WO2009009908A1

WO2009009908A1 - A recombmase based screening method for detecting molecular interactions comprising a single plasmid vector with two unique recombmase sites

Info

Publication number: WO2009009908A1
Application number: PCT/CA2008/001349
Authority: WO
Inventors: Jian Ping Lu; Paula C. Byrne; Jehonathan H. Pinthus
Original assignee: Mcmaster University
Priority date: 2007-07-19
Filing date: 2008-07-18
Publication date: 2009-01-22

Abstract

The present invention is directed to a novel, high-through-put technology for screening for molecular interactions. The invention includes a receiver vector that can receive two open reading frames (ORF), molecules (nucleic acid sequences) or control operators linked to fluorescent or other signal tags in the receiver vector. A recombinase based system is used to control insertion of the ORFs. The method can be used to screen for various types of molecular interactions, such as protein-protein, protein-nucleic acid, nucleic acid-nucleic acid, protein-carbohydrate, etc.

Description

A RECOMBMASE BASED SCREENING METHOD FOR DETECTING MOLECULAR INTERACTIONS COMPRISING A SINGLE PLASMID VECTOR WITH TWO UNIQUE RECOMBMASE

SITES

FIELD OF INVENTION

[0001] The present invention relates to methods, compositions and assays for detecting molecular interactions in living cells.

BACKGROUND OF THE INVENTION

[0002] The sequencing of the human genome led to the identification of numerous gene sequences. However, the function of many of these putative genes is still unknown. The major post-genome sequencing task is to determine the function of each of the genes from the human genome. There is a growing demand for high throughput technology platforms that will enable large scale screening of various types of molecular interactions, such as interactions between gene products (proteins) or between gene product and gene itself, etc. The current in vitro (e.g. protein array) and in vivo methods (e.g. yeast two-hybrid) for screening protein interactions have limitations. These systems require multiple complicated steps and are time consuming. Proteins in an array are not in the native state. In addition, it is difficult to filter out non-specific interactions.

SUMMARY OF THE INVENTION

[0003] Protein interactions at the molecular level can be measured by various methods.

[0004] For example, fluorescence resonance energy transfer (FRET) using a pair of fluorescent molecules, such as cyan fluorescent protein (CFP or ECFP) and yellow fluorescent protein (YFP or EYFP), in which the emission spectrum of CFP significantly overlaps the excitation spectrum of YFP can be used. The resulting energy given off from the donor CFP protein can directly excite the acceptor YFP protein when the proteins are closely approximated (FRET). During FRET, the fluorescence emission of the acceptor is enhanced by the excitation of the donor molecule, accompanied by a reduction in the donor emission. The efficiency of energy transfer is dependent on the molecular distance at an inverse sixth power. (Biophysical Journal Volume 81 ; 2001 : 2395).

[0005] In addition, bimolecular fluorescence complementation (BiFC) can be used. BiFC is based on the reconstitution of the YFP or other fluorescent protein from two nonfluorescent fragments when they are brought into close proximity by a physical interaction between proteins fused to each fragment (Cancer Res 2005; 65: 7413. Nature Methods 2007; 3597).

-l- [0006] Protein fragment complementation (PFC) can also be used to detect protein interactions. Methods that allow direct detection of interactions based on the fusion of complementary fragments of a reporter protein to two putative interacting proteins are utilized. Functional complementation between the reporter protein fragments, mediated by the interaction of the two fusion proteins, results in a quantifiable signal that can be used to investigate the localization and regulation of protein interactions in their normal environment (Nature Method 2006; 3: 969).

[0007] Several attempts have been made to develop systems fro studying protein interactions. For example, United States Patent Application 20030013142 discloses a method to screen for and identify proteins that interact with a protein of interest. The method comprises: transforming an expression system, wherein the expression system comprises a plurality of cells that express a protein of interest tagged with one member of a fluorescent protein pair by a plurality of DNA molecules encoding a protein to be screened tagged with the other member of the fluorescent protein (FP) pair, wherein individual members of the plurality of cells are transformed by different DNA molecules encoding said protein to be screened; screening said transformed expression system by fluorescence resonance energy transfer (FRET) to determine whether an interaction has occurred; and identifying any cells within said transformed expression system where there has been a transfer of fluorescence between the fluorescent protein pair as indicating that an interaction between the protein of interest and a protein being screened has occurred.

[0008] United States Patent 6,911 ,311 discloses a method for detecting an interaction between a first test agent and a second test agent. The method comprises: providing a first fusion construct and a second fusion construct, said first fusion construct having an N- intein and said first test agent, said second fusion construct having a C-intein and said second test agent, wherein at least one of the two fusion constructs has an inactive reporter capable of being converted to an active reporter upon trans-splicing through said N-intein and said C-intein, and wherein said N-intein and said C-intein do not interact with each other; allowing said first test agent in said first fusion construct to interact with said second test agent in said second fusion construct in vitro; and detecting said active reporter thereby detecting an interaction between the first and second test agent.

[0009] The optimal situation for detecting molecular interactions is in living cells. While FRET and techniques based on FRET related technologies, as well as BiFC, PFC, are widely used for examination of interactions among proteins in living cells, these technologies are only applicable to monitoring one or several pairs of interacting molecules at each time point. The current systems are not feasible for high throughput analysis since the plasmids are custom-constructed. There is no commercially available high-throughput or automated platform for screening/verification of protein interactions in vivo.

[0010] Thus, while methods exist to identify one-to-one interactions of known proteins, there remained a need for a rapid throughput system to identify novel molecular interactions such as protein-protein, protein-nucleic acid and protein-carbohydrate interactions.

SUMMARY OF THE INVENTION

[0011] The present invention provides a high-throughput, recombinase based single vector system (platform) for detecting molecular interactions. These interactions may be protein- protein, small molecule-protein, aptamer-ligand, enzyme-substrate, antigen-antibody, receptor-ligand or any other molecular interaction that may occur in a cell.

