peptide are chosen to be robust and relatively small proteins or peptides; especially preferred in the context of the invention are leucine zippers, most preferably leucine zippers which associate to an anti-parallel coiled coil (interacting proteins fused to 3′-terminus of the first subdomain and the 5′-terminus of the second subdomain, or vice versa, respectively). However, for specific embodiments, a parallel orientation may be preferred, e.g. for testing membrane proteins which most commonly exhibit both the N- and the C-terminus to one and the same site.
According to a further embodiment said first fusion subsequence and said second subsequence are created by blunt end ligation.
Blunt end ligation is the method of choice for the construction of said fusion subsequences, as due to the evolutionary, random approach of library generation no predictable, specific sticky-end ligation can be performed. Although blunt-end ligation leads to the creation of statistical amounts of ligation products which are out of the reading frame, this approach still proved sufficiently efficient for the identification of suitable fragmentation sites according to the invention.
Moreover, in another especially preferred embodiment said first fusion subsequence and said second fusion subsequence each comprise
Linker sequences commonly prove useful in the art of construction of fusion proteins in order to both allow for proper folding of both components of the fusion protein individually or cooperatively, and/or to achieve sufficient spatial integrity of both components of the fusion protein.
The use of tag sequences that allow for the detection of transcription of a gene sequence is also routinely applied in the art. In the context of the present invention, tag sequences may be applied to any of the N- and C-terminus of the first subdomain and/or the N- and C-terminus of the complementary second subdomain. It is especially preferred to provide differently recognizable tag sequences both at the N- and the C-termini of each transcription product. Cornmonly applied tags are e.g. the HA tag, the flag tag or the like. Detection of correct expression of these tags, and thereby of the fusion protein(s), may be performed e.g. by Western-blotting according to routine procedures.
According to an especially preferred embodiment, an oligonucleotide is inserted by homologous recombination in between said first subsequence and said second subsequence, encoding for:
An especially advantageous way of carrying out the present invention is to simply initially provide said first and said second subsequence continuously, preferably rearranged, and thereafter to separate them by introducing a transcription terminating sequence succeeding the first subsequence, and a transcription promoting sequence preceeding the second subsequence. Thereby, separate expression is secured of both the first subdomain and the complementary second subdomain, or their fusion domains, respectively. This goal may be especially advantageously achieved by homologous recombination at a predefined site in between said first and said second subsequence (c.f. Oldenburg, K. R., Vo, K. T., Michaelis, S., and Paddon, C. (1997), Nucleic Acids Res 25, 451-452, incorporated herein by reference).
In order to eliminate the otherwise high risk of isolating subdomains, that are fragmented at fragmentation sites nearby the N- and C-termini of the wild-type reporter protein, it is especially preferred to not provide the DNA sequence of said reporter protein according to step (a), vide supra, in its wild-type configuration, but rather already with the wild-type N- and C-termini connected with each other and being an internal part of the DNA sequence of said DNA sequence. Thereby, artificial new N- and C-termini are created in the starting material. Most preferably, a unique restriction site RE2 is introduced in between the wild-type N- and C-terminus. A further restriction site RE1 is advantageously introduced at the new artificial N- and C-terminus of the DNA sequence of said reporter protein according to step (a), allowing for easy and convenient cloning and construction of libraries according to step (b), vide supra. Due to the unique restriction site RE2, homologous recombination in a suitable host cell can be performed in between the wild-type N- and C-terminus of the reporter protein. Due to the necessary overlap for successful homologous recombination, isolation of subdomains with fragmentation sites at or nearby the wild-type N- and C-terminus is suppressed. Most preferably, the oligonucleotide used for homologous recombination comprises a selection marker such as e.g a gene involved in antibiotic resistance in order to check for successful homologous recombination.
