# Patent

## Title
System for detecting protease

## Abstract

Disclosed is a system for detecting a protease inside a cell. In one embodiment, the system includes a chimeric protein that comprises as covalently linked components: 1) at least one optionally masked signal protein; 2) at least one protease-specific cleavage site; and 3) at least one detectable amino acid sequence. The invention has a wide spectrum of applications including use in the detection of novel protease inhibitors inside cells and tissue.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
The present application is a continuation of Korean patent application No.10-2001-0048123 as filed on Aug. 10, 2001 and entitled A System For In Vivo Screening Of Protease Inhibitors, the disclosure of which is hereby incorporated by reference.
FIELD OF THE INVENTION
The present invention generally relates to system for detecting protease inside a cell. A preferred system includes a chimeric substrate protein that includes as covalently linked components: 1) at least one optionally masked signal protein; 2) at least one protease-specific cleavage site; and 3) at least one detectable amino acid sequence. The invention has a broad spectrum of important applications including use in screens to detect compounds that block proteases produced by one or more human pathogens.
BACKGROUND
Protease is an enzyme that cleaves a specific peptide bond of proteins. In biological organisms, proteases having specific proteolytic activities, and their inhibitors are involved in regulation of various biological functions. In diverse biological processes, biologically necessary functions can be activated and regulated by proteolytic cleavage of a polyprotein precursor by a protease that results in formation of active proteins. Examples include blood coagulation, immuno-defensive processes, selective transports of proteins through intracellular membranes, viral proliferation in a host cell, etc. Therefore, protease is a major target in the development of specific protease inhibitors as new drugs.
Viral protease inhibitor is a representative example of a protease inhibitor developed as a new drug. Since the viral protease participates in the activation of polyprotein precursors via proteolytic cleavage, the protease is an essential element for the initiation of the viral proliferation and thus for the correct capsid assembly of replicated viruses in the host cell.
Protease inhibitors have been developed to block the proliferation of HIV that causes the acquired immune deficiency syndrome (AIDS). For example, amprenavir, nelfinavir, indinavir, ritonavir, and squinavir have been approved by FDA as drugs for inhibiting the HIV protease, and lopinavir and efavirenz are under clinical studies. Patients who were administered those medicines showed that the number of HIV particulates decreased to about 10% of that before the medicinal treatment. This shows that the protease inhibitor can be used as an efficient medicine. However, several side effects were reported during such treatments (Miller, T. L. et al., 2001), and mutants having mutated protease genes were reported in the cases of prolonged treatments (Jacobsen, H. et al., 1996; Cote, H. et al., 2001). Therefore, more diverse protease inhibitors that can specifically block the proliferation of various mutant HIV viruses need to be developed.
Protease inhibitors have been studied to inhibit other human and animal viruses such as HCV (Kasai, N. et al., 2001) and HERV (Kuhelj, R. et al., 2001). Researches for plant virus diseases have been also performed based on the same concept. For example, inhibition of the proteolytic cleavage of the polyproteins produced by TEV (tobacco etch virus) and PVY (potato virus Y) has been studied by expressing a recombinant protein as a protease inhibitor in a transgenic plant (Gutierres-Campos, R. et al., 1999). A study to identify proteolytic sites of a protease from a plant virus has been also performed (Yoon, H. Y. et al., 2000).
There have been attempts to develop protease inhibitors. For example, in many cases, screening of protease inhibitors has been performed by measuring cleavage of a substrate using electrophoresis, after a protease, its substrate peptide or protein, and a candidate chemical were mixed to react in vitro. As the importance of the proteolytic site has been recognized, peptides having amino acid sequences that are similar to the proteolytic site have been synthesized and used to find protease inhibitors (Kettner, C. A. and Korant, B. D., 1987). As it becomes easier to determine tertiary structures of proteins and also possible to design chemicals using computer simulation, many researches have been conducted to design and synthesize molecules that specifically bind to the active site of the enzyme (Wlodawer, A. and Erickson, J. W., 1993; Rodgers, J. D. et al., 1998; Mardis, K. L. et al., 2001). Also, attempts were made to use fluorescence-labeled substrates in order to increase the efficiency of the protease activity measurement (Ermolief, J. et al., 2000), and to use fragments of antibody expressed in the periplasm of E. Coli as protease inhibitors in order to enlarge the skeletal structure of the protease inhibitor (Kasai, N. et al., 2001).
Most of the protease activity screening methods used currently are performed in vitro. However, in the in vitro screening method, it is not possible to examine various complicated effects such as the transport efficiency of drug candidates into the cell, the stability and cytotoxicity of drug candidates in the cell, etc. Many additional time-consuming experiments are thus necessary before examining the drug candidates selected by the in vitro screening method in a living body. Therefore, a simpler and more generalized in vivo screening method needs to be developed to examine the cellular functions of the protease inhibitor candidates and also to screen more specific protease inhibitors.
There have been efforts to detect proteolytic cleavage and screening of protease inhibitors in more in vivo or in vivo-like conditions.
For example, there has been a report of a method using protease present in isolated vesicles (Hook, V. Y., 2001). Other disclosed methods include in situ zymography using a tissue section (Yi, C. -F. et al., 2001), and a method of treating cells with a polypeptide substrate of a protease (Kuhelj, R. et al., 2001).
However there is increasing recognition that these and related methods are associated with shortcomings.
For example, many of the methods are believed to only approximate in vivo environments. Accordingly, such methods do not always reflect intracellular environments that may substantially impact protease function.
Moreover, many of the prior methods are believed to be limited in terms of sensitivity, selectivity, and convenience. These and other drawbacks are believed to have lowered the efficiency and reliability of past screening attempts.
It would be desirable to have better in vivo methods for detecting protease inside cells that are more sensitive and easier to use. It would be especially desirable to have in vivo methods that can be readily adapted to detect inhibitors of mammalian and viral proteases.
SUMMARY OF THE INVENTION
The present invention relates to a system for detecting protease inside a cell or tissue. In one embodiment, the system includes at least one chimeric protein that includes as covalently linked components: 1) at least one optionally masked signal protein; 2) at least one protease-specific cleavage site; and 3) at least one detectable amino acid sequence. A preferred chimeric protein functions as a “molecular beacon” that changes position inside the cell in the presence of the protease. The invention has a wide range of applications including use in in vivo screens to detect compounds that inhibit or block proteases associated with a human pathogen.
Preferred use of the invention entails that the subject cells or tissue include at least one active protease therein. Suitable proteases include those that are endogenous to the cell, for instance, what are known as “housekeeping” enzymes. Additionally suitable proteases include those that are not naturally-occurring to the cell or tissue. For instance, such a protease can be a consequence of a pathogen infection. Alternatively, presence of the protease inside the cells or tissue can be a result of an experimental manipulation intended to introduce the protease therein. In these embodiments, a change in the subcellular position of the chimeric protein (or a detectable component thereof) is taken to indicative of the presence of the protease inside the cells or tissue. Thus the invention provides as a spatially sensitive “molecular beacon” whose location inside the cells or tissue is indicative of the presence (or absence) of the subject protease.
As will become apparent, the invention is one of general application. That is, it can be used to detect a wide spectrum of proteases inside the cells or tissue. Preferred proteases are capable of cleaving (hydrolyzing), preferably specifically, a cleavage site within the chimeric protein. Sometimes the chimeric proteins will be referred to herein as chimeric substrate proteins to denote cleavage potential by the protease of interest. Site specific cleavage is understood to break the chimeric molecule, generally at or near the specific cleavage site, and release at least one of components therefrom e.g., the optionally masked signal protein, cleavage site, or the detectable amino acid sequence. Preferred release involves at least one of the detectable amino acid sequences but it may involve other components of the chimeric molecule depending on use.
More preferred release of one or more of the components from the chimeric molecule is intended to provide the spatially sensitive molecular beacon. For example, and in one embodiment, the detectable amino acid sequence is released from the chimeric substrate protein and diffuses essentially freely throughout the host cells or tissue. That diffuse signal is readily detectable and can be taken as indicative of presence of the protease. However in another invention embodiment, release of one or more of the detectable amino acid sequences is associated with guidance of the molecule to another subcellular location by the optionally masked signal protein. In this example of the invention, a more focused and higher intensity signal serves as indication of the protease.
In some embodiments of the invention, it will be useful for the chimeric molecule to retain at least one of the detectable amino acid sequences, preferably as an in-frame fusion, even in the presence of the protease inside the cells or tissue. In this instance, localization of the chimeric molecule can be monitored by reference to the detectable amino acid sequence when the protease is present. In most cases, the release and subsequent subcellular localization of the detectable amino acid sequence (alone or in combination with another component of the detectable chimeric protein) is readily visualized in situ by one or a combination of conventional detection strategies.
It is thus an object of the invention to link presence and preferably activity of the protease of interest to a change in localization of the chimeric protein. That change is readily detectable as increase or decrease in signal location and, preferably, intensity. As an example, the subcellular distribution of a detectably labeled chimeric protein (or one or more of the components) can be initially confined to a relatively small location such as an cell organelle. That confinement produces a relatively high signal intensity. However in the presence of protease that specifically cleaves the chimeric protein, the distribution can be much less constrained and even diffuse. That lack of confinement produces a relatively low signal intensity. In this example, the specific cleavage by the protease can be associated with movement of the chimeric protein (or labeled component) from the organelle to a larger space such as the cytosol. Alternatively, signal intensity can increase sharply in embodiments where presence of active protease is linked by the invention to subcellular movement of labeled protein from the cytosol to a more confined space (e.g., organelle or vacuole). In yet another embodiment, the presence of active protease can be associated with little or no change in signal intensity. Instead, the change is monitored by labeled protein moving from one sub-cellular location to another as in, for instance, movement of the chimeric substrate protein from one organelle to another organelle or vacuole.
Practice of the present invention provides a number of important advantages.
For example, the invention provides chimeric proteins that are cleaved specifically by the subject protease to produce labeled (and unlabeled) component proteins. Preferred practice of the invention links the subcellular location and, preferably, the signal intensity of the labeled proteins to the presence (or absence) of the subject protease. This “two-factor” detection strategy provides for highly sensitive and reliable protease detection. That is, both the location of labeled protein and its signal intensity within the cells or tissues can be taken to be indicative of the presence of the active protease. The invention is also highly selective ie., it can readily discriminate between presence of different proteases or isozymes as well as presence of inactive and active versions of the same protease inside cells. Preferred chimeric proteins of the invention can be made with available reagents and standard recombinant manipulations making the invention easy to use.
Additionally, the invention is flexible and can be used to detect active protease in a wide range of cells, typically eukaryotes, including those derived from plants, yeast, fungi, animals, and insects. Preferred chimeric proteins have minimal impact on the gene expression in the cells or tissue, thereby avoiding potentially complicating genetic effects. The invention is also compatible with a variety of suitable protease cleavage sites and detectable amino acid sequences.
Accordingly, and in one aspect, the invention provides a system for detecting a protease inside a cell which system preferably includes at least one of the foregoing chimeric proteins. With respect to the chimeric protein, the order of linkage of each protein component (optionally masked signal protein, protease cleavage site, and detectable sequence) is not important so long as intended results are achieved. Typically however, the linkage order starts from the N-terminus of one component and ends at the C-terminus of another component.
As mentioned, the chimeric protein includes as covalently linked components: 1) at least one optionally masked signal protein; 2) at least one protease-specific cleavage site; and 3) at least one detectable amino acid sequence. Preferably, the chimeric protein includes less than about 10 optionally masked signal proteins, more preferably less than about five of same, typically about 1, 2, or 3 of such signal proteins. By the phrase “optionally masked” is meant that an intended function (typically a trafficking signal) of the signal protein is masked or it is not masked. By the term “masked” is meant that an intended function of the signal protein is substantially reduced or preferably blocked completely, either reversibly or irreversibly, by covalently linking at least one masking sequence to the signal protein. Typically preferred signal proteins that are masked include about 1 to about 2 of such masking sequences. A generally preferred masking sequence consists of less than about 200 amino acid residues, preferably less than about 50 of same, with between from about 3 to about 20 residues being preferred for many applications. A specifically preferred masking sequence for many invention uses is at least one site specific protease cleavage site e.g., 1, 2, 3 or 4 of such sites.
Additionally preferred chimeric proteins in accord with the invention include, as covalently linked components, less than about 10 protease specific cleavage sites, preferably less than about 5 of same with about 1, 2, 3, or 4 of such sites being often preferred.
Still further preferred chimeric proteins include, as covalently linked components, less than about 10 of the detectable amino acid sequences, preferably less than about 5 of same with about 1, 2, 3, or 4 of such detectable amino sequences being preferred generally. In one embodiment, the sequences are fluorescent, phosphorescent or chemiluminescent proteins or functional fragments thereof. A functional fragment of the detectable amino acid sequence is capable of being detected with substantially the same sensitivity as the full-length sequence. In another embodiment, the amino acid sequence is an enzyme or catalytic fragment thereof that can be made fluorescent, phosphorescent or chemiluminscent upon contact with a suitable substrate. Methods for detecting and optionally quantifying signal from the detectable amino acid sequences are known in the field and explained in more detail below.
In another aspect, the invention provides a substantially pure chimeric protein that includes as covalently linked components: 1) at least one optionally masked signal protein; 2) at least one protease-specific cleavage site; and 3) at least one detectable amino acid sequence.
In yet another aspect, the invention features a nucleic acid that includes a sequence that encodes the chimeric protein. In one embodiment, the encoded chimeric protein includes as covalently linked components: 1) at least one optionally masked signal protein; 2) at least one protease-specific cleavage site; and 3) at least one detectable amino acid sequence.
Further provided by the present invention is a vector that includes a nucleic acid encoding the chimeric protein as disclosed herein. Also provided are cells such as plant, yeast, animal, fungi or insect cells, that include and preferably also express the chimeric protein.
Also provided by the present invention is a kit for detecting a protease inside a cell. In one embodiment, the kit includes the system described herein, which system preferably includes at least one of: a) a chimeric protein as described herein; and b) a vector comprising any of the nucleic acids encoding the chimeric proteins disclosed herein. Optionally, the kit may further include a vector comprising a nucleic acid encoding a protease specific to the chimeric protein and/or the cells that include and preferably also express the chimeric protein and the protease. The kit can also be used to screen inhibitors for the protease.
The invention provides additional uses and advantages. For instance, it is also an object of the present invention to provide an effective and generalized method for in vivo screening of inhibitors that are specific to a protease. Thus in one embodiment, the invention provides:

    (1) a chimeric substrate protein comprising at least one signal protein directing transport to a subcellular organelle, at least one proteolytic cleavage site specific to a protease, and at least one fluorescent protein label, and generalized methods for constructing the chimeric substrate protein;
    (2) a recombinant gene comprising a nucleic acid sequence encoding the chimeric substrate protein, which can be used to transform a cell to express the chimeric substrate protein;
    (3) a system wherein the chimeric substrate protein and its specific protease co-exist in a living cell so that the proteolytic cleavage of the substrate by the protease can take place in a living cell;
    (4) an efficient stepwise method for determining the protease activity in vivo by directly identifying the cleavage of the chimeric substrate protein by the protease in the living cell, via direct observation of the cell with the fluorescence signal emitted from the fluorescent protein label(s) conjugated to the substrate; and
    (5) an effective stepwise method for screening inhibitors specific to a protease by using the above system constructed for determination of the protease activity in the living cell.