[0012] In one aspect of the invention, a vector comprising at least two unique recombinase recognition sites is provided. In a preferred embodiment, a single plasmid comprising loxP and Att recombinase sites is provided.

[0013] In a further preferred embodiment, the vector further comprises selection element, such as an antibiotic resistance gene(s). In another preferred embodiment, the vector comprises cell or tissue specific promoter. In yet another embodiment, the vector includes at least one inducible promoter and may also contain other operators and express other desired characteristics.

[0014] In another aspect of the invention, a method of preparing a vector comprising at least two ORFs of the molecules (fluorescent proteins or sequences that can form FRET, BiFc, PFC and the like) that can emit specific detectable signals in vivo with at least two recombinase-recognizing sequences for in-frame insertion of the ORFs of interest is provided. The method comprises:

i. inserting a first open reading frame sequence into a first supplier plasmid at a first supplier recombinase target site; ii. inserting a second ORF sequence into a second supplier plasmid at a second supplier recombinase target site;

iii. inserting said first supplier plasmid sequence into an receiver expression vector through a first recombinase reaction; and

iv. inserting said second plasmid sequence into said receiver expression vector through a second recombinase reaction, wherein said first and second recombinase reactions are mediated by two different recombinases.

[0015] In another aspect of the invention, a method of screening for a molecular interaction is provided. The method comprises:

i. inserting a first sequence encoding a first protein sequence to couple said protein sequence to a first signaling molecule in a receiver expression plasmid thereby forming a first tagged molecule in the receiver expression vector;

ii. inserting a second sequence encoding a second protein sequence coupled to a second signaling molecule, forming a second tagged molecule in the same receiver expression vector of step i;

iii. wherein the first tagged molecule and the second tagged molecule are translated into fusion molecules (e.g. fusion proteins) and interaction of those fusion molecules provides a signal.

[0016] In a preferred embodiment, the first and second signaling molecules comprise a FRET and/or BiFC or PFC pair. In another embodiment, the first and second tags comprise a fluorophore-quencher pair.

[0017] In another embodiment, more than two tagged molecules comprising serial FRET and/or BiFC or PFC reactions can be utilized to detect the interaction of more than two molecules.

[0018] In further embodiments, the system can be used to monitor the interaction and function of tagged protein with DNA operators in a gene and to determine the expression of a gene product and its interaction with the first tagged protein. [0019] In another embodiment, the system is useful to screen and monitor protein-protein and/or protein-DNA or nucleic acid - nucleic acid interactions and modulators of those interactions. For example, the compositions and methods of the invention can be used to screen and monitor the effect of a protein (ORF inserted in one of the recombination loci) on the activity or function of regulators of a specific gene and to screen for their modulators.

[0020] In yet another embodiment, the system can be used to screen and monitor DNA interactions and modulators of such interactions. For example, compositions and methods of the invention can be used to screen and monitor the function of microRNA (miRNA) or RNA interference (RNAi, inserted in one of the recombination loci in the form of cording area of shRNA) and to evaluate the role of the postranscriptional regulators or to determine the role of RNAi on regulation of postranscriptional processing of the gene of interest, the ORF of which is inserted into the second recombination loci.

[0021] This summary of the invention does not necessarily describe all features of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] These and other features of the invention will become apparent from the following description in which reference is made to the appended drawings wherein:

[0023] FIGURE 1 illustrates the successful preparation of a dual expression recominbinase based (DERB) single vector system.

[0024] FIGURE 2 illustrates a dual receiver prokaryotic expression vector in accordance with an embodiment of the present invention;

[0025] FIGURE 3 illustrates a dual receiver prokaryotic expression vector expressing two fusion proteins generated after two recombination reactions with supplier vectors.

[0026] FIGURE 4 illustrates an exemplary eukaryotic expression vector in accordance with a further embodiment of the present invention involving FRET;

[0027] FIGURE 5 illustrates a eukaryotic expression plasmid in accordance with a further embodiment of the present invention involving BiFC; [0028] FIGURE 6 illustrates a eukaryotic expression plasmid in accordance with a further embodiment of the present invention involving PFC;

[0029] FIGURE 7 illustrates an exemplary eukaryotic expression plasmid after recombination reactions for FRET signal detection;

[0030] FIGURE 8 illustrates an exemplary eukaryotic expression plasmid after recombination reactions for BiFC signal detection;

[0031] FIGURE 9 illustrates an exemplary eukaryotic expression plasmid after recombination reactions for PFC signal detection;

[0032] FIGURE 10 illustrates exemplary results obtained after high throughput screening of signals in FRET plate reader in cells co-expressing two different proteins;

[0033] FIGURE 11 shows results of FRET detection of protein interactions using a plate reader and validated with confocal microscope analysis.

[0034] FIGURE 12 illustrates flow cytometric analysis of cells co-expressing two different proteins;

[0035] FIGURE 13 is a series of fluorescent micrographs of transfected prokaryotic cells after FRET in accordance with one embodiment of the invention;

[0036] FIGURE 14 is a series of fluorescent micrographs of transfected eukaryotic cells after FRET in accordance with one embodiment of the invention;

[0037] FIGURE 15 is a series of fluorescent micrographs of transfected eukaryotic cells after (BiFC) in accordance with one embodiment of the invention;

[0038] FIGURE 16 shows protein interactions detected using BiFC and validated by flow cytometry (top) and fluorescent microscopy (bottom).

[0039] FIGURE 17 is a series of bright field micrographs of transfected eukaryotic cells after (PFC) in accordance with one embodiment of the invention:

[0040] FIGURE 18 is a schematic illustrating various types of molecular interactions. [0041] FIGURE 19 shows a expression vector in accordance with a further embodiment of the present invention illustrating a Protein-DNA interaction and;

[0042] FIGURE 20 shows an expression vector in accordance with a further embodiment of the present invention illustrating a RNA-DNA interaction.

