JP2002512808A - Transduction method of fusion protein - Google Patents

Abstract

  (57) [Summary] The present invention is a highly efficient method of transducing a cell or a group of cells containing a desired cell or tissue or organ with a fusion molecule, the method comprising causing the fusion molecule to misfold; Contacting a cell with a fusion molecule having the following conditions under conditions sufficient to transduce the cell with the fusion molecule:

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for efficiently introducing a fusion molecule into a cell or a group of cells. The methods of the present invention have a variety of uses, including introducing the therapeutic fusion molecule into cells with high efficiency.

2. Background There has been much interest in developing methods for expressing heterologous proteins in cells, tissues, organs, and the like. Most methods for achieving such objectives focus only on introducing a nucleic acid into a cell and then expressing the nucleic acid to produce a protein (generally described by Sambrook et al.). "M
olecular Cloning: A Laboratory Manual (2nd ed., 1989) "and Ausubel et al.," Current Protocols in Molecular Biology, (1989) John Wiley & Sons, N.
ew York ").

[0003] However, many cells are essentially impermeable to nucleic acids. Based on such recognition, methods for transferring nucleic acids and other high-molecular substances into cells have been improved.

[0004] For example, a standard method includes the steps of making a hole in a cell and transferring nucleic acid from the hole by diffusion or concentration gradient. There are various methods for making holes in cells, such as physical perforation. Some examples of such transfer methods have already been disclosed (eg, Cocket et al., Bio / Technology 8: 662 (1990), Au).
See subel et al. (supra) and Sambrook et al. (supra).

[0005] Other nucleic acid transfer methods include those using bacteriophages or viruses. In this case, the introduction of the nucleic acid into the cells can be performed by a “transformation” method or “transformation” method.
It is performed by an infection method also called a "transfection" method. Once the nucleic acid enters the target cell, it is typically expressed as part of the bacteriophage or viral lysate cycle (see, eg, Ausubel et al., Supra, and Samb.
rook et al. (see below)).

[0006] However, existing methods for introducing macromolecules, especially nucleic acids, into cells have significant drawbacks.

For example, in most transformation methods, only a limited range of nucleic acids (typically about 20-50 kb or less) can be used for transfer. Transformation methods with larger nucleic acids are not always successful.

[0008] In addition to this, the undesirable properties of the methods for transferring nucleic acids into cells, such as overexpression of nucleic acids, heterogeneity of protein expression levels, and the percentage of cells actually targeted by these methods ( %, And various reports have been made (eg, Chen et al., Mol. Cell Biol. 7,2745-2752 (1987), Friedman's Nat. Gene 2:
93 (1992), and Bar-Sagi, Meth. Enzy. 255,436-442 (1995)).

[0009] Furthermore, the pharmacologically relevant peptidyl mimetics and / or proteins have a problem that if their molecular size exceeds about 1 kDa, their bioavailability decreases.

[0010] With respect to the introduction of macromolecules into cells, tissues, organs, and the like, other alternative methods have been developed. One approach is to fuse the amino acid sequence with a specific import molecule by a genetic method, and to introduce this fusion construct into cells.

For example, as a specific approach, a method has been attempted in which a target protein is fused with a transducing protein by a genetic technique and the resulting protein is transferred to cells. It has been reported that transduced proteins carry fusion molecules to target cells via a process called protein "transduction" (eg, Frankel, AD and CO
Pabo Cell 55: 1189 (1988), Fawell et al. PNAS (USA) 91: 664 (1994), Chen et al.
Anal. Biochem., 227: 168 (1995); U.S. Patent No. 5,652,122 and WO / 04686).

[0012] However, such known introduction methods have the disadvantage of lacking versatility.

For example, many conventional methods of introduction do not allow a sufficient amount of fusion protein to be introduced into target cells. Therefore, the amount of the fusion protein introduced into the cell and the value of its biological activity are only low. Issues such as how to introduce a large amount of fusion protein into target cells and the problems associated therewith are being studied.
However, such methods can sometimes result in lethality in the cells that receive the transduction. Furthermore, the protein transduction methods tried so far are not very fast in most cases with regard to the speed of transduction.

[0014] Furthermore, known transduction methods usually have the constraint that the structure of the fusion protein must be kept in a native state. Such methods tend to be complex because care must be taken to maintain the native structure during use and storage of the fusion protein.

As described above, establishment of a more efficient method for introducing a desired fusion molecule, particularly a fusion protein, into cells is expected.

SUMMARY OF THE INVENTION The present invention provides a highly efficient method for introducing a fusion molecule into a cell or group of cells. Such methods usually involve misfolding of the fusion molecule and the fusion protein having this misfolded structure (misfolde).
d) a step of bringing a fusion molecule having an incorrectly folded structure into contact with a cell under conditions capable of introducing d fusion protein). The present invention further provides fusion molecules with misfolded structures and methods for preparing the molecules. The uses of the present invention are diverse, including those that introduce therapeutic fusion molecules in vivo or in vitro with high efficiency.

With respect to transduction of cells, it has been found that a fusion molecule having a misfolded structure is more effective than a fusion molecule having a natural structure. More specifically, it has been found that a fusion molecule having a misfolded structure is introduced 10 to 1000 times more efficiently than when the same fusion molecule is in its native state (structure). In the present invention, the phrase "the fusion protein has a misfolded structure" means that the fusion protein has at least a partially denatured structure and a refolded structure, and the biochemical Activity and / or biological activity is reduced.

The present invention can be used for the purpose of increasing the efficiency of introducing various fusion molecules. For example, the effective dose of this method is one half or one third of that of the existing transduction method.
, 1/5, 1/10, 1/50, or 1/100 or less of the therapeutic fusion protein, the same amount of protein as the existing method (for example, per cell) The number of molecules introduced). Importantly, such a low effective dose does not affect the therapeutic effect of the cell-transduced fusion molecule. In other words, even when a relatively small amount of the fusion molecule is used, the method of the present invention can achieve a therapeutic effect equivalent to or similar to that of the existing method in cells into which the molecule has been introduced. As will be described in more detail below, the subject of the present invention can be achieved by causing the fusion molecule to assume an incorrectly folded structure, thereby significantly increasing the introduction efficiency.

The present invention significantly increases the efficiency of introducing fusion molecules of high molecular weight for therapeutic purposes, such as consisting of one or more full-length proteins.

The present invention provides an introduction method that can be performed without any particular limitation on a fusion molecule, a target cell, a target cell group, and the like. Any fusion molecule having a misfolded structure in the present invention can be used for introduction into a wide variety of cells in vitro or in vivo.

A fusion molecule can be misfolded in the present invention by a single technique or a combination of techniques. For example, a first condition is set such that the fusion molecule forms a partially or completely unfolded state (ie, a denatured state), and the fusion molecule is subjected to that condition. This can cause an incorrect folding structure. In turn, this denatured molecule is exposed to second conditions that favor the misfolding.

[0022] Fusion molecules with misfolded structures can also be obtained in single or multiple ways. In one such method, the first condition for denaturing the fusion molecule comprises contacting the fusion molecule with one or more protein denaturants. Prior to exposure under the first condition, the fusion molecule is almost always in its native or near-native structural state. Also, in most cases, a fusion molecule having a natural or near-natural structure is energetically stable, and at least in theory, the Gibbs free energy (ΔG) is the Gibbs free energy of a fusion molecule having a misfolded structure. Lower than energy.

[0023] Preferably used denaturing agents are those that partially or completely denature the fusion molecule. The composition of denaturing agents that denature proteins and denaturing conditions are known in the art (described below). That is, the denaturing agent is generally used to break intramolecular bonds such as hydrogen bonds and ionic bonds. Intramolecular binding contributes to the secondary, tertiary or quaternary structure that proteins take to stabilize in solution or buffer. In this case, the solution or buffer comprises salts and other components at physiological concentrations, and "physiological" refers to the natural solvent for proteins and polypeptides (i.e., Cytoplasm, blood,
Plasma, interstitial fluid, etc.). Denaturants are typically not strongly bound to the fusion molecule and, if bound, can also be used if they can be substantially separated from the modified fusion molecule during purification. be able to. Typical denaturing agents include at least one of an organic compound, salt, surfactant, temperature, or sound waves.

While not wishing to be limited to a particular theory, increasing the Gibbs free energy (ΔG) of the denatured fusion molecule can cause the fusion molecule to assume a misfolded structure, thereby introducing It is possible to create a fusion molecule with significantly enhanced ability. Therefore, it is preferred to expose the modified fusion molecule under a second condition that is preferably used to increase the energetic instability of the modified molecule. It is believed that such an increase in energetic instability can be achieved by contacting the denatured molecule with a solution that suitably increases the energetic instability of the denatured fusion molecule. An example of such a solution is at least about 2 to 100 times, or even about 1000 times or more, greater than the Gibbs free energy of a fusion molecule having a native or near-native structure. Solutions that increase the free energy (ΔG) of the Gibbs. Such preferred solutions are those that can significantly reduce or eliminate denaturing agents that come into contact with denaturing molecules. Even more preferred are aqueous solutions, particularly buffers, as disclosed herein.

As used herein, exposing a denatured fusion molecule under a second condition is referred to as “shock”.
May cause misfolding, which implies such exposure.

As described above, the modified fusion molecule is made to accept the second condition so that the fusion molecule forms a misfolded structure. In one embodiment, the denatured molecule is contacted with an aqueous buffer (or, if necessary, a plurality of aqueous buffers) capable of causing the fusion molecule to form a misfolded structure, The second condition has been achieved. Such contact can be suitably made in various ways. For example, the denatured molecule can be subjected to at least one type of chromatography. Alternatively, the denatured molecules can be precipitated and then suspended in an aqueous buffer, or the denatured molecules can be filtered into an aqueous buffer. When it is preferable to use chromatography for contacting, one or more kinds of eluates are used. Generally, these eluates contain or are the aqueous buffers described above. A preferred eluate is a solution that can elute and elute a fusion molecule (denatured or one having an incorrectly folded structure) from the chromatography apparatus used.

In the denatured fusion molecule, a method for rapidly performing a desalting treatment of replacing the denaturation solution with an aqueous solution is performed, for example, using a chromatography apparatus, which is particularly preferable for many applications.

As described above, in order to enhance the energy instability of the denatured fusion molecule to promote the formation of a fusion molecule having a misfolded structure when transferred to an aqueous buffer, the second condition Is preferably used. Therefore, if this instability is not significantly increased, such a method is not preferably used.

In another aspect of the invention, contacting the denatured fusion molecule with an aqueous buffer (one or more buffers) is accomplished by diluting the denatured fusion molecule with the aqueous buffer. Will be Generally, depending on the intended use, the dilution is about 1:10 (v / v)
From 1: 100,000 (v / v) or higher. The diluted (mis-folded) fusion molecule can be concentrated, if necessary, by conventional means such as filtration or precipitation.

The invention also features a substantially pure preparation of a fusion molecule having a misfolded structure that is suitable for various uses, including pharmaceutical and research uses. Desirably, such misfolded fusion molecules are provided as a sterile composition, optionally with other additives. For example, a fusion molecule with a misfolded structure may contain one or more denaturing agents, for example, upon storage, to maintain the misfolded state. Further, the fusion molecule having an incorrectly folded structure may contain one or more stabilizers known to contribute to the stabilization of proteins, such as albumin, glycol, and sulfoxide.