By addressing the technical problems involved, the present invention provides more realistic in vivo methods for analyzing the activity of a specific protease and screening inhibitors for the protease, in which the protease and its specific substrate co-exist in a living cell so that the cleavage of the substrate by the protease can take place in a living cell and the result can be directly observed from the living cell.
In a related aspect, the invention provides a highly useful method for detecting a protease inside a cell or tissue. In an illustration of the invention, the method includes at least one and preferably all of the following steps:

    
    
        a) introducing, into a subject cell or tissue, a first vector comprising nucleic acid encoding a chimeric protein comprising as covalently linked components: 1) at least one optionally masked signal protein; 2) at least one protease-specific cleavage site; and 3) at least one detectable amino acid sequence,
        b) incubating the cell or tissue under conditions conductive to expressing the chimeric protein encoded by the first vector; and
        c) detecting a change in at least one of the subcellular localization and signal intensity of the chimeric protein (or detectably labeled component thereof) as being indicative of the presence of the protease inside the cell.
    
    


As discussed, the method is flexible and can be readily adapted to suit an intended use. For example, if needed, the method can further include the step of introducing, into the subject cell or tissue, a second vector comprising nucleic acid sequence encoding the protease; and expressing the second vector in the cell or tissue to produce the protease inside the cell or tissue.
The invention also provides a method for detecting and optionally quantifying the in vivo activity of a protease inhibitor. Preferred inhibitors can be endogenous to the cell or tissue of interest. However, in many invention embodiments the inhibitor will be administered to same and include naturally-occurring, synthetic, and semi-synthetic molecules. Such molecules can be obtained from chemical libraries and may include those having known, suspected or completely unknown inhibitor activity. For example, in embodiments in which the activity of a particular protease inhibitor is established, the invention can be used to confirm the activity of the protease in a particular cell, tissue type, or culture conditions. In other embodiments, the invention can be used to screen candidate compounds from the chemical libraries. In one example of the invention, the detection method includes at least one and preferably all of the following steps:

    
    
        a) introducing, into a subject cell or tissue, a first vector comprising nucleic acid encoding a chimeric protein comprising as covalently linked components: 1) at least one optionally masked signal protein; 2) at least one protease-specific cleavage site; and 3) at least one detectable amino acid sequence,
        b) introducing into the cell or tissue a second vector encoding at least one subject protease, preferably one of same,
        c) contacting the cell or tissue with candidate compound,
        d) incubating the cell or tissue under conditions conducive to expressing the chimeric protein encoded by the first vector and the protease encoded by the second vector; and
        e) detecting a change in at least one of the subcellular localization and signal intensity of the chimeric protein (or a detectably labeled component thereof) as being indicative of the presence of the protease inhibitor.
    
    


The foregoing detection method is flexible and can be readily adapted to screen candidate compounds in stand alone, low or high throughput modes. For example, and in one embodiment, the method further includes use of an automated or semi-automated device that is preferably intended to detect the change in subcellular localization and signal intensity of the detectably labeled chimeric protein or labeled component protein thereof. A more particular device includes an optical system adapted to detect the detectable sequence inside the cell or tissue which system can provide output to a user in real-time or as stored output.
If desired, the detectable change in the subcellular localization, signal intensity (or both) of the chimeric protein (or a detectably labeled component thereof) can be monitiored by reference to a suitable control. One suitable control is addition of water, saline or buffer instead of the compound to be tested in step c). Of course, use of a control may not be needed in embodiments in which the characteristics of the chimeric protein, the host cells or tissue, etc. are already established for a particular method.
As discussed, the invention is well-suited to detect protease inhibitors. Known protease inhibitors in accord with the invention are recognized viral disease inhibitors. Because the viral protease is essential for replication and reassembly of viral capsids, the protease inhibitor can be used for treatment of viral diseases by inhibiting the protease to suppress the viral proliferation. Animal viruses can cause diseases such as AIDS and hepatitis etc., and plant viruses reduce crop yield by causing wilting leaves and mottling.
In one aspect, the present invention provides a framework for effectively developing inhibitors specific to various proteases by supplying the substrates and the method for in vivo screening of inhibitors specific to various proteases. This method can be also used to determine the efficiency of the drug candidates screened by the conventional in vitro method.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows schematic diagrams of signal proteins having trafficking signals to subcellular organelles and labeled with fluorescent proteins.
FIGS. 2(a)–(h) show fluorescence photographs visualizing the localized distributions of the signal proteins labeled with the fluorescent proteins when they are expressed correctly in a cell.
(a) shows that Arabidopsis outer envelope protein (AtOEP7) labeled with green fluorescent protein (GFP) is localized in chloroplast envelope;
(b), (c), and (d) show that Rubisco small subunit (RbcS), Chlorophyll a/b binding protein (Cab), and Rubisco activase (RA) labeled with GFP are localized in chloroplast stroma;
(e) shows that F1-ATPase labeled with GFP is localized in mitochondria;
(f) shows that peroxisome targeting motif (SKL) labeled with GFP is localized in peroxisome; and
(g) and (h) show that H+-ATPase and Pleckstrin homology domain (PH) labeled with GFP are localized in the plasma envelope.
FIGS. 3(a)–(b) show schematic diagrams of recombinant genes for NIa protease and an in vivo substrate of NIa protease, RFP:PS(NIa):AtOEP7:GFP:
(a) shows the structure of the recombinant gene constructed to express the chimeric protein RFP:PS(NIa):AtOEP7:GFP used as an in vivo substrate of NIa protease in Example 2 of the present invention, wherein RFP, GFP, PS(NIa), AtOEP7, and 35S indicate red fluorescent protein, green fluorescent protein, the proteolytic cleavage site of the protease, Arabidopsis outer envelope protein, and CaMV35S promoter, respectively; and
(b) shows the structure of the recombinant gene constructed to express NIa protease used in Example 2, wherein NIa represents the coding region of NIa protease from TVMV.
FIG. 4 shows fluorescence photographs observed after expressing the chimeric protein RFP:PS(NIa):AtOEP7:GFP in the Arabidopsis protoplast transformed with the recombinant gene shown in FIG. 3(a):
(a), (b), (c), and (d) are images of green fluorescence signal, red fluorescence signal, and overlap of green and red fluorescence signals, and an image captured under bright field, respectively. The red fluorescence signal observed in chloroplast is auto-fluorescence signal of chloroplast and the red fluorescence signal observed in cytosol originates from the red fluorescent protein.
FIG. 5 shows fluorescence images observed after co-expressing the chimeric protein RFP:PS(NIa):AtOEP7:GFP and NIa protease in the Arabidopsis protoplast transformed with the recombinant genes shown in FIGS. 3(a) and 3(b), showing that the cleavage of RFP:PS(NIa):AtOEP7:GFP by NIa protease can be visualized:
(a), (b), (c), and (d) are images of green fluorescence signal, red fluorescence signal, and overlap of green and red fluorescence signals, and an image captured under bright field, respectively. The red fluorescence signal observed in chloroplast is auto-fluorescence signal of chloroplast and the red fluorescence signal observed in cytosol originates from the red fluorescent protein.
FIG. 6 is representation of a Western blot showing that the cleavage of the chimeric protein RFP:PS(NIa):AtOEP7:GFP by NIa protease takes place in the Arabidopsis protoplast. The case (+) of co-expressing RFP:PS(NIa):AtOEP7:GFP shown in FIG. 3(a) and NIa protease shown in FIG. 3(b) is compared with the case (−) of expressing RFP:PS(NIa):AtOEP7:GFP alone. The protein bands observed at 70 kD and 35 kD correspond to the intact chimeric protein RFP:PS(NIa):AtOEP7:GFP and the fragment protein AtOEP7:GFP produced by the proteolytic cleavage, respectively.
FIG. 7(a)-(j) are drawings showing plasmid maps of various constructs used to express the fusion proteins shown in FIGS. 1: (a) AtOEP7:GFP, (b) AtOEP7:RFP, (c) RbcS:GFP, (d) RbcS:RFP, (e) Cab:GFP, (f) RA:GFP, (g) F1-ATPase:GFP, (h) GFP:SKL, (i) H+-ATPase:GFP, (j) GFP:PH.
FIGS. 8(a) and (b) show plasmid maps for NIa protease and its chimeric substrate protein RFP:PS(NIa):AtOEP7:GFP, respectively. An important part of the nucleic acid sequence for the chimeric substrate protein is noted.
FIGS. 9(a) and (b) show plasmid maps for HIV-1 protease and its chimeric substrate protein RFP:PS(HIV-1):AtOEP7:GFP, respectively. Nucleic acid and protein sequences for the proteolytic cleavage sites (SEQ ID NOs: 57–74) are noted.
FIGS. 10(c)–(d) show schematic diagrams of recombinant genes encoding examples of optionally masked chimeric substrate protein in which trafficking signal of one signal protein remains active. PS indicates the proteolytic cleavage site sequence. FP-1 and FP-2 indicate coding sequences for fluorescent proteins having different fluorescence wavelengths.
FIGS. 11(a)–(c) show fluorescence images observed after expressing a chimeric substrate protein RFP:PS(HIV-1):AtOEP7:GFP in the Arabidopsis protoplast transformed with one of the recombinant plasmids shown in FIG. 9(b). FIGS. 11(a), (b), and (c) are images of green fluorescence signal, red fluorescence signal, and overlap of green and red fluorescence signals, respectively. The weak red fluorescence signal observed in the green fluorescence image is auto-fluorescence of chloroplast.
FIGS. 12(a)–(c) show fluorescence images observed after co-expressing HIV-1 protease and a chimeric substrate protein RFP:PS(HIV-1):AtOEP7:GFP in the Arabidopsis protoplast transformed with the recombinant genes shown in FIGS. 9(a) and (b). FIGS. 12(a), (b), and (c) are images of green fluorescence signal, red fluorescence signal, and overlap of green and red fluorescence signals, respectively. The weak red fluorescence signal observed in the green fluorescence image is auto-fluorescence of chloroplast.
FIGS. 13(a) and (b) shows images observed after expressing a chimeric substrate protein H+-ATPase:PS(NIa):GFP. FIGS. 13(c) and (d) show images observed after co-expressing NIa protease and a chimeric substrate protein H+-ATPase:PS(NIa):GFP. FIGS. 13(a) and (c) are images of green fluorescence signal, and FIGS. 13(b) and (d) are images obtained under bright field. The red fluorescence observed in the green fluorescence images are auto-fluorescence of chloroplast.