DETAILED DESCRIPTION

[0043] The present invention provides compositions, methods and assay systems for screening molecular interactions in cells. A single vector system including at least two recombinase recognizable loci for simultaneous expression of at least two tagged or labeled molecules. The interactions of various types of molecules, such as antibody- antigen, receptor-ligand, protein-nucleic acid, nucleic acid- nucleic acid, protein-protein and protein-carbohydrate interactions, can be studied using the vector system of the invention.

[0044] The present invention provides a new technology platform that is a dual expression recombinase based (DERB) single vector system for high throughput screening to detect novel molecular interactions. At least two supplier plasmids and a receiver plasmid are used in the system. Each of the supplier vectors has unique recombinase recognizable sites to facilitate high throughput cloning. For example in a two- supplier system, supplier vector 1 could have the LoxP sites (whilst supplier vector 2 could have the Att sites. It is clearly apparent that other recombinase recognizable sequence/site(s) can also be used. Preferably these are different recombinase recognizable sites in each of the supplier plasmids. Each of the supplier plasmids includes an ORF associated with the recombinase based system. The ORF may encode a known or unknown molecule.

[0045] Another element of the platform technology is the receiver vector. The receiver vector comprises recombinase recognition sites for each of the recombinase recognizable sites in the supplier vectors. The ORFs from each of the supplier vectors are inserted into the receiver vector via the recombinase based reactions. Various recombinases can be used. Table 1 lists some examples of site /sequence-specific recombinases. Table 1.

Phage/ Amino add name Hα;»t length Ov^lap region

Tyrosine integras-es λ Escherichia ωli 356 TTTATAC

HK022 Escherichia cύli 357 AGGTGAA

P22 Salmonella 387 TTC€TAA typhi murium

HPl Haemophilus inβu- 337 TTTTAAA en∑ae

L5 Mycobacterium 371 CTTCCAΛ smegma tis

Other tyrosine Crc (Pl) Escherichia coti 343 ATGTΛTGC recombinases

FLP Sacchamtnyces 423 TCTAGAAA cetvvisiae

XwC Escherichia coti 298 TGTACA

Serine integrases ΦC31 Streptomycin 613 TTG liviώms

R4 Strephmtyce≤ 469 GAΛGCAGTGGTA parwtlus

TP901 Lictococcus taciis 485 TCAAT

Other Mϊrine recomγδ Escherichia cali 183 TΛTTATAAAT binants

TnJ Klebsiella 185 TΛTTΛTAAΛT pneumoniae gin (Phage Escherichia coti 193 GA

Mu)

[0046] The receiver vector is designed so that the inserted ORFs will be synthesized independently from their own promoter and tagged. Each of the ORFs is tagged with a different tag whereby when the two tags come into close proximity or interact (e.g. when two tagged proteins interact, or one nucleic acid interact with another nucleic acid molecule modulate its structure and/or function) a reporter signal is generated. Various types of tag pairs can be used. For example if one protein is tagged with a fluorophore and the other is tagged with a quencher, there will be a decrease in fluorescence when the two proteins are in close proximity.

[0047] It is clearly apparent that other reporting signal systems may also be utilized. For example, the BiFC, PFC, luciferase system may be used as well as fluorophores that yield a FRET signal or other signal. [0048] In the method of the present invention, each ORF is preferably tagged with a fluorescent protein or tagged with special sequences/structures facilitating further labeling of fluorescent molecule(s) (FP) or other signal molecules. The FPs are capable of generating FRET or other signals the cells when brought in close proximity in the event that the differentially tagged proteins or the molecules of interest interact with each other. A single receiver vector is produced that results in the production of two or more independent FPs. The use of the recombinase based system allows the process to be highly precise and effective which, in turn, allows for high throughput cloning.

[0049] A series of receiver vectors can be created whereby the proteins under investigation can be tagged with FPs or other signal molecules either internally or at the C- or N- terminal. This allows the user to investigate the different possibilities of interaction: e.g. head-head, head-tail, tail-tail, tail-head etc.

[0050] ] Both prokaryotic and eukaryotic receiver vectors are encompassed within the scope of the invention. The receiver vector may include promoters and selective elements (antibiotics and likewise chemicals) for selective growth of transformed host prokaryotic cells or it may have promoters and selective elements for propagation in eukaryotic cells. Receiver vectors can optionally include conditional operators (e.g. tet on/off) for induction or suppression of expression in the presence or absence of special chemicals (e.g. tetracycline).

[0051] The utility of the invention is demonstrated in the attached figures and discussed further in the examples below.

[0052] Figure 1 illustrates a schematic of the system. The proteins of interest present in individual donor vectors were sequentially introduced into the recombinase recognizable loci of a DERB destiny vector (Fig. 1ai). Two independent recombination reactions brought the destiny vector to its dual protein expression vector formation and enabled protein interaction detection through FRET or BiFC following introduction into the cells. The initial LR Clonase recombination integrated ORF1 between the Att sites of the destiny vector while the subsequent CRE recombinase recombination inserted ORF2 into the LoxP site. The LR Clonase mediated reaction product was introduced into Escherichia coli (E. coli) DH5α for negative selection of the recombination byproduct and unsuccessful insertion constructs. The ccdB gene was either introduced into the reaction byproduct plasmid, devoid of ORF1 , or remained in the destiny vector and prevented growth of both plasmids in DH5α. Confirmation of the Att recombination with ORF1 was achieved through polymerase chain reaction (PCR) with primers specific to the ORF1 sequence and Att insert boundary (Fig. 1aii). The subsequent reaction mixture of the CRE recombination was directly transformed into E. coli DH5α whereby successful insertions manifested in large concentric clones and unsuccessful clones succumbed to chloramphenicol and sucrose. Examination of the putative dual expression clones with electrophoresis identified effective ORF2 introductions evident by the increased plasmid size, proportional to the length of insertion, attained above the size without insertion (Fig. 1aiii). Collectively the SucB and antibiotic selection securities prevented the growth of clones with unsuccessful recombination vectors and provided the foundation of the platform's cloning efficiency. Finalized dual expression vectors were introduced into prokaryotic (E. coli BL21(DE3)) or eukaryotic (HeLa, HEK293) cells dependent upon the specified promoter of the destiny vector utilized. Induction of the system translated the two proteins individually fused to either the Yc Yn (Fig. 1bi) or ECFP EYFP (Fig. 1bii) set of tags for BiFC or FRET protein interaction detection, respectively.