The present invention has a number of important advantages, for example, when a fusion molecule with a misfolded structure is used, the transduction efficiency is about 2 compared to transduction of a suitable control molecule. It can be increased by a factor of 5, 5, 10, 20, 50, 100 or 200 or more. A preferred control molecule is a fusion molecule having a native structure, ie, a fusion molecule that has not been treated to form a misfolded structure at a high energy state. As described above, the transduction method of the present invention is considerably more efficient than the conventional method using a fusion molecule having a conventional natural structure or a structure close to nature.
As will be described in more detail below, such improved transduction efficiency can be proved using several parameters such as the transfection rate and the biological activity of the transfused fusion protein.

As noted above, the methods of the present invention allow for the introduction of a variety of fusion molecules, such as fusion proteins with potential therapeutic activity, especially large fusion proteins, with high efficiency. Transduction into a cell or group of cells can be achieved using a lower but more effective amount of the misfolded fusion molecule. In this way, toxicity can be reduced or avoided for cells receiving the transduction.

The method of the present invention has further advantages besides these. For example, the transduction methods described herein also accommodate fusion proteins of a fairly wide range of molecular weights, for example, molecular weights of about 5, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 100 kDa.
, 200 kDa, 300 kDa, 400 kDa, or 500 kDa or larger proteins can be introduced. A fusion protein having a lower molecular weight can be easily introduced by the present method. In contrast, in most conventional transfer methods, only low molecular weight fusion proteins are targeted for transfer. Therefore, the widespread use of existing methods necessarily comes with the problem of limiting the size of the protein to be introduced. In particular, it is difficult to transduce many types of therapeutic proteins and especially large therapeutic proteins by conventional methods.

Furthermore, in the method of the present invention, it is not necessary to cut out only a main domain (for example, an active site or a binding site) of a protein and make it smaller. In general, in conventional methods, only small truncated protein domains (fragments) and / or peptidyl mimetics can be introduced into a desired target cell. However, there are also high molecular weight protein domains, sometimes their size can even exceed about 15 to 500 kDa. Thus, the present invention provides a method for introducing a very large (and intact) protein into a cell, which has not been possible with the conventional method. Furthermore, in the case of the present invention, the uneconomical and time-consuming cutting of protein domains into smaller sizes can be minimized, or such waste can be completely eliminated.

The method of the present invention also has advantages with respect to the purity of the fusion molecule and its yield. For example, in a preferred method, one or more denaturing agents are used to separate and purify the fusion protein from cell components that naturally contain the fusion protein. This cellular component usually consists of a large amount of denatured insoluble protein of the recombinant protein, and is often present in inclusion bodies in the cells. Facilitates separation. Thereby, both the purity and the yield are significantly improved.

As an example of the preparation method according to the present invention, a method including a step of expressing a desired fusion protein in a host cell and a step of isolating the fusion protein from cell fractions can be mentioned. Isolation of the fusion protein from this fraction, particularly from the insoluble fraction, has several advantages, such as protecting fusion proteins with misfolded structures from degradation by proteolytic enzymes from host cells. In particular, when preparing a fusion protein having a misfolded structure, a denaturant is suitably used, and a time-consuming and expensive protein regeneration method is not required.

Furthermore, the method of the present invention has flexibility for general use, and if necessary, a fusion protein having a misfolded structure can be prepared in various amounts. Such methods use a wide range of preparation scales (ie, from micrograms) to produce fusion proteins from cultures grown in various incubators such as spinner bottles, spinner flasks, tissue culture plates, bioreactors, or fermenters. Kilogram amount). A preparation of the fusion protein prepared in any preparation amount on a wide range of preparation scales can cause misfolded structures to be formed by any of the methods described herein.

As noted above, the methods of the invention can be used to facilitate the introduction of a desired fusion molecule into a target cell or group of cells consisting of such cells. Therefore, when it is not necessary to prepare or use a large amount of the fusion molecule, such treatment may not be performed. This feature of the present method is of great advantage where the preparation of the fusion molecule of interest is difficult and where large amounts of the fusion molecule cause cell damage in vitro or in vivo.

The method may be used to amplify fusion molecules with misfolded structures (eg, to provide fusion molecules as one component in suitable kits for medical, research, household, or commercial use). In addition, the method of preparing a large amount of a fusion molecule having a misfolded structure has advantages such as simplifying the structural analysis of the fusion molecule, supporting dynamic analysis of molecular transduction, It is also useful for further purification and / or testing as needed.

The misfolded fusion molecules of the present invention are fluorescent, phosphorescent (photopho
recent) or has a variety of additional uses, such as introducing a tag (marker) such as an immunological tag into a cell or group of cells. Alternatively, or concomitantly, fusion molecules with this misfolded structure can be utilized to introduce specific activities, such as therapeutic activity, into cells. If desired, fusion molecules with misfolded structures can be used both in vitro and in vivo.

Examples of therapeutic activity include the delivery of one or more radionuclides or therapeutic molecules, including cytotoxic agents.

As mentioned above, the present invention is not limited to the preparation and use of a particular type of fusion molecule. A typical fusion molecule usually comprises a transduction domain and the desired molecule.
One or more molecules, including those linked by a covalent bond. Preferred fusion molecules usually include a transduction domain covalently linked to an amino acid sequence so as to constitute a genetically in-frame fusion protein. As shown below, it may be preferable to make the fusion molecule by chemical cross-linking, but in many cases, the fusion protein will be made by standard genetic manipulations. Estimated by SDS-polyacrylamide gel electrophoresis or centrifugal sedimentation analysis, etc., the molecular weight of the fusion molecule is about 50 to 500 kDa or more.

Examples of transduction domains include TAT, Antp, or their specific transduction sites, such as the basic region of TAT. Also, transduction domains consisting of synthetic peptides should be taken into account. Frankel, AD and Pabo, CO (
Supra), Falwell et al. (Supra), Chen et al. (Supra), U.S. Patent No. 5,652,122 and
See WO 04686.

As described above, the fusion molecule having a misfolded structure of the present invention is suitably used for introducing a molecule into a desired cell or a cell group including a desired cell including a tissue or an organ. The method can be used for in vitro applications, for example, those involving primary cultures or culturing of immortalized cells, and, where necessary, in vivo applications.

Another aspect of the present invention is disclosed by:

DETAILED DESCRIPTION OF THE INVENTION As noted above, the present invention provides a highly efficient method of introducing a fusion molecule into a desired cell or group of cells. The method generally involves denaturing the fusion molecule, followed by reacting the denatured fusion molecule under the specific conditions described herein. Further, a fusion protein having a misfolded structure is brought into contact with these cells in order to introduce the cells into a target cell or cell group. As described above, the method of the present invention is a method that is highly efficient and can significantly improve the efficiency of introducing various fusion molecules. The present invention further provides fusion molecules having misfolded structures and methods for preparing fusion molecules having misfolded structures.

The efficiency of introduction can be determined by a single method or a combination of methods.
For example, for a fusion protein to be examined, the transduction efficiency can be determined by examining the ratio of the fusion protein transferred to a cell or cell group. A more preferable method for examining the transduction efficiency is a method for measuring the biological activity of the fusion protein that has entered the inside of the cell. Biological activity can be measured in various ways as set forth herein.

The preferred misfolded fusion molecules of the invention, when measured by the assays specified below, comprise at least about 20%, 30%, 40% of the total number of target cells.
%, 50%, 60%, 70%, 80%, preferably at least about 90%, more preferably at least about 95%, or about 100%.

The efficiency of introducing a target fusion molecule having an incorrectly folded structure can be measured and quantified by various commonly used methods. Usually, such methods include the step of measuring a parameter for transduction relative to a suitable control molecule. In most cases, the control molecule is introduced under the same or nearly the same conditions as those used when introducing a fusion molecule having a misfolded structure. The control molecule is often a fusion molecule with a native structure. That is, it refers to a fusion molecule that, when isolated, was not exposed under conditions of denaturing conditions as described herein, resulting in the formation of "shock" misfolds. In some cases, it may be better to compare the efficiency of introduction of a fusion molecule with a misfolded structure with the efficiency of two control molecules, particularly a fusion molecule with a native structure and a fusion molecule with a structure close to nature. Transduction efficiency can be expressed in a variety of formats, such as% increase in transduction efficiency as compared to the desired control molecule.

“Near-natural state” refers to a state in which the fusion molecule has been substantially regenerated after being exposed to a denaturing agent to cause denaturation. Denatured fusion molecules are usually regenerated by dialysis against a physiologically acceptable buffer such as phosphate buffered saline (PBS). Many, if not all, of the intramolecular bonds (eg, hydrogen bonds, ionic bonds, etc.) of the regenerated fusion molecule are the same as those of the naturally-occurring molecule,
Or similar. In the present specification, such a fusion molecule is referred to as "
Sometimes referred to as "denatured and dialyzed" fusion molecules.

“Native state” refers to the shape and / or form of a protein that is taken up by a substantially physiological environment (eg, a cytoplasm and / or a buffer such as a physiologically acceptable buffered saline solution). Means structure. In contrast, "denaturing" refers to the shape and / or structure that proteins take in a chaotropic environment (eg, one or more denaturing agents as described herein). Most of the protein molecules described herein have sufficient activity when in their native form, but have no or almost no activity when denatured. For example, about 20%, 30%, 40%
%, 50%, 60%, 70%, 80%, 90%, 95% or more of the activity is lost. Denaturation involves, for example, breaking a folded natural structure of a molecule to change a protein from a natural structure to a random loop (coil) structure. Denaturation can be determined by a number of known methods, such as measuring protein activity, measuring viscosity, and / or analyzing optical rotation spectra.

The term “increases efficiency” or like terms, when used in reference to the introduction of a desired fusion protein, refers to at least one of the following: That is, (
1) an increase in the percentage (%) of molecules delivered to the target cell into the cell,
2) the rate at which the protein is transferred into the target cells increases, (3) when the protein is brought into contact with the cells, the percentage (%) of the cells that accumulate the protein increases, (4) in situ And / or the intracellular refolding stage is increased, and (5) the biological activity of the protein is increased.

As described, the transduction efficiency is determined by measuring the rate at which the fusion molecule with the desired misfolded structure is transferred into the cell, more preferably by measuring the biological activity of the protein. be able to.

The determination of such a rate can be made by standard kinetic (kinetic) analysis using one or more control molecules. Preferred misfolded fusion proteins are proteins that can enter target cells faster than control molecules, and are generally about twice as large as native structure control molecules.
Preferably, the transfer is 3x, 4x, 5x, 10x, to about 100x faster. Particularly preferred fusion proteins having misfolded structures are those that have been introduced into cells at least within about 1-2 hours, more preferably within 1 hour, even more preferably about 5 minutes, 10 minutes, 15 minutes, 20 minutes or less. Min, 30 minutes, 35 minutes to about 40-50 minutes, the fusion protein such that the intracellular protein concentration reaches a maximum in the cells that have received the transfection. The analysis in this regard is usually carried out by means of a suitable microscope as described below or by a cell sorting method. When the fusion molecule has a molecular weight of at least about 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa or more, it is often useful to determine the rate of introduction.