DETAILED DESCRIPTION OF THE INVENTION
As discussed, the invention provides a highly useful system for detecting a protease inside a cell or tissue. If desired, the system is readily adapted to detect more than one protease, preferably less than about 3 of same, usually about 1 protease. Preferably, the system includes a chimeric protein that includes as covalently linked components: 1) at least one optionally masked signal protein; 2) at least one protease-specific cleavage site; and 3) at least one detectable amino acid sequence. Typically preferred chimeric proteins consist of less than about 20 components, more preferably less than about 10 of same, with between from about 3 to about 6 components being generally preferred for most of the proteins. The invention has a wide spectrum of important applications including use in screens to detect candidate compounds that reduce or completely block protease activity in vivo.
A “system” according to the invention includes one or more of the chimeric molecules described herein as well as any additional components which may be added thereto such as those which may facilitate solublization or stability of same. Examples include but are not limited to a serum protein such as bovine serum albumin, a buffer such as phosphate buffered saline, or an acceptable vehicle or stabilizer. See generally Reminington's Pharmaceutical Sciences, Mack Pub. Co., Easton, Pa., 1980, for a discussion of acceptable vehicles, stabilizers, etc. Typical systems in accord with the invention will also include at least one of the nucleic acids, vectors, manipulated cells or tissue described herein. In such invention embodiments, the chimeric protein can serve as a useful experimental control. A preferred system includes from between about 1 to 10, preferably less than about 5 and more preferably about 1 of the chimeric proteins dissolved in an acceptable carrier such as water or buffered saline. Preferably, the system is provided sterile.
By the phrase “signal protein” is meant a polypeptide sequence that has either a specific trafficking signal targeting to a subcellular organelle or a specific property related to its localization in a cell such as aggregate formation. Preferred signal proteins can be found throughout this disclosure including the Examples section.
Preferred chimeric proteins according to the invention include an optionally masked signal protein, protease specific cleavage site, and detectable amino acid sequence that are covalently linked together (i.e. fused) by recombinant, chemical or other suitable method. In most embodiments, recombinant approaches will be preferred. Although not generally needed for most invention embodiments, one or more of the components can be fused at one or several sites through a peptide linker sequence. Particular peptide linker sequences will less than about 30 amino acids, more preferably less than about 15 amino acids, still more preferably from about 1 to about 5 amino acids. That peptide sequence can include one or more sites for cleavage by a pathogen induced or host cell induced protease. Alternatively, the peptide linker may be used to assist in construction of the chimeric protein. Specifically preferred chimeric proteins can be referred to as “in-frame” fusion molecules.
As noted, components of the chimeric proteins disclosed herein can be organized in nearly any fashion provided that the protein has the function for which it was intended. And as mentioned, each component of the chimeric protein can be spaced from another component by at least one suitable peptide linker sequence.
For instance, any one of the components of the chimeric protein can include the N-terminus of the protein. Additionally, any one of the components can include the C-terminus of the chimeric protein which terminus may include another component such as a purification tag sequence as discussed below. Unless specified otherwise, the phrase “covalently linked in sequence” means, with respect to an amino acid sequence, peptide bonds bound together in the N to C direction. With respect to a nucleotide sequence, the phrase is meant to denote joining of one nucleoside to another in a 5′ to 3′ direction.
As a more specific example of the system, the chimeric protein includes covalently linked in sequence: 1) the signal protein; 2) the protease-specific cleavage site; and 3) the detectable amino acid sequence. Alternatively, the chimeric protein can include covalently linked in sequence: 1) a masking sequence; 2) the protease cleavage site; 3) the signal protein; and 4) the detectable amino acid sequence. In another embodiment, the chimeric protein for use with the system features covalently linked in sequence: 1) the signal protein; 2) the protease cleavage site; 3) the masking sequence; and 4) the detectable amino acid sequence.
In invention embodiments in which more than one signal protein is needed in the chimeric protein, such a protein can include covalently linked in sequence: 1) a first signal protein; 2) the protease cleavage site; and 3) a second signal protein; and 4) the detectable amino acid sequence. More particularly, such a protein can include covalently linked in sequence: 1) the first signal protein; 2) a first protease cleavage site; 3) the masking sequence; 4) the second signal protein; and 5) the detectable amino acid sequence.
In some instances, it will be helpful to have a system in which the chimeric protein includes more than one protease cleavage site eg., 1, 2, 3 or 4. In such a case, the chimeric protein can include covalently linked in sequence: 1) the masking sequence; 2) a first protease cleavage site; 3) a first signal protein; 4) a second protease cleavage site; 5) a second signal protein; and 6) the detectable amino acid sequence. Alternatively, the chimeric protein can include covalently linked in sequence: 1) a first signal protein; 2) a first protease cleavage site; 3) a second signal protein; 4) a second protease cleavage site; 5) a masking sequence and 6) the detectable amino acid sequence.
In another invention embodiment, the chimeric protein include covalently linked in sequence: 1) the protease-specific cleavage site; 2) the signal protein; and 3) the detectable amino acid sequence. Alternatively, the chimeric protein can include covalently linked in sequence: 1) a first signal protein; 2) a first detectable sequence; 3) the protease cleavage site; and 4) a second detectable sequence. In this invention example, the protein can further include a second signal protein covalently linked between the C-terminus of the protease cleavage site and the N-terminus of the second detectable sequence.
The invention provides for still further chimeric proteins that include covalently linked in sequence: 1) a first signal protein; 2) the protease cleavage site; 3) a second signal protein; and 4) a second detectable sequence. Alternatively, the chimeric protein can include covalently linked in sequence: 1) a first detectable sequence; 2) the protease cleavage site; 3) the signal protein; and 4) a second detectable sequence.
More preferred chimeric proteins in accord with the invention will have a molecular size of less than about 250 kDa, preferably less than about 200 kDa, more preferably a molecular size of between about 25 to about 175 kDa as determined by standard SDS PAGE gel electrophoresis using appropriate molecular weight markers.
A “polypeptide” refers to any polymer preferably consisting essentially of any of the 20 natural amino acids regardless of its size. Although the term “protein” is often used in reference to relatively large proteins, and “peptide” is often used in reference to small polypeptides, use of these terms in the field often overlaps. The term “polypeptide” refers generally to proteins, polypeptides, and peptides unless otherwise noted.
As used herein, the term “cell” is intended to include any primary cell or immortalized cell line, any group of such cells as in, a tissue or an organ. Preferred cells include mammalian cells such of those of human origin, plant cells, yeast, fungi and insect cells. A “host cell” in accord with the invention can be an infected cell or it can be a cell such as E. coli that can be used to propagate a nucleic acid or vector as described herein.
It will be appreciated that particular uses of the invention will often require a specific chimeric protein configuration. Choice of a particular chimeric protein component or group of components will be guided by recognized parameters including the signal protein(s), protease specific cleavage site(s), and detectable amino acid sequences selected, the protease(s) to be monitored, and the level of sensitivity or selectivity required for an application.
By way of example, the invention encompasses embodiments in which the chimeric protein includes one signal protein, one detectable sequence and one protease specific cleavage site. In this example, it will often be helpful to include a masking sequence linked to the N- or C-terminus of the signal protein. That is, it is envisioned that linkage of the masking sequence to the detectable sequence will be less preferred for some of invention uses.
Other specific uses of the invention will typically require other specific chimeric protein configurations. For instance, where a particular protein has one signal protein, and one protease specific cleavage site positioned between two detectable amino acid sequences, it will often be desirable to remove one of the detectable amino acid sequences to optimize the system.
Still other uses of the invention will be facilitated by having multiple masking sequences which can be a signal sequence, detectable amino acid sequence or other suitable sequence such as a protease specific cleavage site. In these embodiments, having one or more additional masking sequences e.g., the protease cleavage site, may not be necessary to achieve maximal use of the system. However in embodiments that include two different signal proteins, one protease cleavage site and two detectable amino acid sequences, one or two masking sequences thereon can be helpful.
Additionally, particular chimeric proteins may include removable tagging sequences that, in some embodiments, may assist identification and/or purification of the chimeric protein. An example is 6×His and MYC tags. Other suitable tagging sequences are well known in the field and can be used with the invention if desired.
Practice of the invention is fully compatible with a wide variety of signal proteins and functional fragments thereof that are masked or unmasked. Preferred examples are generally sufficient to localize the chimeric protein (or detectable component of that protein such as the detectable sequence) to an organelle or other subcellular compartment. By the phrase “compartment” is meant an internally limited space such as a vacuole, peroxisome, mitochondrion, etc.
Preferred plant signal proteins localize the chimeric protein or at least one of its components to the nucleus, golgi body, lytic vacuole, storage vacuole, peroxisome, mitochondrion, endoplasmic reticulum, plasma membrane, or chloroplast of a plant cell. More preferred plant signal proteins include AtOEP7; RbcS; Cab; RA; SKL; F1-ATPase; PH; FAPP; H+-ATPase; or a functional fragment thereof. Preferred animal signal proteins localize the chimeric protein or at least one of its components to the nucleus, golgi body, storage vacuole, lysosome, peroxisome, endoplasmic reticulum, plasma membrane, or mitochrondrion of an animal cell. Examples include human peptide methionine sulfoxide reductase (MSRA), cytochrome b2, 11-beta-hydroxysteroid dehydrogenase (11β-HSD), G9-AKL, peroxisomal integral membrane protein 47 (PMP47); or a functional fragment thereof. See the Examples below for sequences of preferred animal signal proteins.
As discussed, practice of the invention is compatible with use of signal proteins that are functional in animal cells. Typical examples include, but are not limited to, the animal signals shown in the following Table I.












TABLE I







Protein
Organelle
Position of signal
Type1









MSRA
Mitochondria
N-terminal
A



Cytochrome b2
Mitochondria
N-terminal
A



11β-HSD
ER
N-terminal
A & B



G9-AKL
Peroxisome
C-terminal
A



PMP47
Peroxisome
A domain in the
B





middle of protein







1A type: Signal protein can be used as a masked signal protein.



B type: Signal protein can be used as unmasked signal protein.