Thus, in a further embodiment, the method comprises the steps of:
The invention further relates to a recombinant DNA sequence for use in securing expression in a prokaryotic or eukaryotic host cell of a polypeptide product having the primary structural conformation of a first subdomain of a reporter protein or a complementary second subdomain of a reporter protein, wherein detectable activity of said reporter protein is restored, when said first subdomain and said complementary second subdomain are brought into close proximity, and wherein said first and said complementary second subdomain are not subdomains of one of the group of proteins consisting of transcriptional activators, ubiquitin, dihydrofolate reductase, β-lactamase, green fluorescent protein and closely related variants such as e.g. ECFP, EGFP or the like, β-galactosidase, inteins, cAMP cyclase, glycinamide ribonucleotide transformylase, aminoglycoside
In the above-mentioned and herewith disclaimed DNA sequences, suitable fragmentation sites for split-protein sensors were already identified by rational design (cf. e.g. Methods Enzymology 238, Michnick et al. 2000). However, the present invention now opens up for the first time the possibility to identify suitable fragmentation sites in any other DNA sequence encoding for a reporter protein by a random library approach, too. Providing this tool to the person of routine skill in the art by the method disclosed herein, suitable fragmentation sites may be now identified with relative ease.
In especially preferred embodiments, said DNA sequence encodes for a subdomain of a (β/α)8-barrel enzyme, such as e.g. Trp1p.
In further embodiments, which proved especially advantageous, said DNA sequence is selected from the group consisting of:
The above-mentioned DNA sequences encode for the split-Trp sensors split-Trp44 (i.e. 44Ntrp and 44Ctrp), split-Trp53 (i.e. 53Ntrp and 53Ctrp), split-Trp187 (i.e. 187Ntrp and 187Ctrp), split-Trp204b (i.e. 204bNtrp and 204bCtrp), which proved to be valuable tools as split-protein sensors (numbering according to the fragmentation site, given as the last amino acid of the N-terminal subdomain). Especially split-Trp44 was successfully applied herein to demonstrate the interaction of membrane proteins.
The DNA- and amino acid sequences of the above-mentioned split-Trp sensors are given in the attached sequenced listing as follows:
In preferred embodiments according to the present invention, said DNA sequences are used in securing expression in a prokaryotic or eukaryotic host cell of a polypeptide fusion product. Such securing of expression may be achieved by any means routinely applied by the person of routine skill in the art, comprising e.g. incorporation of said DNA sequences into suitable expression vectors or integration of said DNA sequences into the genome of said host.
The invention further relates to a first subdomain of a reporter protein or a complementary second subdomain of a reporter protein, wherein detectable activity of said reporter protein is restored, when said first subdomain and said complementary second subdomain are brought into close proximity, and wherein said first and said complementary second subdomain are not subdomains of one of the group of proteins consisting of transcriptional activators, ubiquitin, dihydrofolate reductase, β-lactamase, green fluorescent protein and closely related variants such as e.g. ECFP, EGFP or the like, β-galactosidase, inteins, cAMP cyclase, glycinamide ribonucleotide transformylase, aminoglycoside phosphotransferase, hygromycin B phosphotransferase, luciferase.
In the above-mentioned and herewith disclaimed proteins, suitable fragmentation sites for split-protein sensors were already identified by rational design. However, the present invention now opens up for the first time the possibility to identify suitable fragmentation sites in any other reporter protein by a random library approach, too. Providing this tool to the person of routine skill in the art by the method disclosed herein, suitable fragmentation sites may be now identified with relative ease.
According to especially preferred embodiments of the invention, a first subdomain of a reporter protein or a complementary second subdomain of a reporter protein are produced by a method of culturing a host transformed with a recombinant DNA sequence as outlined above, wherein said molecules further comprises an expression control sequence, said expression control sequence being operatively linked to said molecule. Said expression control sequences comprise especially those which are commonly referred to as tags which are recognizable e.g. by Western-blotting procedures routinely applied in the art.
The invention further relates to a fusion protein comprising a first subdomain of a reporter protein or a complementary second subdomain of a reporter protein as outlined above, and a further peptide or protein connected thereto in a naturally not occurring combination. By creating such artificial fusion proteins, said further protein of peptide may then be tested for interaction with e.g. a specifically chosen counterpart or against a library of possible counterparts. Moreover, library-library screening assays may also be applied, e.g. genome-wide library screenings as e.g. already performed in the art of traditional two-hybrid assay.
The invention further relates to a prokaryotic or eukaryotic host cell line, transformed with recombinant DNA sequences as outlined above.