In a further alternative, the concentration of the fusion molecule in cells that have received the transduction can be measured relative to a suitable control molecule. Methods for determining the concentration of the fusion protein in cells that have received the transduction usually include standard methods for detecting the protein in living cells. Such methods include Western blot analysis of cell extracts using antibodies to the protein of interest, microscopy (eg, fluorescence microscopy), cell sorting using fluorescence activated cell sorting (FACS), and the like. , But is not limited to only these methods. Usually, in order to facilitate the measurement, a label for detection is attached to the control molecule, for example, by attaching a tag for detection. Examples of suitable detection tags include certain amino acid sequences, and certain fluorescent proteins as described below. Particularly preferred as the misfolded fusion molecule of the present invention is that the intracellular concentration of the introduced fusion molecule is about 2-fold, 5-fold, or 10-fold when compared to a suitable control molecule having a native structure.
Molecules that can increase 2-fold, 20-fold, 30-fold, 50-fold, 70-fold, 100-fold, 200-fold or more.

A preferred method of measuring transduction efficiency is to measure the biological activity of the fusion molecule inside the cell. In many cases, the molecular weight of the fusion molecule is about 45 kDa, 40 kDa,
Biological activity measurements are useful when less than 35 kDa, 30 kDa, 25 kDa, 20 kDa, or 15 kDa.

Typical misfolded fusion molecules of the invention include misfolded fusion proteins. Examples of such fusion proteins include those that exhibit a particular biological activity, particularly when introduced into a target cell or group of cells consisting of such cells.

By measuring and quantifying such activities, it is possible to determine, if necessary, the efficiency of introducing a fusion protein having a specific misfolded structure with reference to an appropriate control molecule. Become. Examples of such assays include cell replication, cell adhesion, oncogenic transformation, cell membrane foaming, cytokinesis, cell division, cell death (apoptosis and necrosis), fertilization, Protein synthesis, including protein expression, transcription, immunoreactivity, translation, protein modification, and secretion;
There are measurement methods for proteolysis, glycosylation, phagocytosis, and the like. Table 1 below
Shows examples of proteins having biological activity introduced in the present invention.

As used herein, the term “fusion molecule” means a molecule obtained by covalently linking (ie, fusing) a desired molecule to a transducing protein by genetic recombination or a chemical method. I have. If necessary, the transduction protein and the molecule can be fused via a peptide linker containing one or more sites that are cleaved by enzymes such as proteolytic enzymes. Examples of transducing proteins will be described later in detail. The target molecule may be as follows. That is, enzymes, polypeptides, amino acids, nucleic acids (RNA or DNA), lipids, glycolipids, carbohydrates such as sugars, glycans, saccharides, polysaccharides, glycoproteins, proteoglycans, drugs or other synthetic and semi-synthetic small molecules, Or hybrids thereof (eg, RNA-DNA hybrid molecules). If desired, the molecule may be dextrorotary (D) or levorotatory (L). The fusion molecules were fused to the conductive protein (including the linker if necessary)
, 8, 10, 20, 50 to about 100 or more molecules as described above. As noted, the fusion molecule preferably adopts a misfolded configuration as set forth in the present invention.

The sequence of the peptide linker is preferably up to about 20 or 30 amino acid residues, more preferably up to about 10 or 15 amino acid residues, even more preferably about 1 to 5 amino acid residues. It is composed of about amino acids. The linker sequence is usually flexible so that the fusion molecule is not tied to a single, less mobile structure. This linker sequence may be used, for example, as a spacer to provide a space between each element in the fusion molecule, if desired. For example, peptide linker sequences are placed between the protein transduction domain and the protein, for example, to chemically link them and at the same time impart mobility to the molecule.

More specifically, if the peptide linker does not significantly affect the function required by the fusion protein, for example, a recognition site for a specific enzyme, particularly a cleavage site for proteolytic enzymes, may be provided. Each element of the fusion protein can be spatially separated from neighboring elements by one or more suitable peptide linker sequences for or for tagging.

Although the molecular weight of this molecule varies depending on the intended use, it is usually about 0.1 to 10
It ranges from 0 kDa or higher to about 500-1000 kDa. When measured by SDS-polyacrylamide gel electrophoresis, centrifugal sedimentation, or other suitable method, the molecular weight of this molecule is preferably about 0.1, 0.2, 0.5, 1, 2, 5, 10
, 20, 30 kDa, or 50 kDa up to about 100 Kda or more. Although the molecular weight of the fusion molecule varies depending on the intended use, it is usually determined by centrifugation or SD.
5, 10, 20, 30, 40, 5 when measured by S-polyacrylamide gel electrophoresis
0, 100, 200, 500 kDa, or 1000 kDa or more.

Further, a preferred fusion molecule of the present invention is a fusion protein consisting of a desired protein fused to a transduction protein (sometimes called a transduction domain). The protein fused to the transduction domain is, for example, a zymogen, an enzyme, a molecule that binds an amino acid or nucleic acid sequence, an immune-related molecule such as an antibody fragment that binds an antigen, or a structural protein. Other suitable fusion proteins for use in the present invention include, for example, "Anti
Provisional Patent Application No. 60/069012, PCT / US98 / 26358, U.S. Patent Application No. 09/208966, filed August 22, 1997, entitled "Pathogen System and Methods of Use" And US Pat. No. 60 / 056,713 entitled “Inducible Regulatory System and use there,” and PCT / US98 / 16887 (WO 99/10376) currently published. All of these disclosed specifications are incorporated by reference.

[0064] Yet another plurality of fusion proteins, including fusion proteins that can adopt a misfolded structure and are compatible with the uses of the present invention, are disclosed in US Pat. No. 5,652,12.
No. 2, No. 5,674,980 and No. 5,670,617. These disclosures are incorporated herein by reference.

[0065] Preferred misfolded fusion molecules of the invention are suitable control molecules,
Preferably, as compared with a fusion molecule having a natural structure corresponding to a fusion molecule having an intended misfolded structure, introduction into a specific cell or a cell group such as a tissue or organ is about twice as large as that of a fusion molecule having a natural structure. X 10, x 20, x 50, x 100, or about 2
It has the ability to increase 00 times or more. A preferred method for determining whether protein transduction is enhanced is described in further detail below.

As mentioned, the term “having a misfolded structure” herein refers to the term once denatured and then exposed to one or more conditions as disclosed herein. A fusion molecule having an incorrectly folded structure. While not wishing to be limited to a particular theory, it is believed that exposure of the modified fusion molecule under such conditions would be energetically unstable as compared to the native or near-native structure. Such energy instability is thought to greatly contribute to promoting the ability to introduce the fusion molecule. In many cases,
The Gibbs free energy (ΔG) of a fusion molecule having a misfolded structure is about 2, 3, 5, 10, 20, or 5 times that of a molecule in a native structure state.
From 0 times or 100 times to about 250 times or more.

The Gibbs free energy of the desired molecule can be determined by various standard thermodynamic methods. As used herein, the expression "molecule in a misfolded state with a high ΔG" refers, at least in theory, to the fact that this molecule has a large negative Gibbs free energy and misfolded. It means that the folding reaction of the molecule having the structure proceeds spontaneously under appropriate conditions.
In contrast, “low ΔG” refers, at least in theory, to a natural structure that is indicative of a substantially stable fusion molecule that does not spontaneously break (or unravel). By comparison, it means that the fusion molecule has a Gibbs free energy of zero or a positive value (generally, Edsa
ll and Gutfreund, Biothermodynamics: The Study of Biochemical Proces
ses at Equilibrium, (Wiley, 1983)).

For example, as a preferable method for obtaining Gibbs free energy, there is a method using the following calculation formula. That is, ΔG = −2.303 RT log keq, where ΔG is the standard energy change and keq is the equilibrium constant for the reaction in which the desired fusion molecule forms the wrong folded structure. Methods for determining the constant keq are known in the art.

From the above description, the fusion protein of the present invention having a natural structural state, a denatured state, and a misfolded structure can be easily distinguished. For example, a fusion protein with a misfolded structure can have a significant facilitating effect on transduction, and can theoretically have a higher Gibbs free energy state than the protein's native structural state It has the property.

In addition, fusion molecules with misfolded structures can be distinguished from native or denatured proteins by various methods such as, for example, viscometry and / or optical rotation spectral analysis ( Edsall and Gutfreun
d (see above)).

The fusion molecule having a misfolded structure of the present invention is characterized in that the biological activity and / or biochemical activity of the fusion molecule having the misfolded structure is compared with that of the native structure of the same protein. The further identification can be advanced by the analysis of.
As described above, in the fusion molecule having a misfolded structure of the present invention, its biological activity and / or biochemical activity is, for example, about 20 %, 30%, 40%, 50%, 60%, 70%, 8
0% or more. The fusion molecule having the misfolded structure of the present invention is completely or almost completely compared with the native structure of the protein.
It may lose its biological and / or biochemical activity. Analysis of whether the biological activity and / or biochemical activity of the fusion molecule having a misfolded structure of the present invention is reduced as compared with that of the native structure of the same protein can be carried out based on the specific organism. It can be performed by standard measurement methods for examining chemical or biochemical properties.

The denaturing agent suitably used in the present invention is a chaotropic agent that can mostly break intramolecular bonds (eg, hydrogen bond, ionic bond or covalent bond) of a protein, polypeptide, or peptide. Any drug may be used. Examples of agents having denaturing action (denaturing agents) include organic compounds, salts, temperature (eg, heating), and sound waves (eg, sonication). In particular,
Organic compounds include urea, especially at a concentration between about 4M and 8M, preferably about 8M. The organic compounds also include guanidine hydrochloride and mercaptans such as β-mercaptoethanol. Although less suitable than these, depending on the application, for example, sodium dodecyl sulfate (SDS)
Surfactants such as ionic surfactants and the like can also be denaturants, and besides, Brij, Nonidet-40, and Triton-X-100
Surfactants such as 100) can also be modifiers. Further denaturing agents that can be used include high concentrations of salts such as NaCl, KCl and the like (eg, in molar order amounts).

Preferred solutions for the fusion molecules of the invention to form a misfolded structure include:
A solution that helps to form the misfolded structure of the fusion molecule. Typically, a water-soluble buffer (or multiple such buffers) of a type known in the art.
It is. Examples of such buffers include those described below and those described in the following references. In other words, "Current Protocol" by Ausubel et al.
ls in Molecular Biology, John Wiley & Sons, New York (1989), Sambrook
"Molecular Cloning: A Laboratory Manual. (2nd ed. (1989))" and Harlow and Lane "Antibodies: A Laboratory Manual, CSH Publicati.
ons, NY (1988) ".

More specifically, suitable aqueous solutions described herein typically include a buffering component (eg, Tris, HEPES) to maintain the pH, and generally have a pH in the range of about 5- 9
And the range of ionic strength is between about 2 mM and 500 mM. Proteolytic enzyme inhibitors or mild nonionic surfactants may be added. In addition, if necessary, a carrier protein such as bovine serum albumin (BSA) can be used at
May be added. Still other examples of aqueous buffers include standard phosphate buffered saline, Tris buffered saline, HEPES buffer, or known buffers, and cell culture media. It is.