See Hansel A et al. FASEB J 2002 June; 16: 911–31 (human peptide methionine sulfoxide reductase; “MSRA”); Bomer U et al. (1997) J Biol Chem 272 30439–30446 (Cytochrome b2); Naray-Fejes-Toth A and Fejes-Toth G (1996) J Biol Chem 271 15436–1544 (11β-hydroxysteroid dehydrogenase (11β-HSD)); McNew J A and Goodman J M (1994) J Cell Biol 127 1245–1257 (G9-AKL); Dyer et al., (1996) J. Cell Biol. 133 269–280 (PMP47 (peroxisomal integral membrane protein 47)).
By the phrase “functional fragment”, when used herein to describe a signal protein, is meant a fragment of a particular signal protein that is capable of providing at least about 70%, preferably higher than about 90% of the shuttling function of the full-length sequence. Methods for detecting and quantifying signal protein function are known and include localization and quantification of signal intensity using the fluorescence imaging techniques described in the Examples section.
As should be apparent, the invention is flexible and not limited to use of any particular protease specific cleavage site. For example, the cleavage site can be specifically cleaved by a mammalian or viral protease. By the phrase “specifically cleaved” is meant that peptide bonds in a specified protease cleavage site are specifically broken (i.e. hydrolyzed) by a subject protease. That is, the protease cleavage sites are not broken by proteases which naturally occur in the host cell including what is generally referred to as housekeeping proteases. Specific cleavage of those protease cleavage sites can be monitored by a variety of techniques including SDS-polyacrylamide gel electrophoretic methods.
Preferred protease cleavage sites are those that are specifically hydrolyzed by a protease associated with a human pathogen e.g., yeast, bacterium, fungus, nematode, virus or protozoan. More specific examples include cytomegalovirus (CMV); herpes simplex virus (HSV); hepatitis virus, preferably type A or C; a plasmodium, human immunodeficiency virus (HIV), Kaposi's sarcoma-associated herpes virus (KSHV), yellow fever virus, flavivirus, rhinovirus, or a plasmodium such as P. falciparum, P. vivax, P. ovale, or P. malariae. Typically, the plasmodia cause malaria or various medical complications relating to malaria. There is recognition that the proteases plasmepsin I and plasmepsin II are implicated. In embodiments in which HSV is of interest, the protease will be the maturational protease of HSV.
A variety of particular HIV-1 and HCV protease specific cleavage sites have been disclosed. See eg., Gluzman, I. Y. et al., J. Clin. Invest., 94:1602 (1994); Grakoui, A. et al., J. of Virol., 67:2832 (1993); Kolykholov, A A. et al., J. of Virol., 68:7525 (1994); and Barrie, K. A. et al., Virology, 219:407 (1996), the disclosures of which are incorporated by reference.
Additional pathogen-specific proteases and specified cleavage sites have been described and can be used in accord with the present invention. For example, an HSV-1 maturational protease and protease cleavage site has been described. See e.g. Hall, M. R. T. and W. Gibson, Virology, 227:160 (1997). Further, the plasmepsins I and II have been found in the digestive vacuole of P. falciparum. The corresponding proteinase cleavage sites have also been disclosed. See e.g., Moon, R. P., Eur. J. Biochem., 244:552 (1997).
Additional protease specific cleavage sites for use with the invention are specifically cleaved by a mammalian protease associated with blood coagulation, apoptosis, or the extracellular matrix. See the Examples and discussion that follows.
Practice of the invention is compatible with use of one or a combination of detectable amino acid sequences e.g., those that are directly or indirectly fluorescent, phosphorescent, luminescent or chemiluminescent. In embodiments in which two or more of such detectable sequences are used, the emission wavelength of one of the detectable sequences will often be different from at least one other of the detectable sequences. For example, a preferred detectable sequence is derived from certain well-known jellyfish fluorescent protein including those that are recognized to emit green, red, and yellow light under appropriate excitation conditions.
As discussed, the invention further provides substantially pure chimeric proteins. Such chimeric proteins can be separated and purified by appropriate combination of known techniques. If desired, such proteins can include one or more purification tags as described herein. These methods include, for example, methods utilizing solubility such as salt precipitation and solvent precipitation, methods utilizing the difference in molecular weight such as dialysis, ultra-filtration, gel-filtration, and SDS-polyacrylamide gel electrophoresis, methods utilizing a difference in electrical charge such as ion-exchange column chromatography, methods utilizing specific affinity such as affinity chromatograph, methods utilizing a difference in hydrophobicity such as reverse-phase high performance liquid chromatograph and methods utilizing a difference in isoelectric point, such as isoelectric focusing electrophoresis, metal affinity columns such as Ni-NTA. See generally Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (1989); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1989); and Ausubel et al., Short Protocols in Molecular Biology, John Wiley & Sons, New York (1999) for disclosure relating to these methods.
It is preferred that the chimeric proteins of the present invention be substantially pure. That is, the chimeric proteins have been isolated from cell substituents that naturally accompany it so that the chimeric proteins are present preferably in at least 80% or 90% to 95% homogeneity (w/w). Chimeric proteins having at least 98 to 99% homogeneity (w/w) are most preferred for many pharmaceutical, clinical and research applications. Once substantially purified the chimeric protein should be substantially free of contaminants for cell culture and related applications. Once purified partially or to substantial purity, the soluble chimeric proteins can be used therapeutically, or in performing in vitro or in vivo assays as disclosed herein. Substantial purity can be determined by a variety of standard techniques such as chromatography and gel electrophoresis.
A suitable host cell can be used for preparative purposes to propagate nucleic acid encoding a desired chimeric protein. Thus a host cell can include a prokaryotic, plant or eukaryotic cell in which production of the chimeric protein is specifically intended. Thus host cells specifically include yeast, fly, worm, plant, frog, mammalian cells, plant cells and organs that are capable of propagating nucleic acid encoding the chimeric protein. Non-limiting examples of mammalian cell lines which can be used include CHO dhfl-cells (Urlaub and Chasm, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)), 293 cells (Graham et al., J Gen. Virol., 36:59 (1977)), myeloma cells like SP2 or NSO (Galfre and Milstein, Meth. Enzymol., 73(B):3 (1981)). Other suitable cells are disclosed in Sambrook et al., supra.
Host cells capable of propagating nucleic acid encoding a desired chimeric protein encompass non-mammalian eukaryotic cells as well, including insect (e.g., Sp. frugiperda), yeast (e.g., S. cerevisiae, S. pombe, P. pastoris., K. lactis, H. polymorpha; as generally reviewed by Fleer, R., Current Opinion in Biotechnology, 3(5):486496 (1992)), fungal and plant cells (eg., Arabidopsis and Nicotinia). Also contemplated are use of certain prokaryotes such as E. coli and Bacillus. 
Nucleic acid encoding a desired chimeric protein can be introduced into a host cell by standard techniques for transfecting cells. The term “transfecting” or “transfection” is intended to encompass all conventional techniques for introducing nucleic acid into host cells, including calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection, viral transduction and/or integration. Suitable methods for transfecting host cells can be found in Sambrook et al. supra, and other laboratory textbooks.
The present invention further provides a production process for isolating a chimeric protein of interest. In the process, a host cell (e.g., a yeast, fungus, insect, bacterial or animal cell), into which has been introduced a nucleic acid encoding the protein of the interest operatively linked to a regulatory sequence, is grown at production scale in a culture medium in the presence of the chimeric protein to stimulate transcription of the nucleotides sequence encoding the chimeric protein of interest. Subsequently, the chimeric protein of interest is isolated from harvested host cells or from the culture medium. Standard protein purification techniques can be used to isolate the protein of interest from the medium or from the harvested cells. In particular, the purification techniques can be used to express and purify a desired chimeric protein on a large-scale (i.e. in at least milligram quantities) from a variety of implementations including roller bottles, spinner flasks, tissue culture plates, bioreactor, or a fermentor.
Thus the invention further provides a nucleic acid sequence encoding the chimeric substrate protein. The nucleic acid encoding the chimeric substrate protein can be used to transform a cell to express the chimeric substrate protein in the cell. In order to transform the cell, the recombinant gene must include a promoter and other regulatory nucleic acid sequences operably linked to the coding region of the chimeric substrate protein. Choice of a promoter will be guided by recognized parameters, typically selection of the host cell.
As discussed, the invention also provides nucleic acid sequences and particularly DNA sequences that encode the present chimeric proteins. Preferably, the DNA sequence is carried by a vector suited for extrachromosomal replication such as a phage, virus, plasmid, phagemid, cosmid, YAC, or episome. In particular, a DNA vector that encodes a desired chimeric protein can be used to facilitate preparative methods described herein and to obtain significant quantities of the chimeric protein. The DNA sequence can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. A variety of host-vector systems may be utilized to express the protein-coding sequence. These include mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used. See generally Sambrook et al., supra and Ausubel et al. supra.
In general, a preferred DNA vector according to the invention comprises a nucleotide sequence linked by phosphodiester bonds comprising, in a 5′ to 3′ direction a first cloning site for introduction of a first nucleotide sequence encoding a chimeric protein as described herein. If desired, the protein can be linked to DNA encoding one or more suitable tag sequences. FIGS. 7(a)–(j); 8(a)–(b); and 9(a)–(c) provide examples of such vectors.
In some invention embodiments, it will be preferred that the chimeric protein encoded by the DNA vector be provided in a “cassette” format. By the term “cassette” is meant that the encoded protein (or a component thereof) can be readily substituted for another component by standard recombinant methods. In particular, a DNA vector configured in a cassette format is particularly desirable when it is useful to “swap” one site specific protease cleavage site or detectable amino acid sequence for another. See FIGS. 7(a)–(j); 8(a)–(b); and 9(a)–(c) in which a core vector is used to express a variety of chimeric proteins.
More specifically, it is envisioned that in some cases, certain pathogen serotypes, especially viral strains, may be associated with individual protease cleavage sites specific for that serotype or strain. In this regard, the emergence of drug resistant HIV serotypes has been particularly problematic. In this case, one or more existing protease cleavage sites in a DNA vector formatted as a cassette can be replaced with other pre-determined protease cleavage sites as needed. Particular protease cleavage sites can be selected in accord with presence of the pathogen in individual human patients.
Significantly, the present invention can serve as an effective “warning system” that can register changes in protease activity in a subject cell or tissue. For instance, in cases where a PCR or hybridization experiment has indicated presence of genomic DNA encoding a pathogen-associated protease, use of the present invention can detect presence of active protease in the cells or tissue. This feature of the invention is useful in a variety of settings including cell culture in which pathogen contamination is known or suspected.
As discussed, the invention can be used to detect the presence of one or more proteases in a host cell or tissue of interest. The methods and compositions described herein are especially useful for detecting and analyzing protease inhibitor molecules, which as discussed, may be naturally-occurring or be part of a pool of such molecules (ie., a chemical library).
A. Use of the Invention to Screen Candidate Protease Inhibitors
1. General Considerations