Said prokaryotic or eukaryotic host cell lines are preferably E. coli or yeast strains. For cloning and storage purposes, mostly E. coli strains such as XL1Blue will be chosen. For the method of identification of suitable fragmentation sites according to the invention, especially involving the step of homologous recombination, a yeast strain may be chosen such as e.g. Saccharomyces cerevisiae, e.g. EGY48, and Schizosaccharomyces pombe. The choice of a suitable host cell line is routinely performed by the person of skill in the art, depending on the specific purpose; such host cell lines are commonly available.
The invention is further related to a kit of parts, comprising a first and a second DNA-based expression vector, wherein
According to a further especially preferred embodiment, such a kit of parts further comprising a suitable prokaryotic or eukaryotic host cell line for expression of said first and second expression vector.
Having provided by the present invention a tool for identifying novel fragmentation sites in reporter proteins, another major aspect of the present invention is related to a method for detecting an interaction between a first test peptide or protein or a fragment thereof, and a second test peptide or protein or a fragment thereof, the method comprising the steps of:
Utilizing split-protein sensors with subdomains identified by a method according to the invention, interaction of said first test peptide and said second test peptide may be identified. Given the tool of identifying suitable fragmentation sites in virtually any reporter protein, the person of routine skill in the art is no more hampered by the limitations of the existing, rationally designed split-protein systems to specific cellular compartments, but rather may now choose a reporter protein depending on his specific test purpose.
In the most preferred embodiment, a library of oligonucleotides or DNA encoding for a set of first test peptides or proteins and/or a library of oligonucleotides or DNA encoding for a set of second test peptides or proteins are fused to said first subdomain of said reporter protein and/or said complementary second subdomain of said reporter protein, respectively.
According to an especially preferred embodiment of the present invention, the interaction between a first test peptide or protein or a fragment thereof and a second test peptide or protein or fragment thereof is mediated by a chemical inducer of dimerization, which binds either covalently or non-covalently to both said test peptides or proteins or fragments thereof.
Comparable systems are commonly referred to in the literature as three-hybrid systems. Chemical inducers of dimerization (CIDs) have been first described by Schreiber and Crabtree (c.f. Spencer D. M, Wandless T. J, Schreiber S. L, and Crabtree G. R (1993), Science 262, 1019-1024, incorporated herein by reference). CIDs are cell-permeable molecules that can simultaneously form a covalent- or non-covalent interaction with two different proteins or peptides, thereby inducing their dimerization. Using split-protein sensors according to the present invention, e.g. robust drug and/or drug target screening assays may easily be established. Towards this aim, e.g. Ntrp may be fused to a protein library and Ctrp to an O(6)-alkylguanine-DNA alkyltransferase (AGT), e.g. human AGT (hAGT). A substrate for hAGT, e.g. Benzylguanine, may be easily covalently linked to a multitude of small molecules (hypothetical drugs), thus allowing for an efficient screening for cellular targets contained in said protein library that react or associate with the corresponding drug.
Moreover, the invention is related to a method for detecting the interruption of an interaction between a first test peptide or protein or a fragment thereof, and a second test peptide or protein or a fragment thereof, the method comprising the steps of:
Comparable systems are commonly referred to in the literature as reverse two-hybrid systems (or split-protein systems, respectively). Exemplarily, 5-fluoroanthranilic acid (FAA) is metabolized in vivo into a toxic product by the tryptophan biosynthetic enzymes. Applying the split-Trp sensors according to the invention, the disruption of protein-protein interaction leading to the spatial separation of the Trp1p fragments (and thus inactivity of the reporter protein) can therefore be linked to the survival of the cells on medium containing FAA. By means of example, libraries of small molecules may be screened for their ability to interact with a pair of fusion proteins. Selection of proteins or peptides that disrupt an interaction can be done by co-expressing two interacting proteins with a random protein or peptide library e.g. on plates containing FAA. The reverse split-Trp sensors may also advantageously be used to determine the binding region of a protein. A random library of the protein carrying mutations is co-expressed with its binding partner on plates containing FAA. Only cells that express a library member with mutations in or affecting the binding region, thus disrupting the interaction of the two proteins, will be able to grow in the presence of FAA.