[0053] Figures 2-9 illustrate exemplary prokaryotic and eukaryotic receiver expression vectors before and after recombination with the supplier plasmids.

[0054] Each of the supplier plasmids includes an open reading frame (ORF) encoding a protein or other molecule of interest. The ORF may encode a known protein or it may encode a putative gene identified by sequencing. For example, supplier vector 1 can be constructed to contain the sequence of a particular protein under investigation, whilst supplier vector 2 could represent a library utilizing PCR products or public and/or commercially available ORF libraries in vectors. Alternatively, both Supplier Vector 1 and Supplier Vector 2 can contain known protein sequences or both may contain a library sequence.

[0055] In an exemplary method of the invention, supplier vector 1 contains protein X sequences in the correct reading frame, supplier vector 2 represents a library of sequences. The recombination recombinase based system is used to produce a library of receiver vectors, where each receiver vector expresses fluorescent tagged protein X and one of the library of proteins that is also tagged. The recombinase based system allow for high-throughput processing. Transformation of the library into prokaryotic cells of the present invention and transfection of a library of receiver vectors into eukaryotic cells is also easily automated using systems such as Amaxa. High throughput screening for

40 protein interaction is achieved by passing transformed/ transfected cells through a flow cytometer and detecting FRET or quenching. FRET is detectable in a single cell that has been transformed with a receiver vector expressing protein X and a protein that interacts with it. Interaction of two or more proteins produced from a single receiver vector can be further confirmed with confocal microscopy by checking for the presence of FRET or quenching. The intensity of interaction and the distance of interacting molecules can be further calculated.

[0056] In another exemplary method of the invention, the receiver vectors may utilize BiFC or PFC as the method for detecting a signal indicative of two or more molecular interactions. Thus, multiple platforms are available for the user to select based upon the availability of facilities (confocal, fluorescent, light microscope, plate reader etc).

[0057] Figure 10-11 illustrate the results from an exemplary experiments demonstrating the utility of the invention. The receiver vector contains the ORFs. The supplier vector was transferred into HeIa cells and the interaction between these two molecules was confirmed using a FRET plate reader.

[0058] Figures 12 through 14 illustrate results from an exemplary experiment demonstrating the utility of the invention, supplier vector 1 encoding spartin (SPG20) and supplier vector 2 encoding tubulin were recombined with a receiver vector. Interaction between these two molecules was confirmed by fluorescence analysis.

[0059] The use of a single receiver vector with at least two promoters and the ORFs of interest in frame with FPs ensure all or nothing expression (transfected or non-transfected) in the cells, avoiding at least 4 groups of cells in double transformations or transfections: single positives, double positives, and negative cells. In addition, selective elements in the vector allow the removal of non-transfected cells thereby facilitating automatic processing of the cells in flow cytometry and confocal microscope. A further advantage of the present system is that the protein interaction detected occurs between proteins in living cells. Thus the number of false positive/negative results is reduced as compared to yeast two-hybrid methods since FRET will only appear when two proteins are at intimate position (less than 4-7nm), while the simple co-localization of two proteins only forms plus effect of the two colors. In addition, the intensity of interaction can be determined by checking the intensity of FRET, and the distance of interacting proteins can also be calculated. A significant advantage of the system is that detection of the protein interactions needs only one

4 1- transfection or transformation step, thereby reducing the artifacts and the costs of multi- step experiments. The recombinase based single vector system ensures the simplest receiver vector library making thereby facilitating high throughput processing (Table2).

[0060] TABLE 2. Receiver expression vector clone selection strategy in a recombinase based single vector system (Prokaryotic expression example)

42 Table 2: Clone selection strategy of present invention (Prokaryotic expression example)

Name Vector Selective ORFs Detail

First recombination reaction (LR clonase mediated):

Donor Vectoii pENTR-ORF1 Kana^R or Resist to kanamycin or

(or pDONR-ORF1) Spn" Resist to Spectinomycin

Destiny Vectori PT7-ECFP-EYFP CmR, Resist to chloramphenicol ccdB Only grow in E. coli with gryA462 allele (e.g. DB3.1 )

Single Expression Vector DT7-ECFP-EYFPORF1 Amp^R Resist to ampicillin

Recombination 1 by-product pENTR-ccdBCm^R Cm^R, Resist to chloramphenicol ccdB Only grow in DB3.1 Culture Medium: LB-Ampicillin Host E. CoIi: DH5a:

Selection strategy: E. coli with donor vector 1 does not grow in ampicillin media

Destiny vector 1, or recombination 1 by-product cannot amplify in E. coli strain without gryA462 allele (DB3.1)

Second recombination reaction (Cre Recombinase mediated):