In addition to the plant protoplasts as used in the examples described in this invention, normal plant cells with cell wall or human, animal, or insect cells can also be used to screen protease inhibitors according to the present invention. Plant cells will often be preferred because transformation and cultivation are easier and also identification of the protein localization can be facilitated due to the regular spherical shape of the protoplast. However, other cells and tissue may be useful e.g., if the protease of interest cannot be expressed as an active form in plant cells. For instance, such a protease may be difficult to express due to inappropriate post-translational modification. Suitable animal or insect cells may be used to overcome this problem if it arises. In such case, signal proteins that can work in the selected cell must be used. For example, if human, animal, or insect cells are to be used for screening, the chloroplast targeting signal proteins such as AtOEP7, RbcS, Cab, and RA cannot be used because chloroplasts are not present in these cells. For human, animal, or insect cells, signal proteins targeting to other subcelluar organelles such as mitochondria, peroxisome, plasma membrane, etc can be used. In addition, the vector system including the promoter and other regulatory elements must be selected appropriately depending on the cells to be used.
For in vivo screening of protease inhibitors according to the present invention, transformed cells that can express both a protease of interest and a chimeric substrate protein specific to the protease are prepared. The most preferred method to prepare the transformed cell is to co-transform the cell with a recombinant plasmid encoding the protease and a recombinant plasmid encoding a chimeric substrate protein provided by the present invention. If the protease is a viral protease, viral infection could also be used to express the protease in the transformed cell.
Methods for transforming a wide variety of plant, animal, yeast, fungi and insect cells are established. For instance, methods for transforming cells by introducing recombinant plasmids include, but are not limited to, chemical-mediated methods using PEG (polyethylene glycol), potassium phosphate, or DEAE-dextran, cationic lipid-mediated lipofection, microinjection, electroporation, electrofusion, and DNA bombardment. Depending on the type of the cell to be used, an appropriate transformation method has to be selected and the conditions need to be optimized to achieve efficient transformation. If protoplasts of plant cells such as Arabidopsis or Tobacco protoplasts are to be used, the PEG-mediated transformation method described in Example 1(c) is a preferred method. The conditions given in Example 1(c) are optimized for Arabidopsis protoplasts. If normal plant cells with cell wall are to be used, DNA bombardment with a gene gun or the PEG-mediated transformation method could be used depending on the type of the plant cell. For human, animal, or insect cells, potassium phosphate-mediated or DEAD-dextran-mediated transfection method or cationic lipid-mediated lipofection method can be used to transform the cells.
To screen protease inhibitors, the transformed cells need to be contacted by a candidate compound during the expression of the protease and the chimeric substrate protein. Typically, a candidate compound is added to the solution containing the transformed cells and the resulting solution is incubated at appropriate temperature. The candidate inhibitor can be one selected from the group consisting of chemical compounds, peptides, mixtures of chemical compounds or peptides, and extracts of natural products. The incubation time to express the proteins can vary depending on the type of the cell and the incubation temperature. It could range about 1 hr to several days. For the Arabidopsis protoplast, the incubation time could be as short as 4 hrs and as long as about a week depending on the incubation conditions. When the Arabidopsis protoplasts are incubated in the W5 solution at 22° C., preferred incubation time ranges from 12 to 48 hrs. The concentration of the candidate inhibitor that can be examined is about 0.1 to 100 μg/ml. In typical screening, concentration of about 1 to a few μg/ml can be used. In the case that a large number of candidate compounds need to be screened, mixtures of about 5–30 candidate inhibitors can be used in the first round of the screening.
Nearly any compound or group of compounds can be screened for anti-protease activity in accord with this invention. Examples include, but are not limited to, cytokines, tumor suppressors, antibodies, receptors, muteins, fragments or portions of such proteins, and active RNA molecules, e.g., an antisense RNA molecule or ribozyme. A preferred compound for screening purposes is a synthetic or semi-synthetic drug (referred to sometimes as a “small molecule”). For example, a pool of derivatives of known inhibitors of human viral pathogens can be readily tested by the present methods. See e.g., U.S. Pat. Nos. 6,420,438; 6,329,525; 6,287,840; 6,147,188; and 6,046,190 (disclosing a variety of testable molecules and derivatives thereof).
It is possible to use the invention to screen additional compounds. See Pillay et al. (1995) Rev. Med. Virol. (disclosing a variety of potential viral protease inhibitor compounds); and Wei et al. (1995) Nature, 373: 117 (disclosing indinavir, ABT-538); Ho et al. (1992) Ann. Intern. Med. 113: 111 (disclosing an anti-herpes agent). Also, derivatives of the forgoing specific compounds can be screened in accord with the invention including, but not limited to, saquinavir and derivatives thereof.
In order to identify inhibition of the protease activity by the candidate compound, it is preferred to monitor the fluorescence images of the transformed cells as a function of time. The expression time could vary depending on the condition of the cells and therefore the proteolytic activity or inhibition could appear in different times. If the Arabidopsis protoplasts are the cells to be used, preferred time sequence to monitor the fluorescence images is 12, 18, 24, 36, and 48 hrs after the expression is started. If the chimeric substrate protein has two or more different fluorescence labels, it is preferred to monitor fluorescence images at two or more fluorescence wavelengths specific to the fluorescence labels. It is preferred to monitor the bright field image because it can facilitate identification of the subcellular organelles. In order to facilitate identification of the protease inhibitors, it is also preferred to monitor the fluorescence images of the transformed cells that are not contacted with the candidate inhibitor for comparison. A standard fluorescence microscope equipped with multicolor fluorescence filter sets such as Zeiss Axioplan fluorescence microscope or Nikon E800 fluorescence microscope can be used to monitor the fluorescence images with magnification of 200×, 400×, or 600×. The scanning confocal microscope can be used to obtain higher resolution images.
It will be apparent that the invention is compatible with the construction and use of a wide spectrum of chimeric substrate proteins. See Example 1, for instance.
2. Illustrative Use of the NIa Protease
To measure the protease activity using the chimeric substrate protein constructed as described above, NIa protease of Tobacco Vein Mottling Virus (TVMV) was used as a model system in the Examples of the present invention. NIa protease is one of the best-characterized viral proteases, and it is known that it cleaves seven specific sites of the polyprotein produced by TVMV. In order to achieve the optimum protease activity, six amino acid residues (P6–P1) on N-terminus and four amino acid residues (P1′–P4′) on C-terminus are needed and four conserved amino acid residues (V-R-F-Q) must be included at P4–P1 of the substrate protein. If any one of the four conserved amino acid residues is mutated to glycine (Gly), the proteolytic cleavage of the substrate protein cannot take place (Yoon, H. Y. et al., 2000).
When the recombinant gene for expressing the chimeric substrate protein, RFP:PS(NIa):AtOEP7:GFP including the proteolytic site (PS(NIa)) of NIa protease was introduced into a protoplast by a polyethyleneglycol-mediated transformation method, GFP and RFP were accumulated as large aggregates in cytosol (FIG. 4). This result corresponds to the case in which the protease does not function. In this case, accumulation of the aggregates in cytosol is suspected to result from hydrophobic interactions among the hydrophobic regions of the AtOEP7 proteins. In the case that the red fluorescence signal of the chimeric substrate protein is not readily distinguishable from auto-fluorescence of chloroplast (FIG. 4(b)), it is possible to clearly determine the reaction of the substrate by using two different fluorescence labels and observing whether or not the green fluorescence coincides with the red fluorescence in the overlapped fluorescence image (FIG. 4(c)). When the plasmid encoding NIa protease and the plasmid encoding RFP:PS(NIa):AtOEP7:GFP were introduced together in the transformation, green and red fluorescent signals were separated into chloroplast outer envelope membrane and cytosol, respectively (FIG. 5). This result suggests that NIa protease successfully cleaved the proteolytic site of the chimeric substrate protein to generate two proteins, RFP and AtOEP7:GFP. The protease reaction can be identified by observing the green fluorescence signal translocated to chloroplast outer envelope membrane by the action of the signal protein (FIG. 5(a)), the red fluorescent signal dispersed in cytosol (FIG. 5(b)), and the overlapped image of both fluorescence signals (FIG. 5(c)).
The efficiency of the present invention for identifying the result of the enzyme reaction can be more clearly observed by comparing with the control experiment. Comparing FIGS. 4(a) and 5(a), it can be observed that the proteolytic cleavage activates the masked trafficking signal of the signal protein, inducing the change in the distribution of the fluorescence. Comparing FIGS. 4(b) and 5(b), it can be observed that the red fluorescent protein used as the signal masking protein plays a role in altering the distribution of the fluorescence signal in addition to the masking of the trafficking signal.
Accordingly, it is proposed that the chimeric substrate protein comprising a chimeric protein including a signal protein and a fluorescent protein, and a signal masking protein including a proteolytic site can be used as a substrate to determine the activity of a protease and also to screen its inhibitors in vivo.
3. High Throughput Screening Assays: General Considerations
The in vivo protease inhibitor screening method provided by the present invention can be easily adapted to use in a high throughput assay. The high throughput screening method comprises contacting the cells transformed to express a protease and its chimeric substrate protein with a candidate compound, incubating the transformed cells, obtaining fluorescence images of the transformed cells, converting the fluorescence images into digital data, and analyzing the digital data to determine whether the candidate compound has inhibited the protease.
In the high throughput screening of the present invention, equal amounts of the transformed cell solution are loaded into arrays of wells in standard microtiter plates with 96 or 384 wells, and different candidate compounds are added to each well. The transformed cells in the microtiter plates are then incubated to express the protease and its chimeric substrate protein at a controlled environment (at appropriate temperature, humidity, and air composition). Fluorescence images of the transformed cells in each well are obtained using a fluorescenece microscope after a preselected incubation times.
The high throughput screening apparatus consists of (1) an incubator for transformed cell sample arrays in multiples of microtiter plates, (2) an automated sampler, (3) an automated fluorescence microscope equipped with a XY-translation sample stage and a high resolution digital camera. After a preselected incubation time, the automated sampler samples a small portion of the transformed cell solution from each well and loads it to an observation plate such as a slide glass. Sampling can be done in a parallel manner using multiple tips or pipets or in a sequential manner using a single tip or pipet. After each sampling, the tip(s) or pipet(s) is subject to be washed with appropriate washing solution. The transformed cell samples are loaded on the observation plate in a form of arrays at preselected positions. The XY translation stage holds and moves the observation plate in the XY direction to locate the each sample under the microscope objective. A Z-axis focus drive moves either the microscope objective or the observation plate in the Z direction for focusing. For each sample, fluorescence images at preselected fluorescence wavelengths are captured using the digital camera, fed into a PC, and stored as a digital data. An automation controller is provided to control the pipet sampler, the XY translation stage, and the Z focus drive. The PC provides a display and a data analysis software.
The hits for the protease inhibitors can be automatically determined by examining the subcellular fluorescence distribution. In general, the extent of the localization or dispersion of the fluorescence signal within the size of the observed cell could be calculated from the digital image data and used as criteria for decision. For some cases, pattern or shape of the fluorescence signal distribution could be used as criteria for decision.
An example of a high throughput screening protocol suitable for use with the invention has been disclosed in U.S. Pat. No. 5,989,835 and PCT Application WO 00/79241 A2.
The following Tables 2 and 3 provide sequence information for use with the invention:







TABLE 2







SEQ ID NOs of PCR Primers










5′ primer
3′-primer













Partial AtOEP7
1
 2


Partial RbcS
3
 4


Partial Cab
5
 6


Partial RA
7
 8


Partial F1-ATPase
9
10


SKL
11 
12


H+-ATPase
13 
14


Substrate protein for NIa protease
15 
16


HIV-1 protease
17 
18


Substrate proteins for HIV-1 protease
19, 20, 21, 22, 23, 24,
28



25, 26, 27
















TABLE 3







SEQ ID NOs of Signal Proteins










Nucleic acid sequence
Protein sequence















Partial AtOEP7
29
30



Partial RbcS
31
32



Partial Cab
33
34



Partial RA
35
36



Partial F1-ATPase
37
38



H+-ATPase
39
40



Partial PH
41
42



Partial FAPP
43
44










The following provides illustrative nucleic acid and protein sequence information for use with the invention. Examples of proteases and their cleavage sites are provided in SEQ ID NOs: 45–50, 55–74, and 77–120. Examples include NIa protease (SEQ ID NOs: 45 and 46) and its cleavage sites (SEQ ID NOs: 47–50), HIV-1 protease (SEQ ID NOs: 55 and 56) and its cleavage sites (SEQ ID NOs: 57–74), HCV NS3 protease (SEQ ID NOs: 77 and 78) and its cleavage sites (SEQ ID NOs: 79–84), HSV-1 protease (SEQ ID NOs: 85 and 86) and its cleavage sites (SEQ ID NOs: 87–90), HTLV-1 protease (SEQ ID NOs: 91 and 92) and its cleavage sites (SEQ ID NOs: 93–96), HCMV protease (SEQ ID NOs: 97 and 98) and its cleavage sites (SEQ ID NOs: 99–102), APP beta-secretase (SEQ ID NOs: 103 and 104) and its cleavage site (SEQ ID NOs: 105 and 106), caspase 3 (SEQ ID NOs: 107 and 108) and its cleavage site (SEQ ID NOs: 113 and 114), the large subunit of caspase 3 (SEQ ID NOs: 109 and 110), the small subunit of caspase 3 (SEQ ID NOs: 111 and 112), human blood coagulation factor II (SEQ ID NOs: 115 and 116) and its cleavage site (SEQ ID NOs: 117 and 118), and human blood coagulation factor XI (SEQ ID NOs: 119 and 120).
Nucleic acid and protein sequences of two NIa protease substrate proteins are provided in SEQ ID NOs: 51–54 and those for a HIV-1 protease substrate protein are provided in SEQ ID NOs: 55 and 56.
The following discussion relates to Korean application No.10-2001-0048123 in which additional uses and advantages of the present invention have been disclosed.
As provided therein, the invention provides important chimeric substrate proteins that can be used to screen protease inhibitors in vivo. As particularly disclosed therein, the invention relates to a system for screening protease inhibitors and it provides: (i) a chimeric substrate protein constructed to induce change in the subcellular localization and distribution of fluorescence by the specific function of a protease, (ii) a recombinant gene comprising a nucleic acid sequence encoding the chimeric substrate protein that can be used to express the chimeric substrate protein in a cell, (iii) a method to identify the activity of the protease by detecting the subcellular localization and distribution of fluorescence under the circumstance that the protease and the chimeric substrate protein are present together in a cell so that a proteolytic cleavage by the protease can take place in the cell, and (iv) a method to screen protease inhibitors in vivo using the chimeric substrate protein and the method described above.
More particularly, the Korean application No. 10-2001-0048123 discloses that such chimeric substrate proteins can be used in a cell in which the signal protein directs trafficking to a subcellular organelle when expressed in a cell. In further detail, the trafficking signal toward a specific subcellular organelle, included in the signal protein, can be inactivated by linking a signal masking protein to the N- or C-terminal of the signal protein. In the present invention, a signal protein is linked to a signal masking protein with a proteolytic cleavage site of a protease so that the trafficking of the signal protein can be activated or inactivated depending on the cleavage at the proteolytic site. In other words, the trafficking of the chimeric substrate protein, in which the signal protein and the signal masking protein are linked with the proteolytic site, does not occur until the signal masking protein is cleaved off by the protease. Such cleavage induces normal trafficking of the signal protein. In the present invention, the signal protein and/or the signal masking protein are labeled with fluorescent proteins so that the activity of the protease can be determined by measuring changes in the localization and distribution characteristics of the fluorescence signal. Therefore, the chimeric protein used as a substrate of the protease in the present invention has the following characteristics:
(1) The chimeric substrate protein includes at least one signal protein that has a trafficking signal directing transport to a specific subcellular organelle.
(2) The chimeric substrate protein includes at least one proteolytic cleavage site for a specific protease.
(3) The trafficking signal of the signal protein clarified in (1) is inactivated by linking the proteolytic cleavage site to the signal protein or by linking a signal masking protein to the signal protein through the proteolytic cleavage site.
(4) The inactivated trafficking signal of the signal protein can be activated when cleavage at the proteolytic site occurs by the protease.
(5) The chimeric substrate protein is labeled with at least one fluorescent protein and the fluorescence signal from the cell changes depending on the proteolytic cleavage by the protease.
As discussed in the Korean application No. 10-2001-0048123, the invention further provides for a method for measuring the protease activity in vivo using the chimeric substrate protein described herein. Also provided is method for screening protease inhibitors using the method for measuring the protease activity.
As further disclosed in the Korean application No. 10-2001-0048123, to measure the protease activity in vivo, a protease and a chimeric substrate protein specific to the protease must co-exist in a cell.
Also, the recombinant gene for the chimeric substrate protein according to one invention aspect is introduced into the cell to express the chimeric substrate protein in the cell. The target protease for screening protease inhibitors can be an endogeneous protease present in the cell or an exogeneous protease expressed by transforming with a recombinant gene or infecting with a virus. However, when an endogeneous protease is a target, the accuracy and efficiency of the screening may be low e.g., due to the difficulties in regulating the expression of the protease and also in detecting under a low level of the protease expression. Therefore, the present invention provides a system for more efficiently determining the protease activity in vivo, wherein a specific protease can be over-expressed or expressed in a regulated manner by transforming the cell with a recombinant gene or infecting the cell with a virus. Viral infection can be used in the case of a viral protease. However, regulation of viral protease expression is not completely understood, it is more preferable to use a protease expressed by transforming the cell with a recombinant gene encoding the protease. Since the expressed protease is located in cytosol, it is necessary to make the chimeric substrate protein located in cytosol as well. Therefore, a system for efficiently measuring the protease activity in vivo can be constructed by using the chimeric substrate protein according to the first aspect of the present invention, wherein the trafficking signal of the signal protein included in the chimeric substrate protein is masked.
Furthermore, inhibitors of the protease can be selected by detecting changes in the localization and distribution of the fluorescence signal, caused by treating the cell with a candidate chemical before, after, or at the same time as the protease and its chimeric substrate protein are expressed in the cell.
In many enzymatic reactions, reactants cannot be completely converted to products. In the case that the reaction is inhibited by an inhibitor, it could also be partially inhibited rather than completely inhibited. Moreover, when multiple cells are observed, the level of the protease activity in each cell could vary considerably. Therefore, there may be a considerable ambiguity in determining the inhibition activity of the protease inhibitor if the method used for determining the protease activity has low sensitivity or low contrast. In order to avoid such ambiguity, it is important in the construction of the chimeric substrate protein to select a signal protein that can induce a clearly distinguishable change in the cellular localization and distribution of the fluorescence signal depending on the proteolytic cleavage. In addition, the efficiency of determining the inhibition activity can be enhanced employing two or more fluorescent proteins having different fluorescence wavelengths. In Example 2 of the present invention, GFP and RFP were employed in the construction of the chimeric substrate protein so that they can be localized in different subcellular organelles upon proteolytic cleavage.
As discussed above, certain signal proteins according to the invention are optionally masked. In this embodiment, the signal protein included in the chimeric substrate protein provided by the present invention is inactivated due to the signal masking by the proteolytic site or the signal masking protein linked to the signal protein and thus the chimeric substrate protein is present in cytosol. The signal protein can be activated by the proteolytic cleavage to direct its trafficking to a subcellular organelle. Different characteristics of signal proteins need to be considered in selecting the signal protein whose trafficking signal is inactivated in the chimeric substrate protein. The endosomal trafficking proteins are translocated to the Golgi body, the lytic vacuole, the storage vacuole, or the plasma membrane as enclosed in the endoplasmic reticulum as soon as they are synthesized. The trafficking signals of the endosomal trafficking proteins are recognized during the translation process. Therefore, it may not be possible to inactivate and activate the trafficking signals of the endosomal trafficking proteins by simply linking and cleaving off the proteolytic site with or without the signal masking protein. Therefore, these endosomal trafficking proteins are less adequate for use in the present invention as the signal proteins whose trafficking signals are inactivated. Proteins expressed in the cytosol and transported directly to the subcellular organelles can be used as the signal protein because their trafficking signals can be masked according to the present invention. Among the latter signal proteins, signal proteins having the nuclear location signal (NLS) are not dependant on the N- or C-terminus and thus it is difficult to control the trafficking of these signal proteins by linking or cleaving off the proteolytic site with or without the signal masking protein. It is thus desirable to select a signal protein that has a trafficking signal targeting to mitochondria, chloroplast, or peroxisome. In the case of plant cells, it is more preferable to use a chloroplast targeting signal protein because chloroplast is relatively big and thus easier to detect its shape and distribution.
The signal masking protein included in the chimeric substrate protein of the present invention inactivates the trafficking signal of the signal protein by being linked to the signal protein through the proteolytic site. The signal masking protein can be an amino acid, a peptide, or a protein that is linked to the signal protein through the proteolytic site. For some cases, it may be possible to inactivate the trafficking signal of the signal protein by linking the proteolytic site alone to the signal protein. The signal masking protein and the proteolytic site must not interfere the binding of the substrate with the protease. In addition to the simple signal masking, the signal masking protein can also be used to change the overall characteristics of the chimeric substrate protein, or to attach an additional trafficking signal or a fluorescent label. For example, if another signal protein is selected as a signal masking protein, this signal protein will move to its target organelle when cleaved off by the protease. In such case, if this signal protein is also labeled with a fluorescent protein, it will be possible to more clearly identify the cleavage of the substrate protein by detecting two different fluorescence signals. In another example, a fluorescent protein can be used as a signal masking protein. In this case, the fluorescent protein formed by the proteolytic cleavage will stay in cytosol. It is thus possible to increase the efficiency of determining the protease activity or detecting the inhibition activity of a protease inhibitor by observing distinctively the fluorescence signal from cytosol and that from the subcellular organelle to which the signal protein formed by the proteolytic cleavage is translocated.
One or a combination of standard recombinant methods can be employed to make the chimeric substrate proteins disclosed herein. That is, the method for constructing the chimeric substrate protein can be characterized by its expandability. For example, if at least two proteolytic sites are included in the chimeric substrate protein of the protease, trafficking of two or more signal proteins can be observed. If proteolytic sites for two or more different proteases are introduced, the activities of two or more proteases can be examined simultaneously.
Detailed methods for selecting the signal protein and the signal masking protein and constructing the chimeric substrate protein are as follows. The mark—↓—indicates the proteolytic site of the protease and M represents the signal masking protein.
(1) In the case that the trafficking signal is present at the N-terminus of the signal protein (nS), the signal masking protein is placed at the N-terminal side of the signal protein: (M-↓-nS).
(2) In the case that the trafficking signal is present at the C-terminus of the signal protein (Sc), the signal masking protein is placed at the C-terminal side of the signal protein: (Sc-↓-M).
(3) In the case that another signal protein S′ is used as the signal masking protein, S′ can be selected to possess the trafficking signal in the opposite side compared to that of S. The trafficking signals of the two signal proteins can be simultaneously masked by constructing the chimeric substrate protein with the trafficking signal parts of the two signal proteins being linked: (Sc-↓-nS′ or S′c-↓-nS).
(4) In the construction as in (3), if two proteolytic sites are to be introduced, the signal masking protein has to be placed between two signal proteins: (Sc-↓-M-↓-nS′ or Sc′-↓-M-↓-nS).
(5) If the trafficking signals of a signal protein (S) and the other signal protein (S′) that acts as a signal masking protein are on the same sides, another signal masking protein that masks the trafficking signal of S′ needs to be linked: (M-↓-nS′-↓-nS or Sc-↓-Sc′-↓-M).
By extending the constructions of (1) to (5) described above, the chimeric substrate protein can be constructed with three or more proteolytic sites: (Scm-↓- . . . -↓-Sc2-↓-Sc1-↓-M-↓-nS1-↓-nS2-↓- . . . -↓-nSn).
The construction methods described above are the representative examples of possible construction methods.
In addition to the cases described above, wherein the trafficking signals of all the signal proteins included in the chimeric substrate protein are masked, there are other construction methods that can provide the chimeric substrate protein with its proteolytic cleavage to occur in cytosol. If the trafficking signal of only one signal protein included in the chimeric substrate protein remains active and all the trafficking signals of the rest of the signal proteins are masked, the chimeric substrate protein will be translocated to a subcellular organelle that is the target of the active signal protein. Herein, in the case that the translocated chimeric substrate protein resides on the membrane of a subcellular organelle, the chimeric substrate protein can be constructed in which at least one proteolytic site and at least one inactivated signal protein is exposed to cytosol so as to achieve the same effect as in the case of using the chimeric substrate protein with all the trafficking signals of the signal proteins being masked. If this chimeric protein having only one signal protein remaining active is used as a substrate, the proteolytic reaction can occur by the protease present in cytosol because the proteolytic site is exposed to cytosol, although the chimeric substrate protein is not freely dispersed in cytosol. In this case, the inactivated signal protein exposed to cytosol becomes activated by the proteolytic cleavage. Therefore, the fragment protein that includes this activated signal protein will be translocated to a specific subcellular organelle that is different from the subcellular organelle where the chimeric substrate protein resided, resulting in alteration in the localization and distribution of the fluorescence signal attached to the activated signal protein.
Another possible method for constructing the chimeric substrate protein with the trafficking signal of only one signal protein remaining active is to link a fluorescent protein such as GFP or RFP, that has no trafficking signal, to the proteolytic site exposed to cytosol, instead of linking a signal protein. In this case, since the fluorescent protein produced by the proteolytic cleavage becomes dispersed in cytosol, the distribution of the fluorescent signal changes from a specific cellular organelle to cytosol. In this case, however, clearness for distinguishing whether the fluorescence signal is located in membrane or cytosol could be low due to incompleteness of the enzyme reaction. In addition, there may be considerable difficulties in constructing the chimeric substrate protein with only one signal protein remaining active, because detailed information is needed not only for the subcellular organelle to which the signal protein is translocated, but also for the orientation and position of the translocated signal protein.