Another aspect of the present invention is related to a use of random circular permutation of a gene and/or the expressed polypeptide derived thereof for the identification of fragmentation sites in a reporter protein for use in a split-protein sensor. To date, random circular permutation has not been used for the identification of such suitable fragmentation sites for separately expressed subdomains, but rather for the identification of proteins of at least approximately wild-type length, but with artificially new N- and C-termini, and with the wild-type N- and C-termini being connected to each other and being an internal part of the sequence. However, this approach now surprisingly proved to be an outstandingly valuable tool for the evolutionary, combinatorial approach of identifying suitable fragmentation sites for subdomains to be expressed separately.
A further aspect of the present invention is related to a use of a host cell line that allows for homologous recombination of DNA for the generation of a recombinant DNA molecule that secures for expression of both a polypeptide product comprising a first subdomain of a reporter protein and a complementary second subdomain of a reporter protein from said recombinant DNA molecule.
To date, homologous recombination has not been used for this purpose, but has now surprisingly found to be an outstandingly valuable tool for simply and conveniently securing for expression of a first subdomain and a complementary second subdomain of a reporter protein.
The invention will now be described in even more detail by means of an example and a specific embodiment, together with the accompanying figures; however, without the invention being limited thereto.
DNA- and protein sequences SEQ ID NO: 1 to SEQ ID NO: 66, as given in the attached sequence listing, are given in the attached sequence listing, incl. all primers and oligonucleotides used for the construction of the vectors.
For any standard molecular biology and especially DNA- and protein manipulation protocols it is generally referred to Sambrook, J. et al., eds., M
Yeast Media. Yeast complete medium containing adenine (YPAD) was used for cultures of Saccharomyces cerevisiae EGY48 and RSY529. Dropout media (YC) were used to select for the presence of pRS315- or pRS316-derived plasmids and for the complementation of tryptophan auxotrophy. Lacking amino acids or components in the resulting medium are indicated by the addition of their one-letter code to the YC-dropout medium. Selective YC-medium used to plate out the yeast cells after transformation by electroporation was supplemented with 1 M sorbitol. For the expression of proteins from the PGAL1-promoter 2% galactose and 0.5% raffinose replaced glucose as carbon source in the YC-medium.
Transformation of yeast cells. The transformation of Saccharomyces cerevisiae strains EGY48 or RSY529 with one or more plasmids was done using a standard protocol for transformation by electroporation. An overnight culture of EGY48 or RSY529 yeast cells in YPAD medium was diluted in 500 ml YPAD to an OD600 of ˜0.3 and grown at 30° C. and 260 rpm to an OD600 of ˜1.4. The culture was harvested by centrifugation at 4300 rpm and washed with 500 ml and 250 ml ice-cold sterile water and with 30 ml ice-cold 1 M sorbitol. The pelleted cells were then resuspended in 300-500 μl 1 M sorbitol and either used directly for transformation or frozen in aliquots of 40 μl at ˜80° C. For the double transformation of two plasmids, competent cells were always prepared freshly. A total amount of 100 ng plasmid DNA was mixed with 40 μl competent yeast cells, and electroporated at 1.5 kV using a Stratagene electroporator 1000 in a 0.2 mm cuvette. The cells were mixed with 500 μl ice-cold 1 M sorbitol immediately after the pulse and plated on the corresponding solid selective YC-medium containing 1 M sorbitol.
Cloning of pRS316-C1/2CUP1. A sequence containing two polypeptides C1 and C2 was first assembled by PCR using a set of primers as described by Stemmer et al (cf. Oakley M. G., and Kim P. S. (1998), Biochemistry 37, 12603-12610; Oakley M. G, and Hollenbeck J. J. (2001), Curr Opin Struct Biol 11, 450-457; Stemmer W. P., Crameri A., Ha K. D., Brennan T. M., Heynecker H. L. (1995), Gene 164, 49-53; all incorporated herein by reference). In short, the primers were mixed in an equimolar concentration (12.5 μM of each primer) and assembled in 55 cycles of denaturation (94° C., 30 s), primer annealing (52° C., 30 s) and extension (72° C., 30 s) using 0.1 unit/μl Pwo polymerase and 0.5 mM of each dNTP in the gene assembly buffer (10 mM Tris-HCl, pH 8.8, 2.2 mM MgCl2, 50 mM KCl and 0.1% Triton X-100). The double gene was then amplified out of this reaction using Pwo polyirerase with the 5′-primer PTP116 that contains an EcoRI site and the 3′-primer PTP111 that contains a SalI site. The PCR product was cleaved with EcoRI and SalI and cloned into pRS316, resulting in pRS316-C1/2 (cf. Sikorski R. S, and Hieter, P. (1989), Genetics 122, 19-27, incorporated herein by reference). The final construct contained the sequences for an N-terminal FLAG tag, the polypeptide C1 followed by a five-residue linker, an HpaI blunt end restriction site and a six-residue-linker followed by the polypeptide C2 with a C-terminal HA tag. C1 and C2 are two peptides that associate into an antiparallel-coiled coil (cf. Oakley M. G., and Kim P. S. (1998), Biochemistry 37, 12603-12610; Oakley M. G, and Hollenbeck J. J. (2001), Curr Opin Struct Biol 11, 450-457). The sequence of the PCUP1-promoter was then cleaved out of the plasmid pAGTM2-Dha with BamHI and EcoRI and positioned upstream of the C1/C2 cassette in pRS316-C1/2, resulting in pRS316-C1/2CUP1.