Donor Vector2 pDNR-ORF2 Amp* Resist to ampicillin

(Or pDNR-Dual-ORF2) Cm^R, Resist to chloramphenicol SucB Sensitive to sucrose

Destiny Vector2 pT7-ECFP- EYFPORF1

(i.e. Single Expression Vector) Amp^R Resist to ampicillin

Recombination 2 by-product pDNR-SucB, Amp' Resist to ampicillin

SucB Sensitive to sucrose

Double Expression Vector pT7-eCFPORF2-EYFPORF1

Amp^R Resist to ampicillin Cm^F Resist to chloramphenicol Resist to sucrose

Culture Medium: LB- chloramphenicol-sucrose Host E. coli: DH5α:

Selection strategy: E. coli with donor vector 2 or recombination 2 by-product does not grow in media with sucrose E coff with Destiny vector 2 does not grow in media with chloramphenicol

[0061] The compositions and methods of the invention can also be used to detect protein interactions using BiFC and PFC signaling technologies by changing the YFP-CFP pair into the N-terminal part of YFP(Yn), C-terminal part of YFP (YC) or, likewise, the N-terminal part of LacZ(ZdC), C-terminal part of LacZ (ZdN) etc. See, for example, Figures 5-6 and 8- 9. The interaction of the molecules of interest can also be detected by evaluating the YFP

43- fluorescence or LacZ enzyme activity as shown in Figures 15-17. The plasmid containing the ORFs of fusion proteins (APPLI-Yc₁ AdipoR1-Yn, or APPLI-ZdN, Adipo RI-ZdC) can be used to transfect eukaryotic cells or transform prokaryotic cells using the promoters of the receiver plasmid vectors. The interaction of the proteins can be detected through fluorescent or light microscopy, (Figures 15-17) or via a plate reader or a flow cytometer.

[0062] While the description has focused on protein-protein interactions, it is apparent that the system can be also used to check interaction of protein with non-protein molecules (DNA, lipid, etc.) or between non-protein molecules.

[0063] It should be understood that the ORF and/or promoter of the plasmid vectors can be replaced with other regulator, reporter and target molecules/sequences that allow one to determine the function and /or interaction of those regulators and/or molecules. Examples of various combinations are shown in Figures 19 and 20.

[0064] This platform technology provides an alternative and unique high throughput method for identifying molecular interactions that overcomes many of the limitations associated with current technologies. The invention is useful for many applications. These include, but are not limited to: high-throughput identification of novel protein interactions in living cells; confirmation of protein interaction in vivo in prokaryotic and eukaryotic cells; identification of protein and /or non protein molecular interactions; and screening potential candidate peptide or other chemicals for interaction with target cellular proteins for the development of new therapeutics or preventatives.

[0065] The technology described herein is also useful for the study of interactions in plants. For example, many important crop traits, such as the solid content of tomatoes, result from the combined interactions of the products of several genes residing at different loci in the genome.

[0066] The above disclosure generally describes the present invention. It is believed that one of ordinary skill in the art can, using the preceding description, make and use the compositions and practice the methods of the present invention. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely to illustrate preferred embodiments of the present invention and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Other

44 generic configurations will be apparent to one skilled in the art. Documents such as patents or patent applications referred to herein are hereby incorporated by reference.

EXAMPLES

[0067] Although specific terms have been used in these examples, such terms are intended in a descriptive sense and not for purposes of limitation. Methods of microbiology and physics referred to but not explicitly described in the disclosure and these examples are reported in the scientific literature and are well known to those skilled in the art.

Example 1. Plasmids

pLP-CFP, pYFP, pDNR, pDNR-CMV, pEYFP-Tub were purchased from Clontech Laboratories lnc (BD bioscience USA). pENTR, pDEST (pT-Rex-DEST30, pDEST 15 etc.), pRSET were the product of Invitrogen ( CA 92008, USA). The preparation of receiver vectors was undertaken according to standard molecular cloning protocols from Molecular Cloning: A Laboratory Manual (Sambruck, J.;Russell DW: Cold Spring Harbor Laboratory Press; 2002.) etc. The materials used for producing supplier vectors are listed in Table 3. Mammalian E. CoIi expression system, gateway-cloning system (Invitrogen, USA), infusion PCR cloning kit, Creator gene cloning and expression system (Clontech Worcester, MA, U.S.A., Cat NO. 631774 PT3650-1 ) were used for subcloning the genes of the interest. A flow cytometric method was used to detect protein-protein interaction in living cells by directly visualizing fluorescence resonance energy transfer (FRET). Cytometry A. 2003 Oct; 55 (2):71-85. PMID: 14505312). The following receiver plasmids were constructed by Lu JP: Plasmids expressing both CFP and YFP in prokaryotic and eukaryotic cells with recombinase sites and other features (pT7C-LoxP-Yatt, PCFP-LoxP- Tet-YFPatt). Plasmid expressing both YFP N-terminal (Yn) and YFP C-terminal (YC) in eukaryotic cells with recombinase sites and other features (pWc-LoxP-Ynatt). Plasmid expressing both LacZ Delete N-terminal (dN) and LacZ Delete C-terminal (dC) in eukaryotic cells with recombinase sites and other features (pVZdNLoxPZdCatt). Routine fluorescent and light microscope techniques were used to detect BiFC and PFC signal. Example expression plasmids For detection of protein interaction through FRET are listed in Tables 4 and 5 (Those through BiFC and PFC are not shown).