As mentioned above, the misfolded molecules of the present invention can be prepared by a single or a combination of multiple methods. For example, a preferred method of causing the fusion molecule to form a misfolded structure comprises one or more of the following steps (a) to (c). (A) contacting a fusion molecule having a natural (or near-natural) structure with one or more types of denaturants under conditions that denature the fusion molecule; (b) presence of a denaturant Applying the denatured fusion molecule to a first chromatography device, such as an affinity column, wherein the denatured fusion molecule is capable of binding to a chromatographic support; and After eluting the denatured fusion molecule from the first chromatography device under such conditions, at least one of the following three steps (ie, the method (1) or the method (2a) to (2d)) ) Or method (3)
). (1) dialyzing the denatured fusion molecule; or (2a) denaturing in the presence of a denaturing agent a second chromatography device, such as an ion exchange column, capable of binding the fusion molecule to a chromatographic support. Applying the fusion molecule; (2b) Contacting the first eluate with a step gradient of aqueous buffer with the bound fusion molecule. The water-soluble buffer is preferably one that can reduce or remove denaturing agents and cause the fusion molecule to form a misfolded structure; (2c) a salt that can elute the fusion molecule from the second column. Contacting a second eluate with a concentration gradient with the fusion molecule to elute the misfolded fusion molecule from the second column; or, if necessary, (2d) removing the misfolded structure. Applying the eluted misfolded fusion molecule to a third chromatography device, such as a desalting column, capable of reducing the concentration of salt in contact with the fusion protein; The modified fusion molecule is applied to a chromatography device capable of removing the denaturing agent, for example, by elution in a desalting column.

This method is schematically shown in FIG.

The misfolded fusion molecule may be purified as described and provided as a substantially pure (preferably sterile) preparation suitable for pharmaceutical or research use. preferable. Further, those preferably used in the present method include:
Specific denaturant, eluate, and concentration gradient conditions disclosed in the examples below.

Among the above-mentioned steps (a) to (c), a particularly preferred fusion molecule having a misfolded structure prepared by a single step or a plurality of steps is a fusion protein having a misfolded structure. is there. The host cell is transfected with a nucleic acid encoding the fusion protein, and the host cell is cultured in an appropriate medium under conditions that allow the fusion protein to be expressed as an insoluble fraction and / or a soluble fraction in the cell. However, a fusion protein having a misfolded structure can be prepared using this method. The insoluble fraction contains inclusion bodies. In general, the nucleic acid can be introduced into a host cell using an appropriate recombinant vector, for example, by any method such as a transformation method, a transfer method using a particle gun, and infection. Examples of such vectors are described below. See Example 1. One or more denaturing agents such as urea or guanidine capable of denaturing the fusion protein are used, contacted with the cell fraction, and the fusion protein is isolated from this fraction. The denatured fusion protein is then subjected to a single or multiple steps of steps (b) to (g) described above such that the fusion protein can form a misfolded structure.

[0079] The fusion molecule with the misfolded structure is preferably provided as a sterile, substantially pure preparation suitable for pharmaceutical use or for cell culture.

A variety of commonly used chromatography devices are suitably used in the present invention,
Such chromatography devices include, but are not limited to, columns for affinity, immunoaffinity, ion exchange, molecular sieving, desalting, and the like.

Another method for producing a fusion protein having a misfolded structure includes a single step or a plurality of steps as follows.
That is, (a) a nucleic acid encoding a fusion protein is introduced into a host cell, and the host cell is cultured in a medium under conditions capable of expressing the fusion protein in the host cell. Preparing a fraction, (c) isolating the fusion protein from the fraction in the presence of one or more denaturants capable of denaturing and solubilizing the fusion protein, (d) solution of the fusion protein Contacting, preferably with an aqueous solution, and (e) converting the denatured fusion protein into a fusion protein having a misfolded structure in the solution.

The formation of the misfolded structure of the fusion protein in the above steps (a) to (e) can be performed by any of the methods described herein. As described above, the cell fraction is insoluble or soluble, or the cell fraction contains insoluble or soluble cellular components.

Although not required to be practiced in the present invention, if necessary, the misfolded structure of the fusion molecule may include, for example, circular dichroism, kinetic analysis of folding, spectroscopic analysis, NMR Analysis can be performed by various methods commonly used such as spectroscopy (including NMR of the solution) and X-ray crystal structure analysis, and further confirmation can be performed. If necessary,
This can also be done by standard calorimetry. In considering the present disclosure, other known methods recognized by those skilled in the art for determining protein structure can also be used. For example, the book "Physical Chemistry" by C. Tanford
See Macromolecules and the method incorporated herein by reference.

Further, a preferred fusion molecule having a misfolded structure of the present invention is one that completely dissolves in an aqueous solution. "Completely lysed" or similar expression means that the fusion molecule, especially the fusion protein, is centrifuged at low gravity in an aqueous buffer such as, for example, cell culture medium (eg, about 30 minutes per minute in a standard centrifuge). , 0
(00 rotations) means that it does not settle easily. Furthermore, at temperatures above about 5-37 ° C. and at or near neutral pH, the fusion protein (precipitated) in the presence or absence of low concentrations of anionic or nonionic detergents. If not) the fusion molecule is dissolved. Under such conditions, the sedimentation coefficient of soluble proteins often shows low values, for example, less than about 10 to 50 Svedbury units.

A “polypeptide” is preferably substantially 20 independent of its molecular weight.
Any macromolecule consisting of the essential amino acids is meant. Although the term "protein" is often used for proteins of relatively high molecular weight and "peptide" is often used for small polypeptides, these terms are often used interchangeably in the art. It is. Generally, the term "polypeptide" refers to proteins, polypeptides, and peptides unless otherwise specified.

[0086] As used herein, the term "cell" refers to a primary cell or an immortalized cell line, such as a tissue or organ consisting of such cells, which is capable of receiving transduction. It includes a group of cells. Such cells may be cells derived from sheep, cows, horses, etc., but are preferably derived from mammals,
In particular, it is of human origin. Particularly preferred are cells, tissues or organs from primates, especially humans. Examples of such cells include peripheral blood lymphocytes, whole blood cells, bone marrow stem cells, diploid fibroblasts, fibroma cells, keratinocytes, leukemic T cells, osteosarcoma, bone There are cancer, glioma, mouse 3T3 cells, hepatocellular carcinoma, and macrophages.

As used herein, the term “cell” refers to, for example, a cell such as Escherichia coli for expressing a desired fusion protein. It also means a cell that is a "target" for introducing the molecule of the present invention.

Suitable cells are from public institutions and the American Type Culture Collection (ATCC: 1
It can be obtained from various sources, including commercial distributors such as 2301 Parklawn Drive, 25).

The “host cell” in the present invention is also a cell such as a bacterial cell (for example, Escherichia coli) that can be used for expression of a desired fusion protein. Other suitable bacterial cells are known in the art.

The present invention relates to fusion proteins, particularly fusion proteins with misfolded structures. The term "fusion protein" as defined above refers to at least two polypeptides that are usually operably linked, but differing in their original origin. With respect to polypeptides, "operably linked" means that the two polypeptides are linked in such a way that each polypeptide can perform its intended function. Usually, the two polypeptides are covalently linked via a peptide bond, but if necessary, they can be separated using a peptide linker. The fusion protein is preferably a standard recombinant
Prepared by DNA technology. For example, a DNA molecule encoding a transduction domain is linked to another DNA molecule encoding a protein or polypeptide, and the resulting hybrid DNA molecule is expressed in a host cell to produce a fusion protein. The DNA molecules are linked together in the 5 'to 3' direction, so that the translational reading frame of the encoded polypeptide does not change (ie, the DNA molecule is in-frame).
frame)). The resulting DNA molecule encodes the fusion protein in frame.

The number, order, and the like of the components in the fusion molecule are not usually important, as long as each of the component molecules is operatively linked and can perform the intended specific function. A multi-component fusion molecule, such as one or more transduction domains,
Alternatively, molecules and the like linked by one or more covalent bonds are also included in the scope of the present invention.

[0092] The transduction domain of the fusion molecule can be used to identify a misfolded fusion molecule.
Almost completely synthesized or natural amino acid sequences may be used as long as they can be introduced or can support the introduction. For example, introduction in the present invention can be achieved using an HIV TAT protein or a fragment thereof covalently linked to a fusion molecule. Alternatively, the transduction protein may be the homeodomain of Antennapedia, or the sequence of HSV VP22, or a suitable transduction domain known in the art or a fragment thereof. Further, a synthetic peptide containing an analog of TAT or ANTP, or a transduction domain containing the same may be used. The fusion molecule is formed into a suitable misfolded structure to facilitate introduction into the target cell. For example, Frankel, AD and Pab
o, CO (supra), Falwell, S. et al. (supra), Chen, et al. (supra), U.S. Pat.
See 652,122 and WO / 04686.

A number of parameters, including the degree of transduction desired, are indicative for determining the type and size of the transducing protein. A preferred transducing protein is at least about 20% when it forms a misfolded structure in accordance with the invention,
Preferably, it has the ability to be introduced into 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even about 100% of target cells. The transduction efficiency of a particular transduction domain can be measured by a single method or a combination of methods, including a method of measuring the ratio (%) of transfected cells to non-transduced cells.
Particularly preferred transduction domains are those that can be introduced into nearly 100% of the total target cell number.

In many cases, the efficiency of introduction of a fusion molecule having an incorrectly folded structure is determined as a specific index when the efficiency of introduction of the fusion molecule in a natural or near-natural structure having the activity is used as a specific index. Determining the transduction efficiency for a fusion molecule with an improperly folded structure of interest can be performed, if necessary, on cells, or consisting of such cells
The fusion can be performed by introducing a fusion molecule into a cell group such as a tissue or an organ. An example of measuring the introduction efficiency will be described later in detail.

Suitable transduction domains also include the rate at which a fusion protein having a misfolded structure capable of introducing at least picomolar fusion molecules into a cell and the rate at which a fusion protein is exported from a cell (k1, respectively) And k2). The import / export rate can be easily measured, or at least estimated, by standard kinetic analysis using a fusion molecule labeled to enable detection. Typically, the ratio of the inflow rate to the outflow rate ranges from about 5 to 100 to about 1000.

As mentioned previously, the fusion molecules of the present invention can also be prepared by a chemical crosslinking method. Such a chemical cross-linking method is described by Means and Feeney in "C.
The method is disclosed in Chemical Modification of Proteins, (1974) Holden-Day. Also, Wong's "Chemistry of Protein Conjugation and Cross-Link
ing, (1991) CRC Press. Preferably, the fusion protein is prepared by standard genetic engineering procedures to create a suitable fusion protein that is genetically in-frame. For general matters, see Ausubel et al. (Supra) and Sambrook et al. (
See above).

As noted, the misfolded fusion molecules of the present invention are preferably substantially pure. That is, the fusion molecule, especially the fusion protein, is isolated from the cellular component that contains it. Usually, the fusion molecule will be at least 90-95
% (W / w) purity. For many medicinal uses, clinical applications, and research applications,
Most preferred are misfolded fusion molecules with a purity of at least 98-99% (w / w), especially misfolded fusion proteins. When purified and substantially pure, the fusion molecule should be substantially free of impurities that are problematic for therapeutic uses. Also, when partially purified or purified to a practical purity, a soluble fusion molecule may be used therapeutically.
Alternatively, it may be used in the in vitro or in vivo assays disclosed herein. Practical purity can be measured by various standard methods, such as chromatography and gel electrophoresis.