Cloning of pRS315CUP1, and of pRS316CUP1. The pRS315-derived vector was constructed for an easy cloning of the different Ntrp-SEC62 constructs, whereas the pRS316-derived vector was constructed for an easy cloning of the different SEC63-Ctrp constructs (cf. Sikorski R. S, and Hieter, P. (1989), Genetics 122, 19-27, incorporated herein by reference). The sequence of the PCUP1-promoter of the plasmid pAGTM2-Dha was amplified by PCR with the primers PTP181 and PTP182. The gene of ECFP was amplified by PCR out of pLP-ECFP-C1 with the primers PTP183 and PTP184. Both fragments were then combined by overlap extension PCR using the 5′-primer PTP181 that contains a BamHI site and the 3′-primer PTP184 that contains a SalI site, so that the PCUP1-promoter is upstream of ECFP (cf. Ho S. N. et al. (1989), Gene 1989, 51-59; incorporated herein by reference). The partially homologous primers PTP182 and PTP183 contain the sequence of the restriction sites EcoRI, BglII and AvrII to allow a versatile cloning of genes downstream of the PCUP1-promoter. The final fragment consisting of PCUP1-promoter and ECFP was then cloned into pRS315 or pRS316 with BamHI and SalI, resulting in pRS315CUP1 or pRS316CUP1, (cf. Sikorski R. S, and Hieter, P. (1989), Genetics 122, 19-27).
To generate split-protein sensors based on Trp1p (split-Trp) we adapted an approach originally developed by Graf and Schachmann for creating random circular permutations of proteins (cf. Graf, R., and Schachman, H. K. (1996), Proc Natl Acad Sci USA 93, 11591-11596, incorporated herein by reference). Using PCR, the TRP1 gene of Saccharomyces cerevisiae was first rearranged so that it started with residue 63 and its former start codon was fused to the stop codon via a linker sequence encoding a unique AvrII restriction site. The N- and the C-terminal domains of TRP1 were therefore amplified separately out of the plasmid pY-ESTrp2 (Invitrogen) with the primers PTP113/115 and PTP112/114, respectively, and recombined using overlap extension PCR with the primers PTP112 and PTP115 (cf. Ho S. N. et al. (1989), Gene 1989, 51-59; incorporated herein by reference). This rearrangement was performed to avoid unwanted isolation of wild-type gene in the subsequent selections. At the same time, a HindIII restriction site was introduced via the PCR primers at the newly generated N- and C-termini by introducing a silent mutation in the gene at around amino acid 63. Since the direct digestion of PCR products in former experiments yielded a product that did not ligate efficiently, the rearranged gene was first inserted into a high-copy plasmid (pAK400) and, after amplification of the vector DNA, cut out with HindIII. The rearranged gene was then incubated with T4 DNA ligase at 16° C. for 14 h at a DNA concentration of 0.14 mg/ml, leading to the formation of circular DNA as well as dimers and higher oligomers. After inhibition of the ligase at 65° C. for 20 min and desalting of the solution using a microcon PCR column, the ligation products were incubated with DNaseI (˜1.2 units/mg DNA) in 50 mM Tris-HCl, pH 7.5, 1 mM MnCl2 at 25° C. for six minutes. The exact conditions for the DNaseI reactions were determined immediately before the digestion in small test reactions. The DNaseI reaction was stopped by phenol extraction and ethanol precipitation. After incubation of the digested DNA with T4 DNA ligase and T4 polymerase to repair nicks, gaps and to flush the ends of the fragments, DNA fragments corresponding to the size of the original gene were isolated by gel electrophoresis. These fragments were ligated into the pRS316-based yeast expression vector pRS316-C1/2CUP1 that was cleaved with HpaI and dephosphorylated according to standard protocols. In the resulting vector, the C-terminal half of TRP1 is fused to a gene encoding for a FLAG tag, a polypeptide C1 and a five-residue linker sequence and is expressed under the control of the PCUP1-promoter. The N-terminal half of TRP1 is fused to a gene encoding for a six-residue linker sequence, the polypeptide C2 and a HA tag. The sequences of the peptides C1 and C2, including epitope tag and linker are:
After transformation in XL1Blue, resulting in a library with about 3×104 independent clones, the bacteria were scratched from the plate, and the plasmids isolated and linearized with AvrII. To insert a terminator for the C-terminal fragment and a promoter for the N-terminal fragment, a DNA fragment was constructed by PCR consisting of the CYC1 terminator, a geneticin resistance gene, the PGAL1-promoter and flanking regions of about 50 base pairs at the 5′-and 3′-ends homol ogous to the original N and C termini of Trp1p. The CYC1-terminator was amplified out of pYESTrp2 with the primers PTP107 and PTP120, whereas the cassette containing the geneticin resistance gene and the PGAL1-promoter was amplified out of pFA6a-GAL1 with the primers PTP108 and PTP121. Both fragments were combined by overlap extension PCR using the primers PTP120 and PTP121 (cf. Ho S. N. et al. (1989), Gene 1989, 51-59; incorporated herein by reference). The linearized vector (0.3 μg) and the PCR fragment (3 μg) were then co-transformed in chemically competent EGY48 cells and plated on plates lacking uracil but containing geneticin (500 μg/ml) to select for insertion of the PCR fragment into the linearized vector through homologous recombination (cf. Oldenburg et al. (1997), Nucleic Acids Res 25, 451-452; incorporated herein by reference). Chemically competent yeast cells were prepared as described by standard protocols. The homologous recombination also suppressed the predominant isolation of TRP1 genes that were cut near the original N or C terminus. In the final construct, the C-terminal fragment fused to C1 (C1-Ctrp) is under the control of the inducible but leaky PCUP1-promoter and the N-terminal fragment fused to C2 (Ntrp-C2) is under the stringent control of the PGAL1-promoter. After 3 days of incubation at 30° C., approximately 1600 colonies were isolated and subsequently replica-plated on plates lacking uracil and tryptophan but containing geneticin (250 μg/ml), galactose (2%) and CuSO4 (0.1 MM). After replica plating, 45 colonies were able to complement tryptophan auxotrophy. Approximately half of those 45 colonies required the presence of galactose and CuSO4 to grow on plates lacking tryptophan and twelve of these clones were then analyzed by DNA sequencing (
DLDQVRHLRRSWRSLSGNCKLLRRRMPSLRRSSRLEVTHMFQITLHFYKSTSRGGPVPSFCSL and in split-Trp180 split-Trp198, split-Trp203 and split-Trp204b with a peptide of 10 residues possessing the sequence (E/Q)RWIWIRSGT. It is assumed that Ntrp and Ctrp of these clones associate spontaneously without the help of interacting proteins. In split-Trp44 and split-Trp204b the mutation Gly8Cys was introduced during the fragmentation procedure. However, the influence of this mutation seems to be of minor importance as the deletion of the first ten amino acids still allowed split-Trp77 to complement tryptophan auxotrophy (
For split-Trp44, split-Trp53, split-Trp187 split-Tr204b and split-Trp77 the sequence encoding Ntrp-C2 was deleted from the plasmid using BglII and SalI and replaced with a PCR fragment encoding only the corresponding Ntrp-fragment. The resulting constructs were then retransformed into EGY48 (
The deletion of C2 abolished the capacity of the four clones split-Trp44, split-Trp53, split-Trp187 split-Trp204b to complement tryptophan auxotrophy (