45 Table 3. Sources of ORFs

Plasmid Proteins Expressed Source Host organism LMGeneNo. UnProtNo.

pDNR-TUBAIB α tubulini B In Lab Homo sapiens NM006082 P68366 pDNR-VIM Vimentin In Lab Homo sapiens NM03380 P08670 pENTR-Vim Vimentin I n Lab Homo sapiens NM03380 P08670 pENTR- TUBAIB α tubuliniB In Lab Homo sapiens NM006082 P68366 pDNR-APPL.1 APPL1 In Lab Homo sapiens NM012096 Q9UKG1 pCFP-YFP CFP-YFP fusion Protein Dr. He LS pCDN A-APPL 1 APPL 1 Dr. Dong LQ Homo sapiens NM012096 Q9UKG1 pENTR-ADIPORI Adponeclin Receptor! Invitrogen Homo sapiens NM015999 Q96A54

PENTR-ADIPOR2 Adponeclri Receptor2 Invitrogen Homo sapiens AK025085 Q86V24 pENTR-APPL1 APPL 1 OpenBiosystems Homo sapiens NM012096 Q9UKG1

Table 4. Platform derivatives for FRET examination in prokaryotic cells

Plasmid Promoter Proteins Expressed

PT7-CFP-YFP T7 CFP, YFP , two proteins pT7-CFPYFP T7 CFPYFP, one fusion protein pT7-CFPVIM-YFPVIM T7 CFPVimentin, YFPVimentin, two tagged proteins pπ-CFPVIIvVYFPTUBA1 B T7 CFPVimentin, YFPαTubulini B, two tagged proteins

PT7-CFPAPPL1-YFPADIPOR1 T7 CFPAPPL1 , YFPAdiponectJn Receptor 1 , Iwo tagged proteins pT7<FPAPPL1-YFPADIPOR2 T7 CFPAPPL1 , YFPAdiponectin Receptor 2, two tagged proteins

PT7-CFPADIPOR1-YFPADIPOR1 T7 CFPAdiponectin Receptor 1 , YFRAdponectin ReceptoM,

46 Table 5. Platform derivatives for FRET examination in eukaryotic cell lines

Plasmid FVomoter Proteins Expressed pCMV-CFP-YFP CMV CFP, YFP, two proteins pCMV-CFPYFP CMV CFPYFP, one fusion protein pCMV-CFPVIM-YFPVIM CMV CFPVimentin, YFPVimentin, two tagged proteins pCMV-CFPVIM-YFPTUBAI B CMV CFPVimentin, YFPαTubutiniB, two tagged proteins

PCMV-CFPAPPL1-YFPADIPOR1 CMV CFPAPPL1 , YFPAdiponectin Receptor 1 , two tagged proteins

PCMV-CFPAPPL1-YFPADIPOR2 CMV CFPAPPL1, YFPAdiponectn Receptor 2, two tagged proteins pFer-CFP-YFP FerH, FerL CFP, YFP, two proteins pFer-CFPYFP FerH, FerL CFPYFP, one fusion protein pFer-YFPVIM-YFPVIM FerH, FerL CFPVimentin, YFPVimentin, two tagged proteins pFer-CFPVIM-YFPTUBA1B FerH, FerL CFPVimentin, YFPαTubuliniB, two tagged proteins pFer-CFPAPPL1-YFPAαPOR1 FerH, FefL CFPAPPL1, YFPAdiponectin Receptor 1, two tagged proteins pFer-CFPAPPLI-YFPADIPOFS FerH, FerL CFPAPPL1 , YFPAdiponedin Receptor 2, Iwo lagged proteins pFer-CFPADIPOR1-YFPADIPOR1 FerH, FerL CFRAdiponeclin Receptor 1 , YFPAdiponectin Receptor 1 ,

Abbreviations: CMV Cytomegalovirus; FerH Human Ferritin heavy chain, FerL Human Ferritin light chain; 5'UTR replaced by the mouse and chimpanzeeEFIα with addition of SV40 and CMV enhancers.

[0068] FIGURE 1 illustrates a schematic of a recombinase based single vector system. Two recombination reactions between two supplier vectors and receiver expression vector generating a dual expression vector for immediate protein-protein interaction detection. (a)(i) Insertion of the ORFs 1 and 2 of interest from supplier vectors with Att and LoxP loci respectively into a receiver vector designated by its promoter presence and interaction detection (BiFC or FRET), (ii) ORF1 introduction into a T7 and FRET receiver vector was mediated with LR Clonase. PCR confirmed the successful single expression vectors by forward primer, specific to the ORF1 sequence, and reverse primer, binding specific to the Att insert boundary, to permit fragment amplification. Insertion was successful in clones present in lanes 2-13 which contrasted the no clonase, no insertion negative control vector in lane 14. (iii) Successful insertions performed ORF2 introduction from the LoxP supplier vector with CRE recombinase. Electrophoresis examination of the plasmid isolated from picked clones revealed successful ORF2 presence in lanes 3-9 and 11-13 by the evident 3kb size increase above the no recombinase control in lane 2and 10 without insertion.(b) Dual expression vectors were introduced into the desired cell model and induced to express the proteins of interest (ORF1 and ORF2) fused to the (i) Yc Yn or (ii) CFP YFP

47 set of reporter tags. Positive interaction partners reconstituted fluorescence through BiFC or generated FRET whereas non-interacting proteins did not generate a signal.

[0069] Figure 2 illustrates an example of prokaryotic expression (here E. CoIi) receiver plasmid. The plasmid contains two T7 promoters for expression of the FP-tagged proteins in E. coli. LoxP and att recombination loci ensure in frame insertion of the ORFs of the interest to form fusion proteins with the FP-tags. Other characteristics include expression of antibiotic resistance genes for selective growth of the recombinant clones and other operators (not shown).

[0070] Figure 3 illustrates the generation of dual expression vector generation from two recombination reactions with supplier vectors, (a) Prokaryotic plasmid derivative comprised of two sets of promoters, recombinase sites and ECFP EYFP fluorescent tags ensured translation of tagged proteins. Supplier vector contained proteins (b) ADIPOR1 and (c) APPL1 were inserted into the respective recombinase recognizable, LoxP and Att, sites and generated (d) pT7-ECFPAPPL1-EYFPADIPOR1 dual expression vector. Other elements like antibiotic resistance gene, chemical resistance or sensitive gene are not shown.