As will be described in more detail below, the fusion protein of the present invention can be separated and purified by a suitable combination of known methods. Examples of such a method include, for example, a method using a change in solubility such as a salting-out method and a solvent precipitation method, and dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. A method using a specific affinity such as affinity chromatography, a method using a difference in its electric charge state such as a method used, ion exchange column chromatography,
There are a method using a difference in hydrophobicity such as reversed-phase high performance liquid chromatography, a method using a difference in isoelectric point such as isoelectric focusing electrophoresis, and a metal affinity column such as Ni-NTA. See Sambrook et al., Supra, and Ausubel et al., Supra, for information on general issues related to these methods. Such fusion proteins usually have a misfolded structure as described herein.

In the following examples, the synthesis and use of various fusion proteins having a folded structure including up to 35 full-length wild-type and mutant TAT fusion proteins are shown (see Table 1). ). These examples also show the results examined for in vivo biochemical and biological reactions (see Table 1). As a result, the use of an in-frame full-length TAT fusion protein that has been misfolded after being expressed in bacterial cells can efficiently transduce both primary and transformed cells. And the transduction was concentration dependent. The fusion protein introduced by this method can bind to the same type of target in the cell and can also induce a biological response.

As described above, the present invention significantly enhances the efficiency of introducing various fusion molecules. A typical example is a 20 amino acid residue fused to the transduction domain of TAT.
The 16 partial peptides can arrest cells at the G1 phase of the cell cycle at a concentration of about 300 μM. However, with the present invention, the concentration of full-length TAT-p16 required to achieve the same effect is only 300 nM. Also, this partial peptide of p16 induces apoptosis as a side effect. However, such induction does not occur with full-length p16. Thus, the introduction of the full-length p16 protein (156 amino acids) causes a specific biological reaction 1000 times stronger than the partial peptide.

The preferred protocol for purification of fusion proteins described below shows several methods for the optimized formation reaction of misfolded structures. For example, in one embodiment, the desired TAT fusion protein was isolated under denaturing conditions that allow for the formation of a fusion protein having a misfolded structure that is considered to have a high energy state. More specifically, the reaction conditions for the formation of the misfolded structure of the TAT fusion protein were optimized by giving the reaction conditions for the formation of the misfolded structure by "shock". That is, the TAT fusion protein was replaced with an aqueous solution after the denaturation reaction, and was placed under conditions of water solubility. An overview of the misfolded formation reaction due to "shock", specifically described below, is shown in FIG.

The purification protocol using the following “shock” -induced misfolding reaction has two purposes. First, it was found that large amounts of recombinant protein expressed in bacterial cells became insoluble and were present in inclusion bodies. As described above, the method of the present invention is advantageous when utilizing a protein in such a state. Second, while not wishing to be limited to a particular theory, it is believed that the TAT fusion protein needs to be unwound and substantially linearized to cross the cell membrane. Thus, the use of an energetically unstable fusion protein with a high ΔG as compared to a fusion protein that is active and correctly folded, and therefore has a low ΔG and is stable, can be introduced into a variety of cells. It can be expected to enhance performance. While not wishing to be limited by any particular theory, proteins introduced into cells, such as chaperones, are known to catalyze the reaction of refolding a misfolded protein into its active site. With the help of the protein, it is thought that it will return to the correctly folded structure again. Preferred chaperones include Hsp90.

FIGS. 10a and 10b schematically show the effect of promoting the introduction efficiency obtained by the present invention.

[0104] The cDNAs described herein can be obtained from a variety of sources, including public institutions and commercial distributors.

For example, one example of such a supplier is the National Center for Biotechnol
ogy Information (NCBI)-There is a Genetic Sequence Data Bank (Genbank). DNA
For a list of sequences, see GenBank (National Library of Medicine, 38A, 8N05, Rockvi
lle Pike, Bethesda, MD 20894). GenBank is also accessible on the Internet (http://www.ncbi.nlm.nih.gov). For general information on GenBank, see the literature such as Nucl. Acids. Res. 25: 1 (1997) such as Benson, DA.

More specifically, sequences of various cDNAs suitable for the use of the present invention can be obtained from GenBank. In selecting a suitable cDNA from GenBank or other suitable sources, for example, the type of fusion molecule for the desired introduction (ie, enzyme, zymogen, nucleic acid, etc.) will guide the selection.

Table A below shows a preferred method of forming a misfolded structure of the fusion molecule by "shock" as described in more detail in the examples further below. The table also describes the thermodynamic parameters of each method, and the relative introduction efficiencies. “Low”, “high”, and “very high” are understood to be relative expressions used to compare the methods described in the tables to the previously recognized methods for introducing fusion proteins. Something to be done.

[Table A]

All documents mentioned herein are cited as references in this specification.

Examples of the present invention will be described below, but the present invention is not limited only to these examples.

Example 1-Construction of pTAT fusion protein expression vector A preferred plasmid for TAT fusion protein expression is described in US Provisional Application No. 60 /
No. 06912. This plasmid map is shown in FIG. 1a of the accompanying drawings.
FIG. 1b shows a pTAT vector encoding a fusion protein comprising HIS, TAT, and protein "X", which is any of the proteins described herein. FIG. 2 shows the nucleotide sequence (SEQ ID NO: 11) and amino acid sequence (SEQ ID NO: 12) of the pTAT linker, and the nucleotide sequence (SEQ ID NO: 6) and amino acid sequence (SEQ ID NO: 13) of the pTAT-HA linker. ing.

The pTAT and pTAT-HA (tag) bacterial expression vectors are created by insertion of an oligonucleotide corresponding to the 11 amino acids of the TAT domain flanked by glycine residues,
Free-bound rotation of the TAT domain (G-YGRKKRRQRRR-G) (SEQ ID NO: 14) to the BamHI site of pREST-A (Invitrogen) became possible. NcoI-K
By inserting a second oligonucleotide containing a pnI-AgeI-XhoI-SphI-EcoRI cloning site into the NcoI site (5 ′ or N ′) and EcoRI site, a polylinker was added to the C ′ end of the TAT domain. (See FIGS. 1A and 1B). This L
CS is followed by the remaining original polylinker of the pREST-A plasmid containing a BstBI-HindIII site.

The pTAT-HA plasmid contains the HA tag (YPYDVPDYA SEQ ID NO: 15) adjacent to glycine.
Oligonucleotides encoding the sequence in FIG. 2 in bold) were inserted into the NcoI site of pTAT. 5 'or N' Nco
The I site was inactivated and only the 3 'or C' of the HA tag was followed by the polylinker described above. The HA tag allows detection of the fusion protein by immunoblot, immunoprecipitation or immunohistochemistry using a 12CA5 anti-HA antibody.

The nucleotide and amino acid sequences of each linker are shown in FIG. pRSET-A
The backbone encodes ampicillin resistance, fl, ori, ColE1 ori (plasmid replication), and transcription is driven by the T7 RNA polymerase promoter.

The pTAT vector contains an N′-terminal 6-histidine leader, followed by minimally basic
It contains an 11 amino acid TAT protein transduction domain, flanked by glycine residues for free bond rotation, followed by a polylinker (FIG. 1a).

Example 2 Generation of TAT-Fusion Proteins To demonstrate the availability of the pTAT construct and demonstrate efficient transduction of full-length proteins, an in-frame pTAT fusion vector of A wild-type ("wt") protein and a mutant ("mut") protein from an inactive tumor,
For example, it was prepared with (R87P) p16 ^INK4a tumor suppressor gene and herpes simplex virus thymidine kinase (HSV-TK) cDNA. For example, see the papers by Hall, M. and Peters, G. (Adv
ances in Cancer Res. 68:67 (1996)).

The pTAT vector was used as a parent vector for producing a fusion protein.
In general, cDNA encoding the desired amino acid sequence was ligated to a multiple cloning site or pTAT vector polylinker, often referred to as "MCS", in a conventional manner to produce an in-frame fusion protein. The vector encoding this in-frame fusion protein was propagated using DH5-α bacteria as host cells. From this vector, a fusion protein was expressed using BL21 (DE3) pLysS cells (Novagen). The pTAT fusion protein was subsequently analyzed by SDS-PAGE gel electrophoresis. The production of a specific TAT in-frame fusion protein is preferably as follows.

1. TAT-p16 fusion protein: pTAT-p16 wild-type (often referred to as p16 ^wt ) and pTAT-p16 mutant vectors are created by ligation of the appropriate cDNA to the pTAT vector polylinker. did. The mutant p16 protein is often referred to as p16 ^mut or p16 ^INK4a because it indicates the R87P mutation of the wild-type p16 sequence. Halls, M. et al. (Advances in
Cancer Res. 68:67 (1996)). These cDNAs were individually inserted into the NcoI / EcoRI sites of the pTAT vector polylinker. The cDNA sequence of the p16 protein is Genba
can be obtained from nk. The molecular weight of the fusion proteins encoded by these vectors was approximately 20 kDa as estimated by SDS-PAGE gel electrophoresis.

2. TAT-HPV E7 fusion protein (HPV16 strain): The pTAT-HPV E7 ^wt and pTAT-HPV E7 mutant vectors were separately cloned into E7 ^wt and E7 ^mut
The cDNA encoding the protein was inserted into a pTAT vector polylinker (NcoI / EcoRI) to prepare. The cDNA sequence of E7 ^wt can be obtained from Genbank. Mutant E
The 7 protein is often referred to as E7 ^mut , meaning a deletion in the E7 ^wt sequence of the wild-type E7 sequence. The molecular weight of the fusion proteins encoded by these vectors was approximately 18 kDa as estimated by SDS-PAGE gel electrophoresis.

3. TAT-13S E1A fusion protein (adenovirus strain 5): The pTAT-13S E1A vector is created by inserting the cDNA encoding the 13S E1A protein into the pTAT vector polylinker (NcoI / EcoRI sites). did. The cDNA sequence encoding the 13S E1A protein can be obtained from Genbank. The molecular weight of the fusion protein encoded by this vector was approximately 61 kDa as estimated by SDS-PAGE gel electrophoresis.

4. TAT-p27 ^Kipl fusion protein: The pTAT-p27 ^Kipl vector was prepared by inserting the cDNA encoding the p27 ^Kipl protein into the pTAT vector polylinker (NcoI / EcoRI). p27 ^Kipl protein D
NA sequences can be obtained from Genbank. The molecular weight of the fusion protein encoded by this vector was approximately 30 kDa as estimated by SDS-gel electrophoresis.

5. TAT-pRB fusion protein: The pTAT-pRb vector was replaced with the cDNA encoding the retinoblastoma (Rb) protein by pT
It was prepared by inserting into an AT vector polylinker (NcoI / EcoRI site). The cDNA sequence encoding the RB protein can be obtained from Genbank. The molecular weight of the fusion protein encoded by this vector was approximately 115 kDa as estimated by SDS-PAGE gel electrophoresis.

6. TAT-HSV-TK fusion protein: The TAT-HSV TK vector was made according to the method disclosed in US Provisional Application No. 60/069012. The HSV-1 TK sequence was obtained from Genbank (accession number J02224).
). PCR primers corresponding to the N 'and C' ends of TK were prepared. After PCR, the DNA fragment was cut with NcoI / EcoRI and inserted into the corresponding polylinker site of the pTAT vector. In this method, the following primers were used:

The molecular weight of the TAT-TK fusion protein encoded by this vector was determined by SDS-PAGE
It was about 40 kDa as estimated by gel electrophoresis.

7. Preparation of TAT-CDK2 fusion protein: The TAT-CDK2 fusion protein was prepared according to the flow of the fusion protein described above.
The CDK2 cDNA sequence can be obtained from Genbank.