Since the structure of Trp1p from S. cerevisiae has not yet been solved, we aligned its sequence with the sequences of the N-(5′-phosphoribosyl)-anthranilate isomerases from E. coli (ePRAI) and Thermotoga maritima (tPRAI), and identified the fragmentation sites in the known crystal structures of the homologous enzymes (
Detection of Membrane Protein Interactions Using Split-Trp Sensors
An important application for new split-protein sensors will lie in the detection and characterization of protein-protein interactions occurring at the membranes of intracellular organelles and the cell membranes. To test whether the split-Trp system operates at the membrane, the interaction-dependent split-Trp pairs were attached to the membrane proteins Sec62p and Sec63p (
To monitor the interaction between Sec62p and Sec63p, trp1 yeast strains expressing pairs of matching Ntrp-Sec62p and Sec63p-Ctrp fusion proteins were spotted on selective plates lacking tryptophan (
In more detail, the gene of SEC62 was amplified by PCR from yeast EGY48 genomic DNA and combined by overlap extension PCR with the N-terminal fragments of split-Trp44, split-Trp53, split-Trp187 and split-Trp204b, yielding 44Ntrp-SEC62, 53Ntrp-SEC62, 187-Ntrp-SEC62 and 204bNtrp-SEC62. At the same time, a 6× His tag was introduced at the 5′-end of Ntrp. The Ntrp genes and SEC62 are connected by a sequence coding for a six-residue linker (GGSGSG). The four Ntrp-SEC62 PCR products were isolated by gel electrophoresis and ligated in a pRS315-derived expression vector (LEU2) (pRS315CUP1) under the control of the PCUP1-promoter. Towards this aim, the vector was cleaved with BglII and SalI and the ECFP gene was replaced by the corresponding Ntrp-SEC62 construct.
The genes of SEC63 and STE14 were amplified by PCR from yeast EGY48 genomic DNA and combined by overlap extension PCR with the C-terminal fragments of split-Trp44, split-Trp53, split-Trp187 and split-Trp204b. At the same time, a 6× His tag was introduced at the 3′-end of Ctrp, yielding SEC63-44Ctrp-His, SEC63-53Ctrp-His, SEC63-187Ctrp-His, SEC63-204bCtrp-His, STE14-44Ctrp-His and STE14-187Ctrp-His. SEC63 and the Ctrp-His genes are connected by a sequence coding for a six-residue linker (GGSGSG). The different SEC63-Ctrp-His and STE14-Ctrp-His PCR products were isolated by gel electrophoresis and ligated into a pRS316-derived vector (URA3) (pRS316CUP1, vide supra) under the control of the PCUP1-promoter. To replace the 6× His tag by the more sensitive HA tag the genes of the different SEC63-Ctrp-His and STE14-Ctrp-His constructs were amplified by PCR with a 3′-primer that contains an HA tag and cloned into pRS316CUP1. All SEC63 and STE14 fusions contained an HA tag fused to the C terminus of Trp1p. The vector was cleaved with BglII and SalI and the ECFP gene was replaced with the corresponding SEC63-Ctrp and STE14-Ctrp constructs. All constructs were verified by DNA sequencing.
Expression of Ntrp-Sec62p fusion proteins. Expression and functionality of the Ntrp-Sec62p fusion proteins was confirmed by complementation of the temperature-sensitive yeast strain RSY529 (MATα his4 leu2-3, 112 ura3-52 sec62-1) (cf. Rothblatt J. A. et al. (1989), J Cell Biol 109, 2641-2652). RSY529 contains an endogenous temperature-sensitive variant of Sec62p. A colony of RSY529 cells transformed with either pRS315 or a pRS315-derived vector expressing 44Ntrp-Sec62p, 53Ntrp-Sec62p, 187Ntrp-Sec62p or 204bNtrp-Sec62p was resuspended in 1 ml water and 5 μl were spotted on YC-L medium containing 0.1 mM CuSO4 to induce the expression of the fusion proteins and incubated at 30° C. and 38° C. for 6 d to control for the complementation of the temperature sensitivity of RSY529.