[0071] Figure 4 illustrates an example of eukaryotic expression (here mammalian cells) receiver plasmid. The plasmid contains two Pcmv promoters for expression of the FP- tagged proteins in mammalian cells. LoxP and att recombination loci ensure in frame insertion of the ORFs of the interest to form fusion proteins with the FP-tags. Other characteristics include expression of antibiotic resistance genes for selective growth of the recombinant clones and other operators (not shown).

[0072] Figure 5 illustrates an example of eukaryotic expression of a receiver plasmid. The plasmid contains two promoters for expression of the tagged proteins in mammalian cells. The tags (N-terminal part of YFP (Yn) and C- terminal part of YFP(Yc)) initiate BiFC when their protein counterparts are in close vicinity. The plasmid may also contain other markers such as antibiotic resistance genes for selective growth of the recombinant clones (Neo) and other operators (Tet).

[0073] Figure 6 illustrates an example of a receiver plasmid for eukaryotic expression. The plasmid contains two promoters for expression of the tagged proteins in mammalian cells. The tags, N-terminal part of LacZ(ZdC) and C-terminal part of LacZ (ZdN) can form PFC when the counterpart sequences of interest are fused and thereby come into close vicinity with each other. The plasmid may include other functionalities such as expression of antibiotic resistance genes for selective growth of the recombinant clones (Neo) and other operators (Tet).

[0074] Figure 7 illustrates an example of a receiver plasmid for eukaryotic expression after recombination reaction. The plasmid contains two FP-tagged proteins: ECFP-Spartin (SPG20) and EYFP-tubulin. Both fusion proteins have Pcmv promoters (Not shown). Other features include expression of antibiotic resistance genes for selective growth of the recombinant clones and other operators (not shown).

[0075] Figure 8 illustrates another example of eukaryotic expression receiver plasmid after recombination reaction. The plasmid contains two tagged proteins: APPLI-Yc and AdipoR1-Yn. Both fusion proteins have their promoters. Other features include antibiotic resistance genes for selective growth of the recombinant clones and other operators.

[0076] Figure 9 illustrates another example of eukaryotic expression receiver plasmid after recombination reaction. The plasmid contains two tagged proteins: APPLI-LacZdN and AdipoRI-LacZdC. Both fusion proteins have their promoters. Other features include antibiotic resistance genes for selective growth of the recombinant clones and other operators.

Example 2. Chemicals

[0077] Restriction enzymes were purchased from New England BioLabs, MBI Fermentas or Roche. Lipofectamine 2000, and culture media (Invitrogen), Antibiotics (ampicillin, gentamicin, chloramphenicol etc.) were from Promega, Sigma-Aldrich. Other chemicals were the product of Sigma Aldrich.

Example 3. Cell Culture

[0078] E. coli [BL21 (DE3) strain] were transformed with plasmids and inoculated in SOB medium, protein expression was induced with IPTG forlθhrs at 25°C.

[0079] HeIa cells were grown in Dulbecco's modified Eagle medium (DMEM) high glucose, Gibco, Invitrogen 41965-039) containing 10% fetal calf serum (FCS), 10U/ml penicillin,

49 IOOpglml streptomycin, 1% non- essential amino acid (Gibco, InvitrogenH 140-035) and 1% GlutaMax-l Supplement (Gibco, Invitrogen 35050-038).

[0080] Electrotransfection: Amaxa GmbH instrument (Nattermannallee 1 D 50829 KoIn Germany) was used for part of the mammalian cell electrotransfections.

Example 4. Analysis of fluorescence

[0081] A SPECTRAmax GEMINI XS Dual-Scanning Microplate spectrofluorometer (Molecular Dewice) was used to measure the FRET of cells in 96 well format. Flow Cytometry: Flow cytometric data were collected using a BD FACS Vantage SE with digital signal processor (FACS Diva) or using a DakoCytomation MoFIo (Fort Collins, CO).

[0082] Confocal microscopy: Zeiss LSM510Meta or LSM510 confocal microscope (Carl Zeiss. Inc, Thornwood, NY, USA) operating with a 40 mW argon laser. The laser was tuned to lines at 458,488 and 514 nm. The confirmation of the interaction followed the method of Karpova et al.

[0083] Figure 10 shows the results from an exemplary experiment demonstrating the utility of the invention. Receiver vectors contain the ORFs. Supplier vectors were transferred into HeIa cells and the interaction between these two molecules was confirmed by FRET plate reader. Receiver vectors included: TW containing Vimentin-CFP, Vimentin-YFP ORFs; TVaT containing Vimentin-CFP, alpha tubulin-YFP ORFs; TLRI containing adiponectin R1- YFP, APPLI-CFP ORFs. TW and TaT were positive and negative controls of the protein interactions and the TLRI is the testing sample. The experiment shows there is interaction between APPLI and adiponectin R1.

[0084] Figure 11 illustrates FRET detection of protein interactions validated with both plate reader and confocal microscope analysis, (a) The 96-well formatted plate reader detected similar FRETN from protein couplets encoded in both prokaryotic (pT7) and eukaryotic (pFer) DERB vector derivatives. Unspecific p-CFP-YFP established background was surpassed by the p-ECFPEYFP fusion in the prokaryotic (p=0.0014) and eukaryotic (p=0.0005) cells. The APPL1 and ADIPOR1 in E. coli (p=0.011 ) and HeLa cells (p=0.0005) as well as the pT7-CFPAPPL1-YFPADIPOR2 (p=0.003) and pFer-CFPAPPL1- YFPADIPOR2 (p=0.00005) FRETN values all constituted positive interaction, (b) Confocal visualization of eukaryotic (HeLa) cells of CFP (top row), YFP (middle row) and FRET (bottom row) fluorescence distribution ensured dual protein presence. The pFer- CFPAPPL1-YFPADIPOR1 and pFer-CFPAPPL1-YFPADIPOR2 transfected cells surpassed the background FRETN fluorescence validating interaction detection.