Signal proteins targeting to outer membranes of mitochondria, chloroplast, and nucleus, peroxisome membrane, and plasma membrane can be used as the signal protein that remains active in the chimeric substrate protein. Signal proteins that can specifically bind to phospholipids can also be used. Examples includes Pleckstrin homology domain (PH) that binds to phosphatidylinositol 4,5-diphosphate (PI(4,5)P2) as shown in FIG. 2(h) and pleckstrin homology domain of FAPP (family A (phosphoinositide binding specific) member 3) that binds to phophatidylinositol 4-phosphate (PI(4)P).
In Example 2, Western blot analysis was performed for the cells transformed to express a protease and its chimeric substrate protein constructed according to the present invention, and it was confirmed that the protease reaction was taking place correctly. Comparing with the Western blot analysis in which the cells were lysed and the crude extract was electrophoresed and identified with antibody, the system provided by the present invention in which the identification can be carried out by simply observing the cell itself is more efficient in terms of both time and cost.
In Example 1 of the present invention, a system was constructed in which trafficking and distribution of a protein can be visually determined in a cell. Chimeric proteins were constructed to visualize the localization of the proteins after translocation by selecting signal proteins that have trafficking signals to subcellular organelles and labeling with a fluorescent protein. It is shown that localization of the chimeric protein can be identified by observing the fluorescent image of the cell transformed with a recombinant plasmid that includes a recombinant gene for the chimeric protein.
Among these chimeric proteins, AtOEP7:GFP was selected and a proteolytic site of a protease was linked to construct a chimeric substrate protein that can be used for screening protease inhibitors in vivo. AtOEP7 is a protein targeting to Arabidopsis chloroplast outer envelope membrane, and it was already mentioned that it is more desirable to select a chloroplast targeting protein for plant cells. A signal masking protein was linked to the N-terminal side of AtOEP7:GFP, because AtOEP7 has its trafficking signal at N-terminus. As a signal masking protein, red fluorescent protein (RFP) was selected. Therefore, the substrate chimeric protein was constructed such that the green fluorescence localizes to chloroplast envelope membrane and the red fluorescence distributes in cytosol after the proteolytic cleavage.
In general, preparation of the fusion molecules of the invention includes conventional recombinant steps involving, e.g., polymerase chain amplification reactions (PCR), preparation of plasmid DNA, cleavage of DNA with restriction enzymes, preparation of oligonucleotides, ligation of DNA, isolation of mRNA, introduction of the DNA into a suitable cell, and culturing of the cell. Additionally, the chimeric proteins described herein can be isolated and purified in accordance with well known techniques including methods that comprise standard electrophoretic, centrifugation and chromatographic manipulations. See generally, Sambrook et al., supra; and Ausubel et al., supra; for disclosure relating to these methods.
DNA and protein sequences described herein can be obtained from a variety of public sources including those specifically mentioned. A preferred source is the National Center for Biotechnology Information (NCBI)-Genetic Sequence Data Bank (Genbank) at the National Library of Medicine, 38A, 8N05, Rockville Pike, Bethesda, Md. 20894. Genbank is also available on the internet. See generally Benson, D. A. et al., Nucl. Acids. Res., 25:1 (1997) for a description of Genbank.
Other reagents used in the examples such as antibodies, cells and viruses can be obtained from recognized commercial or public sources such as Linscott's Directory (40 Glen Drive, Mill Valley Calif. 94941), and the American Type Culture Collection (ATCC) 12301 Parklawn Drive, Rockville, Md. 20852.
All documents mentioned herein are incorporated herein by reference.
The present invention is further illustrated by the following Examples. These Examples are provided to aid in the understanding of the invention and are not construed as a limitation thereof.
EXAMPLE 1
Detection of Chimeric Proteins and Trafficking to the Subcellular Organelles
(a) Construction of Recombinant Plasmids for Expression of the Chimeric Proteins
The coding region of the outer envelope membrane protein of Arabidopsis, AtOEP7, that is a homolog of OEP14 of pea, was amplified by polymerase chain reaction (PCR) from Arabidopsis genomic DNA using two specific primers (5′-GACGACGACGCAGCGATG and 5′-GGATCCCCAAACCCTCTTTGGATGT) designed to remove the natural termination codon. Then, it was ligated in frame to the 5′ end of the coding region of the green or red fluorescent protein to construct recombinant plasmids for AtOEP7:GFP and AtOEP7:RFP, respectively. The ligated genes were regulated by the 35S promoter in the recombinant plasmids. The same method was used for construction of other recombinant plasmids described hereafter.
For expression of the chimeric protein of Rubisco (ribulose bisphospate carboxylase) complex protein, the coding region for the transit peptide of the small subunit of the Rubisco complex was amplified by PCR from a λZAPII cDNA library using two specific primers (5′-CCTCAGTCACACAAAGAG and 5′-ACTCGAGGGAATCGGTAAGGTCAG). The resulting PCR product was subcloned into pBluescript and subsequently ligated in-frame to the 5′ end of the coding region of GFP or RFP to construct recombinant plasmids for RbcS:GFP or RbcS:RFP, respectively.
For expression of the chlorophyll a/b binding protein, the corresponding gene was amplified by PCR from a λZAPII cDNA library using two specific primers (5′-TAGAGAGAAACGATGGCG and 5′-GGATCCCGTTTGGGAGTGGAACTCC) to construct a recombinant plasmid for Cab:GFP.
The coding region for the transit peptide of rubisco activase (RA) was amplified by PCR from a λZAPII cDNA library using two specific primers (5′-TCTAGAATGGCCGCCGCAGTTTCC and 5′-GGATCCATCTGTCTCCATCGGTTTG) and ligated to the 5′ end of the coding region of GFP to construct a recombinant plasmid for RA:GFP.
The coding region for the transit peptide of F1-ATPase-(accession number: D88374) was amplified by PCR from a λZAPII cDNA library using two specific primers (5′-CTTTAATCAATGGCAATG and 5′-CCATGGCCTGAACTGCTCTAAGCTT) and ligated to the 5′ end of the coding region of GFP to construct F1-ATPase:GFP.
A recombinant plasmid for the peroxisome targeting protein, GFP:SKL, was constructed by PCR amplification with 326GFP (Davis, S. J. and Viestra, R. D., 1998) as a template using two specific primers (5′-CCGTATGTTACATCACC and 5′-TTATAGCTTTGATTTGTATAGTTCATCCAT).
The full length H+-ATPase (AHA2 of Arabidopsis) was amplified with two specific primers (5′-GAGATGTCGAGTCTCGAA and 5′-CTCGAGCACAGTGTAGTGACTGG) using the above method and ligated to the 5′ end of the coding region of GFP to construct a recombinant plasmid for H+-ATPase:GFP.
A recombinant plasmid for the chimeric protein of the PH domain (Pleckstrin homology domain), GFP:PH, was constructed according to the method described by Kost, B. et al. (1998).
Schematic structures of the chimeric proteins expressed from the recombinant plasmids constructed according to the above method are shown in FIG. 1.
(b) Preparation of Protoplasts
Leaf tissues (5 g) of 3–4 week-old Arabidopsis plants grown on soil in a green house were cut into small squares (5–10 mm2) with a new razor blade and incubated with 50 ml of the enzyme solution (0.25% Macerozyme R-10, 1.0% Cellulase R-10, 400 mM mannitol, 8 mM CaCl2, 5 mM Mes-KOH, pH 5.6) at 22° C. with gentle agitation (50–75 rpm). After incubation, the protoplast suspension was filtered through 100 μm mesh and protoplasts were collected by centrifugation at 46×g for 5 min. The pelleted protoplasts were resuspended in 5 to 10 ml of the W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5 mM glucose, 1.5 mM Mes-KOH, pH 5.6), overlaid on top of 20 ml of 21% sucrose, and centrifuged at 78×g for 10 min. The intact protoplasts at the interface were transferred to 20 ml of the W5 solution. The protoplasts were pelleted again by centrifugation at 55×g for 5 min, resuspended in 20 ml of the W5 solution, and then incubated on ice for 30 min.
(c) Isolation of the Recombinant Plasmid DNAs and Transformation of the Protoplast
Recombinant plasmids were purified using Qiagen columns (Valencia, Calif.) according to the manufacture's protocol. To transform the protoplasts with the DNA, the protoplasts were pelleted again at 46×g for 5 min and resuspended in the MaMg solution (400 mM Mannitol, 15 mM MgCl2, 5 mM Mes-KOH, pH 5.6) at a density of 5×106 protoplasts/ml. The recombinant plasmid constructs were introduced into the Arabidodsis protoplasts by PEG (polyethylene glycol)-mediated transformation method (Jin et al., 2001). About 20–50 μg of the plasmid DNA at a concentration of 2 μg/μl was mixed with 300 μl of the protoplast suspension, and 325 μl of the PEG (polyethylene glycol) solution (400 mM Mannitol, 100 mM Ca(NO3)2, 40% PEG 4000) was added and gently mixed. The mixture was incubated for 30 min at room temperature. After incubation, the mixture was diluted with 10 ml of W5 solution. The protoplasts were recovered by centrifugation at 50×g for 5 min, resuspended in 3 ml of the W5 solution, and incubated at 22° C. in the dark.
(d) Expression of the Chimeric Proteins and Observation of Their Subcellular Localizations
The recombinant plasmid DNAs constructed in Example 1(a) were used to transform the protoplasts according to the method described in Example 1(c). The expression of the chimeric proteins after the transformation was monitored as a function of time by capturing images using a fluorescence microscope (Axioplan fluorescence microscope, Zeiss, Germany) equipped with a cooled charge-coupled device camera. The filter sets used were XF116 (exciter: 474AF20, dichroic: 500DRLP, emitter: 510AF23), XF33/E (exciter: 535DF35, dichroic: 570DRLP, emitter: 605DF50), and XF137 (exciter: 540AF30, dichroic: 570DRLP, emitter: 585ALP) (Omega, Inc, Brattleboro, Vt.) for GFP, RFP, and auto-fluorescence of chlorophyll, respectively. Data were then processed using Adobe (Mountain View, Calif.) Photoshop software and presented in pseudo-color format.
Green fluorescence of the chimeric protein of AtOEP7:GFP was observed at the outer envelope membrane of the chloroplast (FIG. 2(a)). This result indicates that the chimeric protein comprising the chloroplast envelope targeting signal peptide and the fluorescent protein label was correctly targeted to the chloroplast envelope membrane.
Localization of the green fluorescence from the chimeric proteins RbcS:GFP, Cab:GFP and RA:GFP are presented in (b), (c), and (d) of FIG. 2, respectively. As shown in the figures, RbcS:GFP was located in the stroma of chloroplast, and Cab:GFP and RA:GFP also emitted the fluorescence in chloroplast. These results indicate that the chimeric proteins comprising the signal peptide of RbcS, Cab, or RA, and the fluorescence protein label were targeted to chloroplast.
The green fluorescence signals of the chimeric proteins, F1-ATPase:GFP, GFP:SKL, and H+-ATPase:GFP were observed in mitochondria, peroxisome, and plasma membrane (FIGS. 2(e)–(g)), respectively. The red fluorescent signals in these results were auto-fluorescence of chloroplast.
The green fluorescence signal of GFP:PH, comprising the PH domain (Pleckstrin homology domain) that specifically binds to a phospholipid, was distributed on the plasma membrane where phosphatidylinositol 4, 5-diphosphate (PI(4, 5)P2) was present (FIG. 2(h)).
FIGS. 7(a)–(j) is explained in more detail as follows. The figure shows plasmid maps of the recombinant plasmids used to express the fusion proteins depicted in FIG. 1. The methods for constructing these recombinant plasmids are described in the present example. Nucleic acid and protein sequences of the signal proteins included in these fusion proteins are provided in SEQ ID NOs: 29–42. In addition, nucleic acid and protein sequences of partial pleckstrin homology domain of FAPP (family A (phosphoinositide binding specific) member 3) are provided in SEQ ID NOs: 43 and 44.
These signal proteins are examples of the signal proteins that can be used as either inactivated signal proteins or active signal proteins according to the present invention. AtOEP7, RbcS, Cab, RA, F1-ATPase, and SKL (peroxisome targeting sequence) are examples of signal proteins that are inactivated by masking in the chimeric substrate protein. AtOEP7, H+-ATPase, PH, and FAPP are examples of signal proteins that remain active in the chimeric substrate protein.
EXAMPLE 2
Detection of Cleavage of the Chimeric Substrate Protein by Protease
(a) Construction of the Recombinant Plasmids
The recombinant plasmid for NIa protease was constructed by placing the coding region of NIa protease under the control of the 35S promoter in a pUC vector.
The recombinant plasmid for Arabidopsis outer envelope membrane protein:green fluorescent protein (AtOEP7:GFP) was constructed by ligating the AtOEP7 coding region without the termination codon to the 5′ end of the coding region of the green fluorescent protein in the 326GFP vector (obtained from Arabidopsis Biological Resource Center, Ohio University, USA). The cleavage site of the protease, VRFQ, was ligated to the N-terminus of AtOEP7:GFP by PCR amplification of this plasmid with two primers (5′ primer, 5′-CCCGGGGTGTGCGCTTCCAGGGAAAAACTTCGGGAGCG and 3′ primer, 5′-GAGCTCTTATTTGTATAGTTCATC). The PCR product (SmaI and XhoI fragment) was then ligated to HindIII (filled in) and XhoI sites of the 326RFP-nt vector to construct the recombinant plasmid for the chimeric substrate protein RFP:VRFQ:AtOEP7:GFP (FIG. 3(a)).
(b) Transformation with the Recombinant Plasmids
Transformation was performed as in (b) and (c) of Example 1.
(c) Detection of the Fluorescent Proteins Using a Fluorescence Microscope
Detection of the fluorescent protein was carried out as in (d) of Example 1. The final recombinant plasmid for expression of the chimeric substrate protein RFP:VRFQ:AtOEP7:GFP was introduced to the protoplast and the subcellular trafficking was examined for 24–36 hrs after transformation. As shown in FIG. 4, the chimeric substrate protein was localized as large speckles or aggregates in the protoplast but not targeted to the chloroplast. Both of the red and green fluorescent signals were observed in the same speckle.
In the next experiment, it was examined whether NIa protease can cleave the cleavage site in the chimeric substrate protein. When the protoplast was co-transformed with the recombinant plasmid for NIa protease, the green fluorescence signal was observed at the envelope membrane of the chloroplast whereas the red fluorescence signal was observed as uniformly dispersed in the cytosol, as shown in FIG. 5. Furthermore, the red and green fluorescence signals no longer overlapped each other, strongly suggesting that NIa protease cleaved the chimeric substrate protein in vivo.
(d) Western Blot Analysis
The transformed protoplasts were harvested and lysed in 50 μl of cell lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM DTT, 1 mM EDTA, 50 mM NaCl). Expression of the chimeric substrate protein RFP:VRFQ:AtOEP7:GFP and cleavage of the chimeric substrate protein into RFP and AtOEP7:GFP by NIa protease were identified by Western blot analysis using monoclonal anti-GFP antibody (Clontech, Inc) and the ECL kit (Amersham, Inc).
As shown in FIG. 6, when NIa protease was not co-transformed, the chimeric substrate protein RFP:VRFQ:AtOEP7:GFP was detected at the expected size of 70 kDa. In contrast, when NIa protease was co-transformed, the anti-GFP antibody detected a protein at 35 kDa, an expected size of AtOEP7:GFP. This result indicates that the chimeric substrate protein RFP:VRFQ:AtOEP7:GFP was cleaved into two proteins, RFP and AtOEP7:GFP. Therefore, this result clearly demonstrates that NIa protease can cleave the chimeric substrate protein in vivo and the cleavage reaction can be easily assayed by detecting the localization of the green fluorescence signal at the chloroplast envelope membrane and the dispersed distribution of the red fluorescence signal in the cytosol.
FIG. 8(a)-(b) are explained in more detail as follows. The figure shows plasmid maps of the recombinant plasmids used in the present example to express NIa protease and its chimeric substrate protein RFP:PS(NIa):AtOEP7:GFP, respectively. Nucleic acid and protein sequences of NIa protease and its cleavage sites are provided in SEQ ID NOs: 45–50. Full nucleic acid and protein sequences of this chimeric substrate protein are given in SEQ ID NOs: 51 and 52, respectively.
EXAMPLE 3
In vivo Screening System for HIV-1 Protease Inhibitors
A convenient in vivo screening system for detecting inhibitors of the human immunodeficiency virus (HIV-1) protease was performed as follows.
(a) Construction of Recombinant Plasmids for Expression of HIV-1 Protease
To construct a recombinant plasmid for HIV-1 protease, the coding region of HIV-1 protease was PCR amplified with two primers: (5′-TCTAGAATGCCTCAGGTCACTCTTTGG-3′ and 5′-CTCGAGTCAAAAATTTAAAGTGCAACC-3′) using pHX2BΔRT as a template. The pHX-2BΔRT is a plasmid clone containing HX2B (GenBank accession number K03455) without the reverse transcriptase coding region. The amplified product was subcloned into pBluescript-T vector and subsequently cloned into XbaI and XhoI sites of a pUC vector under the control of the 35S promotor.
The plasmids maps for HIV-1 protease is shown in FIG. 9(a) and the nucleic acid and protein sequences are given in SEQ ID NOs: 55 and 56, respectively.
(b) Construction of Recombinant Plasmids for Expression of Chimeric Substrate Proteins for HIV-1 Protease
Recombinant plasmids for total of 9 chimeric substrate proteins were constructed.
Primers used are as follows: Forward primers used were