8. Preparation of Additional pTAT Fusion Proteins: In addition to the above-identified examples of TAT fusion proteins, the preparation and use of other fusion proteins is described in US Provisional Patent Application Ser. No. 60/069012. In particular, this U.S. Provisional Application discloses the generation of a fusion protein comprising a domain of TAT and a potential toxic zymogen, proenzyme or preproenzyme. As described above, these fusion proteins can form misfolds in accordance with the present invention to enhance transduction in a desired cell or group of cells.

Example 3-Preparation of a TAT fusion protein with a misfolded structure Some of the TAT fusion proteins described in Example 2 were purified from host cells,
Following the protocol below, a deliberately incorrect fold was formed. This protocol is often referred to herein as the "misfolded structure" or "misfolded structure due to shock" structure formation, or similar words. Fusion proteins made according to the misfolded protocol are often referred to herein as "shocked" misfolded fusion proteins or similar terms.

Protocol for protein misfolding: TAT fusion protein was obtained from transfected BL21 (D
E3) pLysS cells (Novagen) were purified by sonication (3 times, 15 seconds each). The culture was inoculated with 100 ml of an overnight culture in 10 ml of buffer A (8 M urea / 2OmM HEPES (pH 7.2 (100 mM NaCl)). The cell lysate was separated by centrifugation and added to buffer A for 20 minutes.
Loading was performed on a Ni-NTA column (Qiagen) in a solution to which mM imidazole was added. The column was then washed with 10 × column volume and eluted with increasing amounts of imidazole in buffer A (incremental).

Fusion molecules with misfolded structures can be processed by, but not limited to, 1) dialysis, 2) desalting on Mono Q / S columns, or 3) rapid desalting. is not.

1. Dialysis Method 1-3 ml of the denatured fusion protein was placed in a dialysis cassette (Pierce). The cassette was then placed in 4 liters of PBS at 4 ° C. and dialyzed. PBS is about 12
Three changes were made every 4 hours for a period of 、 24 hours, causing salt reduction and misfolding. The protein with the misfolded structure is 20 mM HEPES (pH 7.2) / 13
Stored in 7 mM NaCl and frozen in 10% glycerol at -80 ° C.

2. Mono Q / S desalting method The denatured fusion protein was added to a FPLC Mono-Q column (Pharmacia) in 4 M urea / 20 mM HEPES (pH 7.2, (50 mM NaCl)) solution. To form proteins into misfolds due to shock, the ion exchange column was stepped from 4 M urea to aqueous buffer and the protein was eluted stepwise with 50 mM to 1 M NaCl. The TAT fusion protein was eluted with a 1 M NaCl step, and PBS on a PD-10 desalting column (Pharmacia).
Or desalted to 20 mM HEPES (pH 7.2) / 137 mM NaCl and in 10% glycerol
Frozen at 80 ° C.

3. Rapid desalting method To rapidly form a misfold of the fusion protein, add 1 ml of the denatured fusion protein in 8M urea to a PD-10 desalting column at 4 ° C and equilibrate with PBS. And
The fusion protein was desalted by eluting with 5 ml of PBS added to the top of the column. About 0.5 ml of column fraction was obtained. The fusion proteins were obtained in PBS fractions 5, 6, and 7
Only recognized during. Urea was found in column fractions 10 to 15. Proteins with misfolded structures were stored in 20 mM HEPES (pH 7.2) / 137 mM NaCl and frozen in 10% glycerol at -80 ° C.

FIG. 3 summarizes these methods.

The TAT fusion protein remained at high concentrations and was resistant to freeze-thaw denaturation when stored at -80 ° C in 10% glycerol (Figure 5, lanes 1 or 2).

The various TAT fusion protein fractions obtained from the misfolded protocol were analyzed by conventional SDS-polyacrylamide gel electrophoresis (FIG. 4,
Lanes 1-10). The TAT fusion protein was found to contain approximately 1-10% of the total bacterial cell lysate; however,> 99% were insoluble and were present in inclusion bodies (
See FIG. 4, lane 1). To solubilize and denature the TAT fusion protein, the bacterial pellet was sonicated in 8M urea and the lysate was applied to a Ni-NTA column (see FIG. 3). The column fraction containing the recombinant TAT fusion protein was
Eluted by increasing the amount of imidazole in 8M urea (FIG. 4, lines 7-10)
. The flow-through and wash fractions from the Ni-NTA column were also analyzed (Figure 4, line 3).
And 5).

While not intending to be bound by any particular theory, efficient transduction of proteins across cell membranes unfolds proteins into linear sequences (u
nfolding), and once inside the cell, the transduced protein appears to be correctly refolded by a chaperone such as Hsp90. See, for example, Lissy, NA et al. (Immunity, 8: 57-68 (1998)). Thus, in this misfolded protocol, the TAT fusion protein is purified by subjecting it to "shock" misfolding from a denaturing environment to an aqueous environment. These results are consistent with transduction that requires the protein to be unfolded into a linear sequence. See Figures 4, 5 and 10.

1. Preparation of FITC-Labeled Fusion Protein FITC-labeled (fluorescent) TAT fusion protein was produced by a conventional method using a fluorescent label (Pierce) of a protein having an incorrectly folded structure. Labeled protein is FPLC (P
Purification was performed by gel purification in PBS on an S-12 column attached to Harmacia). A misfolded and FITC-labeled fusion protein is
Typically, it was added directly to the culture medium.

Example 4 Transduction of TAT-Fusion Proteins into Cultured Cells and Primary Cells Several TAT fusion proteins made in Example 2 above were analyzed to efficiently transduce cultured cells and primary cells. Determined ability to do. In general, all
The TAT fusion protein was found to transduce these cells efficiently.

About 0.1-100 nM of FITC-complexed TAT-p16 ^wt or TAT-HSV-TK fusion protein was added to Jurkat
T cells and / or human whole blood cells were added to the culture medium. Approximately 15, 30, or 45 minutes thereafter, flow cytometry analysis (FACS) was performed on the labeled protein (FIGS. 6A-E; 7A, 7B). These results indicate that both fusion proteins target> 99 cells.
% Rapid transduction, indicating that maximum intracellular concentrations are reached within 40 minutes (FIGS. 6A, B). It was also observed that there was a narrow intracellular concentration range within the population, measured as the FACS peak width between control and transduced cells. Similar results were observed with the TAT-p16 ^mut FITC protein.

It has also been found that the TAT fusion protein is capable of transducing primary cells. As an example, whole blood was treated with TAT-HSV-TK protein conjugated to a fluorescent dye and analyzed by FACS one hour after addition (FIGS. 6C, D). The TAT-HSV TR-FITC protein was homogeneously transduced into all cells present in human whole blood, including both nucleated and enucleated cells. In addition, TAT-p16 ^wt , TAT-p16 ^mut and TAT-HSV
TK proteins are also diploid (fibroblasts), fibrosarcoma cells, keratinocytes, bone marrow stem cells, peripheral blood lymphocytes (PBL), leukemic T cells, hepatocellular carcinoma cells, osteosarcoma and
NIH 3T3 cells were also shown to transduce efficiently.

In FIGS. 6A-F, concentration-dependent transduction and narrow intracellular concentrations are seen as measured by the near equivocal FACS peak width between control and transduced cells. Was. Approximately 10,000 cells were analyzed for each histogram.

The following TAT fusion protein was found to efficiently transduce all test cells after forming a misfold: TAT-HPV E7 ^{WT / MUT} protein,
TAT-p27 ^Kipl protein, TAT-13S E1A ^WT protein, and TAT-pRB protein. These results indicated that it was possible to transduce all cells of the cell population with the fusion protein of approximately 18-110 kD.

Table 1 below shows the biochemical and biological responses induced by transduction of a misfolded TAT fusion protein having a misfolded structure according to the present invention.

TABLE 1 Biochemical and biological responses induced by transduction of TAT fusion protein a. ND = undecided

1. Cell culture and flow cytometry, p16 ^INK4a (−) Jurkat T cell culture conditions and centrifugation elutriation were performed as described, and fractions containing G1-phase cells were 〜1 × ¹⁰⁶ was re-seeded at cells / ml. See Lissy, N.
A. TCR-antigen-induced cell death arising from late G1 cell cycle checkpoint
(AID) (1998), Dowdy, SF, (1997), Cell Entrainment by Elution: Cell Cycle Analysis of Specific Gene Expression, Human Genome Methods, edited by KW Adolph (in press) (1997).
Cell cycle location was determined by staining EtOH-fixed cells in propidium iodide and analyzed by flow cytometry (FACS; Bec
ton Dickinson). Uptake of FITC-labeled TAT-p16, -HSV TK, -EtA proteins into Jurkat cells and / or whole blood cells was determined by FACS analysis of 10,000 viable cells. Confocal microscopy was performed on paraformaldehyde-fixed Jurkat transduced with TAT-p16-FITC and TAT-HSV TK-FITC complex protein.

Example 5 Concentration-Dependent Transduction of TAT Fusion Protein To determine whether protein transduction occurs in a concentration-dependent manner, the fluorescent dye-conjugated TAT-p16 and TAT-HSV TK proteins were , Jurkat T cells were added at final concentrations of 4 nM, 16 nM, 40 nM and 025, 1.25, 10 nM, respectively, and analyzed by FACS after equilibration (1 h after addition) (FIGS. 6E and 6F). TAT-p16-FITC and TAT-HSV T
Both proteins of K-FITC showed a concentration-dependent transduction and the ability to modulate the intracellular concentration of the transduced protein. In addition, washing and replating cells preloaded with the TAT fusion protein resulted in a concentration-dependent transduction from the cells.

Example 6 Confocal Microscopy of Transduced TAT-p16 Fusion Protein FIGS. 7A and 7B show confocal microscopic analysis of transduced TAT-p16-FITC fusion protein. These results demonstrate the presence of the fusion protein in the nucleus and cytoplasm of the cell and little binding to the cell wall. TA
Intracellular fluorescence of T-p16 ^wt -FITC protein and stained periplasmic membrane ring (peric
ellular membrane ring) was observed to be absent (see FIGS. 7A-B).
.

Example 7-Detection of Folded TAT-Fusion Protein in Transduced Cells The formation of a complex between binding proteins depends on whether the protein is correctly folded (ie, native or nearly native structure) Usually the best one. Using this principle, it was shown that the transduced TAT-p16 and TAT-HSV E7 fusion proteins were correctly refolded after transduction into target cells.

1. Labeling and immunoprecipitation Jurkat T cells were labeled with 250 μCi ³⁵ -methionine (NEN) for 4 hours in the presence of TAT-p16 protein, and primary antibody, anti-p16 antibody as described. Immunoprecipitated with anti-E7 antibody, anti-E1A antibody, then polyclonal antibody, anti-Cdk6 antibody or anti-
Secondary immunoprecipitation with pRb antibody (Santa Cruz Biotechnology).

In FIGS. 8A and 8B, lysates of transduced cells were immunoprecipitated with anti-p16 antibody and re-immunoprecipitated with anti-Cdk6 antibody, and then separated by SDS-PAGE gel electrophoresis.