Expression of Sec63p-Ctrp and Ste14p-Ctrp fusion proteins. The expression of the different Sec63p-Ctrp and Ste14p-Ctrp fusion proteins was verified by immunoblotting using antibodies against the HA tag at the C terminus of Trp1p. Towards this aim, an overnight culture of yeast EGY48 cells containing one of the Sec63p-Ctrp or Ste14p-Ctrp fusion proteins was diluted in 10 ml selective medium YC-U to an OD600˜0.8 and grown for 3 h at 30° C. and 220 rpm. Protein expression was induced by adding CuSO4 to a final concentration of 0.1 mM. After 3 h of expression at 30° C. and 220 rpm, the cell solution (same volume at same OD when different samples were compared) was centrifuged at 4300 rpm for 10 minutes and the pellet resuspended in 150 μl yeast lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100) containing 1% (v/v) protease inhibitor cocktail and 0.5 mM PMSF. 200 gl glass beads were added and the solution was vortexed at full speed for 3×30 s and cooled on ice in between the vortexing steps. The glass beads and the cell debris were pelleted by centrifugation for 30 s at 13000 rpm and the supernatant was mixed with an appropriate volume of 5×SDS sample buffer (50% glycerol, 7.5% SDS, 250 mM Tris-HCl, pH 8.0, 0.5% Bromphenol blue, 12.5 mM 2-Mercaptoethanol). Proteins were denatured for 3 min at 95° C. Aliquots were analysed by Western blotting (12% SDS-PAGE) as described by standard protocols. After blotting, the nitrocellulose membrane was incubated with 3% dry milk in TBST (10 mM Tris-HCl, 150 mM NaCl, pH 7.9, 0.05% Tween 20) to block unspecific antibody binding. Expression of Sec63p-Ctrp or Ste14p-Ctrp fusion constructs was controlled by incubation of the membrane with the primary anti-HA antibody 1:7500 in TBST (10 mM Tris-HCl, 150 mM NaCl, pH 7.9, 0.05% Tween 20). An anti mouse-HRP antibody conjugate was used 1:7500 in TBST (10 mM Tris-HCl, 150 mM NaCl, pH 7.9, 0.05% Tween 20) as secondary antibody. Detection was done on a Kodak Image Station 440CF using the NEN Renaissance kit, a luminol-based chemiluminescence system.
The present data demonstrate that in particular split-Trp44 is well suited for the detection of protein-protein interactions between membrane proteins. Interestingly, yeast cells co-expressing 44Ntrp-Sec62p and Sec63p-44Ctrp require lower growth temperatures for the complementation of tryptophan auxotrophy than the cells expressing the corresponding C1 and C2 coiled coil fusions. This effect might be due to a more favorable orientation of the N- and C-terminal Trp1p fragments in the antiparallelcoiled coil than in the Sec62p/Sec63p complex.
In conclusion, we have used directed evolution to convert N-(5′-phosphoribosyl)-anthranilate isomerase into a split-protein sensor. In coupling the interaction of cytosolic and membrane proteins to a simple growth assay, the split-Trp system possesses all the necessary features to complement already existing systems to measure and screen for new protein interactions. This split-Trp approach may be used in identifying partners of medically relevant targets, e.g. in three-hybrid assays and protein/small molecule interaction assays. Furtherrmore, the evolutionary approach introduced here is generally applicable to other enzymes. By generating novel split-protein sensors that are based on proteins functioning in the matrix of e.g. the mitochondrium, the peroxisome or the lumen of the secretory path, this evolutionary approach will help to overcome the lack of techniques to measure protein interactions in the interior of these organelles. Finally, the analysis of the different split-Trp pairs that either spontaneously assemble into a functional (β/α)8-barrel or need to be fused to interacting proteins to yield folded protein supports the hypothesis that a large N-terminal subdomain of Trp1p is an important intermediate in the folding of the (β/α)8-barrel.
Further Experimental Details
For the various PCR- and gene assembly reactions, if not already noted explicitly above, the following primers and templates were used.
Primers used for Ntrp-constructs (cf. attached sequence listing for details):
Primers used for Ctrp-constructs (cf. attached sequence listing for details):
Primers used for zipper construction (cf. attached sequence listing for details):
Primers used for the copper promoter (cf. attached sequence listing for details):
Primers used for circular permutation of Trp1p (cf. attached sequence listing for details):
Primers used for homologous recombination (cf. attached sequence listing for details):
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP04/11289 | 10/8/2004 | WO | 1/3/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60510231 | Oct 2003 | US | |
| 60510231 | Oct 2003 | US |