[0085] Figure 12 illustrates histograms obtained upon flow cytometry examination of cells Co-expressing CFP and YFP (left image) proteins. In the control, shown on the left, there is no obvious FRET (Area Rl), indicating no obvious interaction between the two proteins in the cells transfected with pCFP-LoxP-YFP-attR, which express CFP and YFP as two separate proteins.

[0086] In the same image (Figure 12) on the right, flow cytometry examination of cells Co- expressing ECFP-Spartin and EYFP-tubulin proteins, demonstrate FRET (Area Rl), indicating interaction between the two proteins, confirming the interaction between Spartin and tubulin.

[0087] Figure 13 illustrates co-expression of Spartin-ECFP (left two circles) and alpha- tubulin-EYFP (right two circled areas) fusion proteins in E. coli. Cells were examined for of EYFP 1 ECFP (upper row) and FRET (lower row). Positive interaction is shown as intensified CFP signal (left two circled areas, arrow) after photobleaching of YFP (right two circled areas).

[0088] Figure 14 shows co-expression of ECFP-Spartin (A, C) and EYFP - tubulin (B, D) fusion proteins in HeIa Cells were examined for of EYFP, ECFP and FRET. Positive interaction is shown as intensified CFP signal (A, C red squared areas) after photobleaching of YFP (B, D red squared areas).

[0089] Figure 15 shows the co-expression of APPLI-Yn and AdipoRI-YC fusion proteins (left panel) and Yn, Yc tag only (right panel) in HeIa Cells, which were examined for YFP(BiFC). Positive interaction is shown as YFP signal (left panel).

[0090] Figure 16 illustrates protein interactions detected with BiFC validated by flow cytometry (top) and fluorescent microscopy (bottom).

(a) The absence of YFP fluorescence was in (i) pFer- YcVI M-YnTUBAI B whereas positive cell populations were prominent with (ii) pFer- YcVIM-YnVIM and (iii) pFer- YcAPPLI- YnADIPORI . (b) The fluorescent microscope background level was established with the (i) pFer- YcVIM-YnTUBAI B and was surpassed by (ii) pFer- YcVIM-YnVIM and (iii) pFer- YcAPPLI -YnADIPORI transfected HeLa cells. [0091] FIGURE 17 shows the co-expression of APPLI-LacZdN and AdipoRI- LacZdC fusion proteins (left panel) and LacZdN, LacZdC tag only (right panel) in HeIa Cells, which were examined for LacZ activity (PFC). Positive interaction is shown as blue staining (left panel).

[0092] FIGURE 18 is an exemplary schematic example illustrating a vector (i) and its use in studying protein-DNA interactions after introduced into the cells (ii-iii). Insertion of Protein A and B into the LoxP and att sites respectively enable the plasmid producing CFP-A, YFP-B, if the A, B interacts, FRET would be observable (ii). If the protein(s) can interact with and enhance the promoter a, increased CFP-A will interact with YFP-B resulted in increased FRET signal (iii) .

[0093] FIGURE 19 shows an exemplary vector in accordance with a further embodiment of the present invention illustrating a Protein-DNA interaction: if the regulator protein expressed can enhance or inhibit the target promoter, a change of CFP signal would be observer. YFP is used as internal expression level control.

[0094] FIGURE 20 shows an example of a vector in accordance with a further embodiment of the present invention illustrating a nucleic acid-nucleic acid interaction. When ShRNA transcribed from the plasmid can promote the degradation of the YFP-target protein coding mRNA, there is be a decrease of YFP signal, CFP is used as internal control.

[0095] The present invention has been described with regard to one or more embodiments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.

Claims

WHAT IS CLAIMED IS:

1 ) A vector comprising at least two unique recombinase recognition sites.

2) A vector according to claim 1 wherein the recombinase recognition sites comprise loxP and Att recombinase sites.

3) A vector according to claim 1 further comprising a selection element.

4) A vector according to claim 3 wherein the selection element is an antibiotic resistance gene.

5) A vector according to claim 1 comprising a tissue specific promoter.

6) A vector according to claim 1 comprising an inducible promoter.

7) A method of preparing a vector comprising at least two open reading frames, said method comprising:

i) inserting a first open reading frame (ORF) sequence into a first supplier plasmid at a first supplier recombinase target site;

ii) inserting a second ORF sequence into a second supplier plasmid at a second supplier recombinase target site;

iii) inserting said first supplier plasmid sequence into an receiver expression vector through a first recombinase reaction; and

iv) inserting said second plasmid sequence into said receiver expression vector through a second recombinase reaction, wherein said first and second recombinase reactions are mediated by two different recombinases.

8) A method of screening for a molecular interaction, said method comprising:

i) inserting a first molecule encoding sequence coupled to be a first tag into a receiver expression plasmid;

ii) Inserting a second molecule-encoding sequence into the same receiver expression plasmid in frame with a second tag; wherein, after introduction of the expression plasmid into a cell, interaction of the first tagged molecule and the second tagged molecule provides a signal.

9) A method according to claim 8 wherein the first and second tag comprise a FRET BiFC or PFC or other reporting/signal pair.

10) A method according to claim 8 wherein the first and second tag comprise a fluorophore/quencher pair.

1 I) A kit for an assay to detect molecular interactions, said kit comprising a vector as defined in claim 1 , a first recombinase and a second recombinase.

12) A system for screening molecular interactions comprising at least two-supplier plasmids each encoding different proteins or molecules and a receiver vector containing at least two unique recombinase recognition sites, which are in frame with signal/reporting molecules that will forming fusion molecules.

13) A system according to claim 12 wherein upon interaction of the two molecules of interest after expression in said receiver expressing vector in a cell, a detectable signal is generated.