1. Transduced TAT-p16 fusion protein The p16 ^INK4a protein, which previously binds to Cdk6, but has a tumor-derived mutant,
For example, the p16 ^mut (R87P) used here has been shown to have lost this property. See, for example, Hall et al., Supra, Koh, J. et al. (Nature, 375, 506) and L.
ukas, J. et al. (1995) (Nature, 375, 503 (1995)), Medema, RH (
PNAS, 92, 6289 (1995)). The 10 nM and 200 nM TAT-p16 ^{WT / MUT} fusion proteins were labeled with S-methionine for cellular proteins and simultaneously with p16 (-) Jurkat.
T cells were transduced for 4-6 hours. From the whole cell lysate, the TAT-p16: Cdk6 complex was immunoprecipitated with an anti-p16 antibody and then immunoprecipitated again with an anti-Cdk6 antibody, and SDS-PAG
Separated on E (FIG. 8A). Co-immunoprecipitation and consequent Cd of TAT-p16 ^WT
In vivo association with k6 was easily detected at 10 nM and increased dramatically at 200 nM, whereas the 200 nM TAT-p16 ^MUT protein failed to associate with Cdk6.

2. Transduced TAT-HPV E7 fusion protein p16JurkatT cells are labeled with ³⁵ S-methionine for 4-6 hours and TAT-13S E1
Transduced with A ^MUT protein or TAT-13S E1A ^WT protein. Immunoprecipitation of the transduced and labeled cells indicates that the TAT-13S E1A protein is capable of associating with the retinal fibroblastoma suppressor protein (pRb) but not the TAT-E7 mutant protein in vivo. (FIG. 8B). These findings indicate that the transduced (mis-folded) protein can be correctly refolded once by intracellular chaperones and then associated with its cognate labeled protein in vivo. ing.

Example 8 Cell Cycle Arrest by Transduced TAT-p16 Fusion Protein Transient transfection of cells with a wild-type p16 expression vector in G1 phase
Cell cycle arrest in For example, Koh, J. et al., Supra, and Lukas,
See J. et al., Supra, Medema, R.H., supra. Therefore, transduction
P16 (-) Jurkat T cells to verify the biological effects of the isolated TAT fusion protein
G1 phase specific cells were centrifuged, re-seeded, 150 nM and 300 nM TAT-p16 ^WT Or TAT-pl6^MUTTransduction of the fusion protein and cell cycling 16 hours after treatment
Phase locations were analyzed by propidium iodide DNA staining and FACS analysis (FIGS. 9A-E). Lissy, ENG GB
See N.A., op.cit., Dowdy, S.F., op.cit. (1997). Centrifugal elution
G1 phase Jurkat T cells were transformed with 150 nM or 300 nM TAT-p16^WTOr TAT-pl6^MUTTampa
Cells for 16 hours, and then propidium iodide DNA staining and FACS
The position of the cycle was analyzed. Control and TAT-pl6^MUTG1 to S / G2 / M phase of treated cells
To TAT-p16^WTA concentration-dependent G1 arrest of the treated cells was observed.

As shown in FIGS. 9A-E, JurkatT cells transduced with the TAT-p16 ^WT protein were still arrested in the G1 phase of the cell cycle in a concentration-dependent manner, but were treated with the TAT-pl6 ^MUT protein. Isolated or untreated cells transitioned from the G1 phase to the S / G2 / M phase. In addition, p16 (+) human HaCaT keratinocytes have also been shown to be arrested by the TAT-p16 ^WT protein. A paper by Ezhevsky, SA (Proc Natl. Acad. Sci. USA, 94: 1069
9 (1997)). These findings demonstrate that the transducible TAT-p16 ^WT protein continues to retain all or nearly all of the previously known properties.

Example 9-Increased Transduction Efficiency of Proteins with Misfolded Structure by Shock The transduction efficiency of the desired fusion protein can be determined at various steps in the misfolded structure formation protocol. See Example 3 and FIG. For example, the transduction efficiency can be calculated after solubilizing the fusion protein from the host bacterium, after denaturation and reconstitution of the fusion protein, or after denaturation of the fusion protein followed by ion exchange chromatography. The transduction efficiency can be calculated, for example, by measuring one or more of the transduction rate, the intracellular amount of the fusion protein or the amount or activity of the transduced protein compared to a suitable control.

A related experiment was performed with the TAT-p27 fusion protein. TAT-p27 has the ability to induce cell cycle arrest and cell scatter (scatte) in HepG2 cells 24-48 hours later.
The ability to induce ring) was measured. Cell scatter was measured with a microscope. In this experiment, TAT-p27 which was denatured and had a misfolded structure due to shock
It was observed that the fusion protein transduced cells more than 20 times more efficiently than the denatured and dialyzed TAT-p27 fusion protein.

Briefly, BL21 (DE3) pLysS cells (Novagen) were transformed with the TAT-p27 vector described above and grown in 1 liter of medium for 5 hours. This culture was inoculated with 100 ml of the overnight culture. The culture was split into two fractions, one denatured and dialyzed as described above, and the other formed a misfold due to shock. An aliquot was mixed with about 10 ml of buffer A (8 M urea / 20 mM HEPES (pH 7.2 (100 mM NaCl)), sonicated, and the "denatured" The denatured fraction was clarified and then loaded onto a Ni-NTA column (Quiagen) in buffer A plus imidazole 20 mM, and the column was then washed with 10 × column volume. And eluted with increasing (gradual increasing) concentration of imidazole in buffer A, followed by 4 M urea / 20 mM HEPES (pH 7.2,
50 mM NaCl)) in a Mono-Q column (Pharmacia) on FPLC. The resulting "denatured and shocked" TAT-p27 fusion protein with misfolded structure was eluted, desalted and stored as described in Example 2.

The p27 fusion protein, which is denatured and has a misfolded structure due to shock, was found to be more than 20-fold more efficient at inducing cell scatter in transduced cells than the denatured and dialyzed fusion protein.

These results indicate that this shock-induced misfolding protocol is more efficient than the soluble fully or partially correctly folded protein for 2,5.
, 10, 20, 50, 100-200-fold, indicating that a TAT fusion protein having a misfolded structure with a transduction efficiency that is greater than -fold can be generated.

The present invention can be used to enhance expression of vectors, proteins, and / or to assist transduction of pharmaceutically important peptidyl mimetics into target cells. The present invention overcomes problems due to low percentage of targeted cells, overexpression, size constraints and bioavailability. In some embodiments, the invention provides for the integration of the transduction domain of an N'-terminal protein from an in-frame bacterial expression vector, pTAT, HIV TAT. Further, there is provided a purification protocol for obtaining an energetically unstable, misfolded protein that can be efficiently transduced. The 15-115 kD full-length TAT fusion protein was transduced in> 99% of all target cells tested: peripheral blood lymphocytes, whole blood cells, bone marrow stem cells, diploid fibroblasts, Fibroblast sarcoma cells, leukemic T cells, osteosarcoma cells, gliomas, mouse 3T3 fibroblasts, hepatocarcinoma cells, macrophages, and keratinocytes. Transduction occurs in a concentration-dependent manner, reaching a maximum intracellular concentration in less than 30 minutes.
Therefore, transduction of proteins directly into cells has wide potential in the manipulation of experimental systems and in the delivery of pharmaceutically relevant proteins.

Example 10-Rapid desalting method for the production of fusion proteins with misfolded structures due to shock To obtain misfolded / denatured TAT fusion proteins, as shown in the previous example. Was purified on Ni-NTA in 8M urea. Denatured TA
PD-1O Sephadex desalting column equilibrated with PBS with T fusion protein (Pharmacia)
Was injected. The protein was separated from urea by size exclusion. Denatured
The TAT fusion protein was found to transition very quickly from a denaturing environment (high urea concentration) to an aqueous environment (relatively low urea concentration). As a result, the TAT fusion protein is misfolded, exists in a high ΔG state, and is completely dissolved in aqueous solution. The rapid transition from a denaturing environment to an aqueous environment is due to the α helix and β-
It is believed to prevent the formation of normal (ie, natural) structures, including pleated sheet structures.

The rapid desalination process of the present invention is easy to perform and can be performed with minimal capital investment. This method is generally applicable to most, if not all, fusion proteins.

Example 11-Analysis of fusion proteins with native and misfolded structures Previous attempts relating to the potential for protein transduction have shown that correctly folded soluble proteins (theoretically low) ΔG) and / or in combination with drugs, such as chloroquine, to treat the cells. In this example, the transduction efficiencies between the native protein and the fusion protein with a misfolded structure were compared. ^Transduction of full-length wild-type p27 ^kipl protein (TAT-p27 ^wt ) induced a biological response of cell migration or “scattering”. 8M urea denatured misfolded TAT with shock-induced misfolding on Mono-S ion exchange column for soluble and correctly folded TAT-p27 ^wt protein made in bacteria The ability of -p27 ^wt protein to induce cell migration was compared (Figure 12)
. Transduction of the denatured and misfolded TAT-p27 ^WT protein is 50n
M, 100 nM and 150 nM concentrations readily induced cell migration; however, soluble, correctly folded TAT-p27 ^wt , even at the highest concentration, failed to induce cell migration. Therefore, we conclude that the use of a TAT fusion protein with a misfolded structure enhances the ability to transduce cells into cells and alter biological methods.

FIGS. 11a-e illustrate the following in more detail: Denaturation of soluble (SL) to correctly folded TAT-p27 ^wt protein (
DN), Direct comparison of cell migration induction of TAT-p27 ^wt protein with misfolded structure. Cell migration is a morphological change in the cell membrane (induction of a "puzzle-piece" morphology)
And by cell migration in colonies away from each other. Positive hepatocyte growth factor (HGF) and negative PBS control (ctrl) were used as indices.

The present invention has been described in detail with reference to these preferred embodiments. However, it will be apparent to those skilled in the art that many modifications and improvements can be made within the spirit and scope of the invention based on the teachings herein.

[0164] All references mentioned herein are all incorporated by reference.

[Brief description of the drawings]

FIG. 1A is a view showing a pTAT expression vector.

FIG. 1B is a diagram of a pTAT expression vector, in which the protein “X
Is fused with 6XHIS (HIS tag) and TAT.

FIG. 2 shows the nucleotide sequence and amino acid sequence of a pTAT linker and a pTAT HA linker. The minimal domain of TAT is shown in bold. The underlined sequence is the minimum TAT domain flanked by glycine residues.

FIG. 3 outlines a protocol for purification and misfolding by “shock”. A triangle indicates that a concentration gradient of imidazole was added. "Desalting" refers to desalting a fusion protein that has formed a misfolded structure by shock.

FIG. 4 shows a Coomassie brilliant blue stained image of an SDS-polyacrylamide gel. On the gel, the various fractions obtained in the purification of TAT-p16 ^Wt performed according to the protocol outlined in FIG. 3 are shown. Ss = start, FT = flow through.

FIG. 5 shows a Coomassie brilliant blue stained image of an SDS-polyacrylamide gel. This gel compares TAT-p16 ^Wt before and after freezing and thawing (lane 1) and after (lane 2) of a sample frozen and stored at -80 ° C in 10% glycerol.

6A-F. Fluorescence-activated cell sorter of FITC-labeled TAT fusion protein (fl
FIG. 3 illustrates a histogram obtained by analysis by uorescence activated cell sorting (FACS). FITC-labeled TAT-p16 ^Wt (6A) and T
AT-HSV TK (6B) fusion protein was added. At 40 minutes and 160 minutes after the addition
FACS analysis was performed. FITC-labeled T on human whole blood (6C) and macronucleated red blood cells (6D)
AT-HSV TK fusion protein was added. One hour after the addition, FACS analysis was performed. Jurka
For equilibrium FACS analysis 1 hour after transfection of t cells, 0.25, 1.25, and 10 nM TAT-p16 ^Wt- FIT
C protein (6E) or 4, 16, or 40 nM TAT-HSV-TK-HTC fusion protein (
6F) was used.