5′-CCCGGGTAGCCAAAATTACCCTATAGTGGGAAAAACTTCGGGAGCG-








3′,



 







5′-CCCGGGTGCAAGAGTTTTGGCTGAAGCAGGAAAAACTTCGGGAGCG-








3′,



 







5′-CCCGGGTGCTACCATAATGATGCAGAGAGGAAAAACTTCGGGAGCG-








3′,



 







5′-CCCGGGTAGACAGGCTAATTTTTTAGGGGGAAAAACTTCGGGAGCG-








3′,



 







5′-CCCGGGTCCAGGGAATTTTCTTCAGAGCGGAAAAACTTCGGGA GCG-








3′,



 







5′-CCCGGGTAGCGTGCCTCAAATAGGAAAAACTTCGGGAGCG-3′,


 


5′-CCCGGGTACTTTAAATTTTCCCATTAGCGGAAAAACTTCGGGA GCG-








3′,



 







5′-CCCGGGTGCAGAAACCTTCTATGTAGATGGAAAAACTTCGGGAGCG-








3′, and



 







5′-CCCGGGTAGGAAAGTACTATTTTTAGATGGAAAAACTTCGGGAGCG-








3′.







Common reverse primer used was 5′-CTCGAGTTATTTGTATAGTTCATC-3′. These primers were designed to contain the proteolytic sites of the HIV-1 protease. The underlined regions in the forward primers correspond to the proteolytic site sequences (SEQ ID NOs: 57, 59, 61, 63, 65, 67, 69, 71, and 73). PCR amplification was performed with one of the forward primers and the common primer described above using the plasmid for the NIa protease substrate protein (SEQ ID NO: 51) as a template. PCR product was restriction digested with SmaI and XhoI and subcloned into HindIII (filled-in) and XhoI digested 326RFP-nt vector. Therefore, each of the 9 resulting plasmids contains a DNA sequence encoding one of the chimeric substrate proteins of the composition RFP:PS(HIV-1):AtOEP7:GFP with PS(HIV-1) being one of the proteolytic site sequences of the HIV-1 protease. Nucleic acid and protein sequences of one of these chimeric substrate proteins are provided in SEQ ID NOs: 75 and 76.
(c) HIV-1 Protease Inhibitor Screening in vivo
Preparation and transformation of the protoplast of Arabidopsis leaf tissue were performed according to the procedures described in Example 1(b) and (c). Detection of the fluorescence images of the transformed protoplasts was carried out as described in Example 1(d) (but using a Nikon E800 fluorescence microscope using the same types of the filter sets).
As a working example for detecting inhibition of HIV-1 protease in vivo, the protoplasts were transformed with a recombinant plasmid for a HIV-1 protease substrate protein, RFP:PS(HIV-1):AtOEP7:GFP. The proteolytic site sequence included in the chimeric substrate protein was RQANFLG (SEQ ID NO: 64). The transformed protoplasts were incubated at 22° C. in the W5 solution for 18–48 hrs to express the chimeric substrate protein, and the subcellular localization of the fluorescence signals from the expressed chimeric substrate protein was monitored using a fluorescence microscope.
As shown in FIG. 11, both the green and red fluorescence signals were observed at the same positions in the cytosol as large speckles or aggregates, but not targeted to the chloroplast. This result indicates that the chimeric substrate proteins were not cleaved and thus they are present in the cytosol as an un-cleaved form. This data corresponds to results when complete inhibition of HIV-1 protease occurs.
Detection of HIV-1 proteolytic activity was performed as follows. Protoplasts were co-transformed with the recombinant plasmid for HIV-1 protease and the recombinant plasmid for a HIV-1 protease substrate protein RFP:PS(HIV-1):AtOEP7:GFP. The proteolytic site sequence included in the chimeric substrate protein was RQANFLG (SEQ ID NO: 64). The subcellular localization of the fluorescence signals from the expressed chimeric substrate protein was monitored 18–48 hrs after the transformation using a fluorescence microscope. As shown in FIG. 12, the red fluorescence signal was observed as uniformly dispersed in the cytosol, while most of the green fluorescence signal was observed around the chloroplasts. These results indicates that the chimeric substrate proteins were cleaved by HIV-1 protease.
This system can be used to detect molecules that decrease the dispersed red fluorescence signal and the chloroplast-trageted green fluorescence signal, and thus block or inhibit HIV-1 protease activity in the protoplasts.
FIGS. 9(a) and (b) are explained in more detail as follows. The figures show plasmid maps for the recombinant plasmids for HIV-1 protease and the chimeric substrate proteins RFP:PS(HIV-1):AtOEP7:GFP. These recombinant plasmids can be used to express HIV-1 protease and the chimeric substrate protein in plant cells such as Arabidopsis thaliana, Tobacco, etc. Nucleic acid and protein sequences of HIV-1 protease and its cleavage sites are provided in SEQ ID NOs: 55–74. Full nucleic acid and protein sequences of the chimeric substrate protein used in Example 3 are provided in SEQ ID NOs: 75 and 76, respectively.
EXAMPLE 4
Preparation and Use of Optionally Masked Chimeric Proteins with One Signal Protein Remaining Active
As discussed above, it is an object of the present invention to provide recombinant chimeric proteins in which at least some of the signal proteins are masked by at least one suitable amino acid sequence. For instance, all the signal proteins of a particular chimeric protein in accord with the invention can be masked or they can be unmasked as needed. Alternatively, a portion of the signal component of the chimeric protein can be unmasked and the remaining signal(s) can be masked. Such “optionally masked” chimeric proteins provide significant flexibility to the invention and have a wide range of important applications.
For instance, such chimeric proteins can be used in screens to detect in vivo protease activity by virtue of a change in subcellular localization of one or more fluorscence signals of the chimeric protein. Such a screen is highly sensitive, namely because it can register slight changes in the spatial distribution of the chimeric protein. Choice of whether to mask or unmask one or more than one signal proteins included within a subject chimeric molecule will be guided by intended invention use.
FIG. 10 provides an illustrative collection of “optionally masked” chimeric proteins. More specifically, the figure shows schematic diagrams of recombinant genes encoding chimeric substrate protein in which the trafficking signal of one signal protein remains active. H+-ATPase is used as an example of the active signal protein whose trafficking signal is not masked in the chimeric substrate protein. Other examples that can be used as the active signal protein include AtOEP7, PH, and FAPP. AtOEP7 is used as an example of the inactivated signal protein whose trafficking signal is masked by linking a proteolytic cleavage site or a signal masking protein through a proteolytic cleavage site. Other examples that can be used as the inactivated signal protein include RbcS, Cab, RA, F1-ATPase, and SKL. These chimeric substrate proteins are designed to induce a change in the subcellular fluorescence signal distribution upon proteolytic cleavage. Nearly any of the protease cleavage sequences disclosed herein can be used to provide the cleavage site (PS) of the chimeric proteins exemplified in FIG. 10. Accordingly, such optionally masked chimeric proteins can be used to detect a wide variety of protease inhibitor molecules. Of course, nearly any of the protease cleavage sequences disclosed herein can be used to provide the cleavage site (PS) of the chimeric proteins exemplified in FIG. 10.
FIG. 10 is explained in more detail as follows. FIG. 10(a) shows a construct where the fluorescence signal (FP-1) is translocated from the plasma membrane to the cytosol upon proteolytic cleavage. In the case of FIG. 10(b), one fluorescence signal (FP-2) is translocated in the same manner as in the case of FIG. 10(a), but the other fluorescence signal (FP-1) remains on the plasma membrane. In the case of FIG. 10(c), the fluorescence signal (FP-1) is translocated from the plasma membrane to the chlorophyll upon proteolytic cleavage. In the case of FIG. 10(d), one fluorescence signal (FP-2) is translocated in the same manner as in the case of FIG. 10(c), but the other fluorescence signal (FP-1) remains on the plasma membrane.
(a) Construction of Recombinant Plasmids for Expression of Chimeric Substrate Proteins with One Signal Protein Remaining Active
Examples of the recombinant plasmids depicted FIG. 10 were constructed as follows.
Two recombinant plasmids encoding H+-ATPase:PS:GFP (FIG. 10(a)) with the proteolytic cleavage site sequences being VRFQ (SEQ ID NO: 48) and RQANFLG (SEQ ID NO: 64) were constructed as follows.
Forward primers 5′-CTCGAG PS ATGAGTAAAGGAGAAGAA-3′ (here PS is GTGCGCTTCCAG for VRFQ NIa cleavage site or AGACAGGCTAATTTTTTAGGG for RQANFLG HIV-1 cleavage site) and a reverse primer 5′-GAGCTCTTATTTGTATAGTTCATC-3′ were used for PCR amplification of 326GFP vector. These PCR products, containing a proteolytic cleavage site for NIa or HIV-1 protease, restriction sites (Xho I and Sac I) for subcloning, and stop codon at C-terminal of GFP, were subcloned into pBluscript-T vetor. Xho I/Sac I fragments of these subclones were ligated into Xho I and Sac I digested pH+ATPase-G vector (FIG. 7(i)).
Two recombinant plasmids encoding H+-ATPase:GFP:PS:RFP (FIG. 10(b)) with the proteolytic cleavage site sequences being VRFQ (SEQ ID NO: 48) and RQANFLG (SEQ ID NO: 64) were constructed as follows.
In order to prepare GFP without stop codon, PCR amplification was performed with primers 5′-CTCGAGATGAAAGGAGAAGAACTT-3′ and 5′-GAGCTCTTTGTATAGTTCATCCAT-3′. The PCR product containing Xho I and Sac I sites was subcloned into pBluescript-T vector and subsequently restriction digested with Xho I and Sac I. This Xho I/Sac I fragment was subcloned into Xho I and Sac I digested pH+ATPase-G vector (FIG. 7(i)).
In order to place proteolytic cleavage site upstream of RFP, forward primers 5′-GAGCTC PS ATGGTGCGCTCCTCCAAG-3′ (here PS is GTGCGCTTCCAG for VRFQ NIa cleavage site, or AGACAGGCTAATTTTTTAGGG for RQANFLG HIV-1 cleavage site) and a reverse primer 5′-GAGCTCCTACAGGAACAGGTGGTG-3′ were used for PCR amplification of 326RFP vector. The constructs containing PS:RFP, which also contained Sac I sites, were restriction digested with Sac I and these Sac I fragments were subcloned into Sac I sites of the H+-ATPase:GFP (without stop codon) subclone prepared as described above to generate recombinant plasminds for H+ATPase:GFP:PS:RFP.
Two recombinant plasmids encoding H+-ATPase:PS:AtOEP7:GFP (FIG. 10(c)) with the proteolytic cleavage site sequences being VRFQ (SEQ ID NO: 48) and RQANFLG (SEQ ID NO: 64) were constructed as follows.
The pSub-NIa1 vector (FIG. 8(b)) was PCR amplified with primers 5′-CTCGAG PS GGAAAAACTTCGGGAGCG-3′ (here PS is GTGCGCTTCCAG for VRFQ NIa cleavage site, or AGACAGGCTAATTTTTTAGGG for RQANFLG HIV-1 cleavage site) and 5′-GAGCTC TTATTTGTATAGTTCATC-3′. Thus, the PCR products contained Xho I and Sac I sites. Sho I/Sac I fragments of these subclones were ligated into Xho I and Sac I digested pH+ATPase-G vector (FIG. 7(i)) to generate recombinant plasmids for H+-ATPase:PS:AtOEP7:GFP.
Two recombinant plasmids encoding H+-ATPase:RFP:PS:AtOEP7:GFP (FIG. 10(d)) with the proteolytic cleavage site sequences being VRFQ (SEQ ID NO: 48) and RQANFLG (SEQ ID NO: 64) were constructed as follows.
Primers 5′-CTCGAGATGGTGCGCTCCTCCAAG-3′ and 5′-GAGCTCTTATTTGTATAGTTCATC-3′ were used to PCR amplify the pSub-NIa1 vector (FIG. 8(b)) and the pSub-HIV4 vector (FIG. 9(b)). Thus, these PCR products contained Xho I and Sac I sites. Sho I/Sac I fragments of these subclones were ligated into Xho I and Sac I digested pH+ATPase-G vector (FIG. 7(i)) to generate recombinant plasmids for H+-ATPase:RFP:PS:AtOEP7:GFP.
(b) A Working Example Using a Recombinant Plasmid Depicted in FIG. 10(a)
Preparation and transformation of the protoplast of Arabidopsis leaf tissue were performed according to the procedures described in Example 1(b) and (c). Detection of the fluorescence images of the transformed protoplasts was carried out as described in Example 1(d) (but using a Nikon E800 fluorescence microscope using the same types of the filter sets).
As a working example for detecting inhibition of NIa protease, the protoplasts were transformed with a recombinant plasmid encoding a chimeric substrate protein H+-ATPase:PS:GFP prepared as described in Example 4(a). The proteolytic site sequence included in the chimeric substrate protein was VRQF (SEQ ID NO: 48). The transformed protoplasts were incubated at 22° C. in the W5 solution for 18–48 hrs to express the chimeric substrate protein, and the subcellular localization of the fluorescence signal from the expressed chimeric substrate protein was monitored using a fluorescence microscope. As shown in FIG. 13(a), the green fluorescence signal was translocated to the plasma membrane. This result indicates that the chimeric substrate proteins were not cleaved as expected, and thus the attached green fluorescence proteins were targeted to the plasma membrane by the trafficking signal of H+-ATPase. This data corresponds to results when complete inhibition of the protease occurs.
Detection of NIa proteolytic activity was performed as follows. Protoplasts were co-transformed with the recombinant plasmid for NIa protease (SEQ ID NO: 45) and the recombinant plasmid for a chimeric substrate protein H+-ATPase:PS:GFP (SEQ ID NO: 53). The proteolytic site sequence included in the chimeric substrate protein was VRQF (SEQ ID NO: 48). The subcellular localization of the fluorescence signal from the expressed chimeric substrate protein was monitored 18–48 hrs after the transformation using a fluorescence microscope. As shown in FIG. 13(c), the green fluorescence signal was observed in the cytosol, but not targeted to the plasma membrane. This data indicates that GFP was cleaved off from the chimeric substrate protein by the protease.
All references disclosed herein are incorporated by reference. The following references are specifically incorporated by reference.
Hook, V. Y. H. U.S. Pat. No. 6,245,884 (2001).
Kettner, C. A. and Korant, B. D. U.S. Pat. No. 4,644,055 (1987).
Cote, H. C. F., Brumme, Z. L., and Harrigan, P. R. (2001). J. Virol. 75, 589–594.
Davis, S. J. and Vierstra, R. D. (1998). Plant Physiol. 112, 833–844.
Ermolieff, J., Loy, J. A., Koelsch, G., and Tang, J. (2000). Biochem. 39, 12450–12456.
Gillooly, D. J., Morrow, I. C., Lindsay, M., Gould, R., Bryant, N. J., Gaullier, J.-M., Parton, R. G., and Stenmark, H. (2000). EMBO J. 19, 4577–4588.
Gutierrez-Campos, R., Torress-Acosta, J. A., Saucedo-Arias, L. et al. (1999). Nat. Biotechnol. 17, 1223–1226.
Jacobsen, H., Hanggi, M., Ott, M., Duncan, I. B., and Owen, S. (1996). J. Infect. Dis. 173, 1379–1387.
Kasai, N., Tsumoto, K., Niwa, S., Misawa, S., Ueno, T., Hayashi, H., and Kumagai, I. (2001). Biochem. Biophys. Res. Comm. 281, 416–424.
Kuhelj, R., Rizzo, C. J., Chang, C.-H., Jadha, P. K., Towler, E. M., and Korant, B. D. (2001). J. Biol. Chem. 276, 16674–16682.
Kost, B., Spielhofer, P., and Chua, N. H. (1998). Plant J. 16, 383–401.
Mardis, K. L., Luo, R., and Gilson, M. K. (2001). J. Mol. Biol. 309, 507–517.
Miller, T. L., Mawn, B. E., Orav, E. J., Wilk, D., Weinberg, G. A., Nicchitta, J., Furuta, L., Cutroni, R., McIntosh, K., Burchett, S. K., and Gorbach, S. L. (2001). Pediat. 107–5, 1–6.
Morise, H., Shimomura, O., Johnson, F. H., and Winant, J. (1974). Biochem. 13, 2656–2662.
Pih, K. T., Yi, M. J., Liang, Y. S., Shin, B. J., Cho, M. J., Hwang, I., and Son, D. (2000). Plant Physiol. 123, 51–58.
Rogers, J. D., Lam, P. Y., Johnson, B. L., Wang, H. S., Ko, S. S., Seits, S. P., Trainor, G. L., Anderson, P. S., Klabe, R. M., Bachelor, L. T., Cordova, B., Garber, S., Reid, C., Wright, M. R., Chang, C. H., and Erickson-Biitanen, S. (1998). Chem. Biol. 5, 597–608.
Wlodawer, A. and Erickson, J. W. (1983). Annu. Rev. Biochem. 61, 543–585.
Yi, C.-F., Gosiewska, A., Burtis, D., and Geesin, J. (2001). Anal. Biochem. 291, 27–33.
Yoon, H. Y., Hwang, D. C., Choi, K. Y., and Song, B. D. (2000). Mol. Cell 10, 213–219.
The invention has been described in detail with reference to preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of this disclosure, may make modifications and improvements within the spirit and scope of the invention.

## Claims

1. A chimeric protein comprising at least one signal protein that has a trafficking signal targeting to a subcellular organelle and at least one proteolytic cleavage site for a protease, which is constructed such that
(a) the trafficking signals of all the signal proteins are inactivated by linking the proteolytic site or a signal masking protein through the proteolytic site to the N- or C-terminus of the signal proteins, and thus the chimeric protein is present in cytosol;
(b) the trafficking signal of at least one signal protein is activated when the proteolytic cleavage site is cleaved by the protease, and as a result at least one fragment protein that includes the activated signal protein is transported to a subcellular organelle; and
(c) the chimeric protein is labeled with at least one detectable amino acid sequence and the position and intensity distribution of the detectable amino acid sequence signal in the cell is altered depending on the cleavage by the protease.

2. A chimeric protein comprising at least two signal proteins that have trafficking signals targeting to subcellular organelles and at least one proteolytic cleavage site for a protease, which is constructed such that
(a) the trafficking signal of one signal protein remains active, and those of the rest of the signal proteins are inactivated by linking the proteolytic site or a signal masking protein through the proteolytic site to the N- or C-terminus of the signal proteins, and thus the chimeric protein is transported to a specific subcellular organelle targeted by the trafficking signal of the active signal protein;
(b) at least one proteolytic site and at least one inactivated signal protein are exposed to cytosol after the chimeric protein is transported to the subcellular organelle;
(c) the trafficking signal of the at least one inactivated signal protein exposed to cytosol is activated when the proteolytic cleavage site is cleaved by the protease, and as a result the fragment protein that includes the activated signal protein is transported to a subcellular organelle that is different from the subcellular organelle to which the chimeric protein was transported; and
(d) the chimeric protein is labeled with at least one detectable amino acid sequence and the position and intensity distribution of the detectable amino acid sequence in the cell is altered depending on the cleavage by the protease.

3. The chimeric protein according to claim 1 or 2, wherein among the fragment proteins produced by the proteolytic cleavage, at least two fragment proteins with different cellular localization characteristics includes different detectable amino acid sequences.

4. The chimeric protein according to claim 1 or 2, wherein among the fragment proteins including a signal protein whose inactivated trafficking signal is activated by the proteolytic cleavage, at least one fragment protein includes the detectable amino acid sequence.

5. The chimeric protein according to claim 1 or 2, wherein the trafficking signal of the inactivated signal protein is a signal targeting to a subcellular organelle selected from the group consisting of mitochondria, chloroplast, and peroxisome.

6. The chimeric protein according to claim 1 or 2, wherein the signal protein that is inactivated is a full length protein selected from the group consisting of Arabidopsis outer envelope membrane protein 7 (AtOEP7), Rubisco small subunit (RbcS), Chlorophyll a/b binding protein (Cab), Rubisco activase (RA), F1-ATPase, and Peroxisome-targeting motif (SKL), or a portion thereof that includes the trafficking signal.

7. The chimeric protein according to claim 2, wherein the trafficking signal of the signal protein remaining active is a signal targeting to one selected from the group consisting of outer membranes of mitochondria, chloroplast, and nucleus, peroxisome membrane, and plasma membrane.

8. The chimeric protein according to claim 2, wherein the signal protein remaining active is a protein that binds specifically to a specific phospholipid.

9. The chimeric protein according to claim 2, wherein the signal protein remaining active is a full length protein selected from the group consisting of Arabidopsis outer envelope membrane protein 7 (AtOEP7), H+-ATPase, Pleckstrin homology domain (PH), and pleckstrin homology domain of FAPP (family A (phosphoinositide binding specific) member 3), or a portion thereof that includes the trafficking signal.

10. The chimeric protein according to claim 1 or 2, wherein the signal masking protein is selected from the group consisting of amino acids, peptides, and proteins.

11. The chimeric protein according to claim 1 or 2 wherein the detectable amino acid sequence is selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), mutants thereof, and derivatives thereof.