7A and B: A control molecule (7A) and a TAT-p16 ^Wt -FITC protein (7B
7 is a photomicrograph showing a confocal microscope image of Jurkat T cells into which J) was introduced.

FIGS. 8A and B: TAT-p16 ^WT and Cdk6 complex, and TAT-I 35 E1A and pR
FIG. 2 illustrates an SDS-polyacrylamide gel electrophoresis image showing that the complex of b is formed in vivo. While labeling p16 (-1-) Jurkat T cells with ³⁵ S-methionine for 4 hours, 10 nM and 200 nM TAT-p16 ^{Wt / mut} fusion protein was introduced during this time (8A). Keratinocytes were labeled with ³⁵ S-methionine in the presence of 100 nM TAT-I3SE1A ^mut or 100 nM TAT-I3SE1A ^wt fusion protein, and then treated in the same manner as described above. This was immunoprecipitated with anti-E7 antibody or anti-E1A antibody, and anti-pRb
Treatment with the antibody and SDS-polyacrylamide gel electrophoresis were performed (8B). Cdk
6, and the position of pRb are shown. The symbol “α” before the protein name indicates that the protein was treated with an antibody specific to that protein. "IP" means immunoprecipitation using anti-p16 ^Wt antibody or anti-Cdk6 antibody. “Mut” means p16 ^mut .

9A to 9E are graphs showing that p16 (−) Jurkat T cells arrest in the G1 phase of the cell cycle by introduction of the TAT-p16Wt protein.

FIG. 10A is a diagram showing the Gibbs free energy (ΔG), and a diagram schematically showing the misfolded structure and the native structure of the fusion protein adjacent thereto. A fusion protein having a misfolded structure is energetically unstable compared to a protein having a natural structure.

FIG. 10B is a model showing that a fusion protein having an incorrectly folded structure is more easily introduced than a protein having a natural structure. In this model, a fusion protein with a misfolded structure is regenerated by the chaperone Hsp90 in the transfected cells.

11A to 11F are photomicrographs showing that a TAT-p27 ^wt fusion protein having a misfolded structure has an activity to promote cell motility. TGF-β = transforming growth factor β, HGF = hepatocyte growth factor.

FIG. 12 is a graph showing cell migration expressed as a percentage in% after introducing TAT-p27 ^Wt in a soluble native structural state (SL) and in a denatured misfolded structural state (DN).

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY , CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG , KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW

Claims (60)

[Claims] 1. A method of transducing a cell with a fusion molecule, the step of forming a misfold of the fusion molecule; and transducing the fusion molecule having a misfolded structure into the cell. Contacting the cell under conditions sufficient for the method. 2. The method of claim 1, wherein the step of forming a misfold comprises exposing the fusion molecule to a first condition sufficient to denature the fusion molecule. 3. The method of claim 2, wherein the step of forming a misfold further comprises exposing the modified fusion molecule to a second condition sufficient to form a fusion molecule having a misfolded structure. Method. 4. The fusion molecule having a misfolded structure has a higher Gibbs free energy (ΔG) than the fusion molecule of its native or nearly native structure.
The method of claim 3. 5. The method of claim 4, wherein the Gibbs free energy (ΔG) is between about 2 to 100 times or more than the fusion molecule of its natural or near-natural structure. 6. The method of claim 2, wherein the first condition comprises contacting the fusion molecule with one or more denaturing agents. 7. The method according to claim 6, wherein the denaturing agent is selected from the group consisting of an organic compound, a salt, a surfactant, heating or sonication. 8. The method according to claim 7, wherein the organic compound is selected from the group consisting of urea or guanidinium salts. 9. The method of claim 3, wherein the second condition comprises contacting the denatured fusion protein with a solution sufficient to form a fusion molecule having a misfolded structure.
The described method. 10. The method of claim 1, wherein the first condition comprises contacting the fusion molecule with one or more denaturing agents sufficient to denature the fusion molecule, and the second condition comprises: 4. The method of claim 3, comprising contacting the denatured fusion molecule with one or more aqueous buffers sufficient to increase energy ([Delta] G). 11. The second condition is that the Gibbs free energy (Δ
11. The method of claim 10, wherein G) is increased to be between about 2 to 100-fold or more relative to the fusion molecule of natural or near-natural structure. 12. The method of claim 10, wherein each contacting step is performed by one or more chromatography devices. 13. The method of claim 12, wherein the chromatography device comprises at least one affinity column and an ion exchange column. 14. The method of claim 13, further comprising a desalting column. 15. The contacting step of the second condition, wherein the denaturation in an aqueous buffer sufficient to increase the Gibbs free energy (ΔG) of the denatured fusion molecule relative to the fusion molecule of native or near-natural structure. 11. The method of claim 10, comprising diluting the fusion molecule. 16. The modified fusion molecule may be added to an aqueous buffer at about 1:10 (v / v) to about 1: 100,000.
16. The method according to claim 15, wherein the method is added in a ratio of (v / v). 17. The method of claim 15, wherein the aqueous buffer is sufficient to reduce or eliminate the presence of a denaturing agent. 18. The method of claim 16, further comprising the use of a device sufficient to concentrate the misfolded fusion molecule from the aqueous buffer. 19. A substantially pure misfolded fusion molecule produced by the method of any one of claims 1-18. 20. A method for producing a fusion protein having a misfolded structure, comprising the steps of: a) expressing a nucleic acid encoding a fusion protein into a host cell in a medium, and expressing the fusion protein in the host cell. B) preparing an insoluble fraction from the host cells; c) in the presence of one or more denaturing agents sufficient to denature and solubilize the fusion protein Isolating the fusion protein from the insoluble fraction; d) contacting the denatured fusion protein with a solution sufficient to produce a fusion protein having a misfolded structure; and e) aqueous buffer from the denatured fusion protein. Generating a fusion protein having a misfolded structure therein. 21. The solution according to claim 1, wherein the Gibbs free energy (Δ
21. The method of claim 20, wherein G) is selected to increase to between about 2 to 100-fold or more relative to the fusion protein of native or near native structure. 22. The method of claim 20, wherein the solution is one or more aqueous buffers. 23. The method according to claim 20, wherein the denaturing agent is selected from the group consisting of an organic compound, a salt, a surfactant, heating or sonication. 24. The method of claim 23, wherein the denaturing agent comprises at least about 4-8M urea. 25. The method of claim 25, wherein step c) further comprises the use of at least one chromatography device sufficient to separate the denatured fusion protein from the insoluble fraction.
Method according to 20. 26. The method of claim 25, wherein the chromatography device is an affinity column sufficient to bind a denatured fusion protein thereto. 27. The method of claim 26, wherein the denatured fusion protein is contacted with a first eluent capable of eluting the denatured fusion protein from the affinity column. 28. The method of claim 27, wherein the first eluent comprises an organic compound. 29. The method of claim 28, wherein the eluent further comprises an imidazole gradient and about 4-8 M urea. 30. The method of claim 20, wherein step b) further comprises the use of at least one chromatography device sufficient to generate a fusion protein having a misfolded structure. 31. The method of claim 20, wherein the at least one denaturing agent is a urea or guanidinium salt. 32. The chromatography device is an ion exchange column, and wherein the denatured fusion protein is from less than or equal to about 8M urea to an aqueous buffer sufficient to produce a fusion protein having a misfolded structure. 31. The method of claim 30, wherein the method is stepped. 33. The method of claim 32, wherein the misfolded fusion protein is eluted from the ion exchange column by contacting the misfolded fusion protein with a second eluent. 34. The method of claim 33, wherein the second eluent is a salt gradient sufficient to elute a misfolded fusion protein from the ion exchange column. 35. The method of claim 34, wherein the salt gradient comprises about 10 mM to about 2M NaCl. 36. The method of claim 35, further comprising reducing the presence of a salt in contact with the fusion protein having a misfolded structure. 37. The method of claim 36, wherein salt reduction is achieved by use of a chromatography device. 38. The fusion molecule having a misfolded structure is capable of enhancing transduction at least about 2, 5, 10, 50, 100 to 200-fold or more relative to a control molecule. 20. The method according to any one of 1 to 19. 39. The control molecule is a fusion molecule that exists in its native structure.
38. The method according to 40. The fusion protein having a misfolded structure is capable of enhancing transduction at least about 2, 5, 10, 50, 100 to 200-fold or more relative to a control protein. The method according to any one of claims 20 to 37. 41. The method of claim 40, wherein the control protein is a fusion protein present in its native structure. 42. The method of claim 1, wherein the fusion molecule comprises a protein transduction domain covalently linked to a molecule, wherein the fusion molecule has a molecular weight of about 0.1-500 kD or more. 43. The method of claim 42, wherein the molecule is selected from the group consisting of nucleic acids, proteins, polypeptides, peptides, amino acids, lipids, glycolipids, proteoglycans, proteolipids, carbohydrates, and glycoproteins. 44. The method of claim 20, wherein the fusion protein comprises a transduction domain of the protein covalently linked to an amino acid sequence, wherein the fusion protein has a molecular weight of about 0.1-500 kD or more. 45. The method of claim 44, wherein the protein transduction domain is a protein transduction domain or another protein transduction domain. 46. The transduction domain of another transduction protein, wherein at least
46. The method of claim 45 comprising amino acids 49-56 of TAT, a synthetic domain or ANTP. 47. The method of claim 44, wherein the amino acid sequence is a protein selected from the group consisting of p16, TK, EIA, p27, E7, GFP, Rb, or a fragment thereof. 48. A substantially pure preparation of a misfolded fusion molecule produced by the method of any one of claims 1-19. 49. A substantially pure preparation of a fusion protein having a misfolded structure produced by the method of any one of claims 20-37. 50. The substantially pure misfolded fusion protein of claim 48 or 49, wherein the misfolded fusion protein is contacted with one or more stabilizing agents. 51. The substantially pure misfolded fusion protein of claim 50, wherein the one or more stabilizing agents is bovine serum albumin, glycol or dimethyl sulfoxide. 52. The substantially pure misfolded fusion protein of claim 51, wherein the glycol is glycerol present in an amount of about 1-15% (v / v). 53. The fusion protein having a misfolded structure comprises about -10 to -10
53. The substantially pure misfolded fusion protein of claim 52, which is stored between 0 ° C. 54. A method for transducing a cell or a group of cells with a fusion protein, the method comprising the step of: A method comprising contacting a cell or group of cells under conditions sufficient to transduce a cell or group of cells having a folded structure. 55. The method of claim 54, wherein the cells are mammalian cells. 56. The method of claim 55, wherein said mammalian cell is an immortalized or primary cell. 57. The group is a mammalian cell comprising a target tissue or organ.
The described method. 58. The method of claim 57, wherein said population of mammalian cells is in vivo. 59. The transduction domain of another transduction protein has at least
46. The method of claim 45, comprising amino acids 49-56 of TAT. 60. The method of claim 20, wherein the host cell is a bacterial cell and the insoluble fraction comprises inclusion bodies.