# Patent

## Title
Protein complex for intracellular delivery and uses thereof

## Abstract

A protein complex comprising an in vitro stabilization protein, a membrane translocation sequence domain, a biologically active molecule, and an in vivo stabilization protein, as well as methods for the use and production thereof.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of Korean Patent Application No. 10-2011-0081160, filed on Aug. 16, 2011 in the Korean Intellectual Property Office, the disclosures of which are incorporated herein in their entirety by reference.
INCORPORATION BY REFERENCE OF MATERIAL ELECTRONICALLY FILED
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 206,551 byte ASCII (Text) file named “2ndReplacement710893_ST25.txt”, created on Jan. 21, 2013.
BACKGROUND
1. Field
The present disclosure relates to protein complexes for intracellular delivery and uses thereof.
2. Description of the Related Art
Many human diseases develop due to abnormal activities of intracellular proteins. Accordingly, number of efforts has been made to develop novel medicines for treating various human diseases by controlling the abnormal activities of such intracellular proteins over the world. In particular, peptide, peptidergic material or protein-type material based on an enzyme-protein or protein-protein interaction that specifically controls biological activities have been rapidly developed. Although a peptide, a polypeptide, and a protein have much better physiological selectivity and efficacy than any other compounds, they cannot easily pass through a cell membrane due to their sizes and biochemical characteristics. Thus, they are not practically used as an effective therapeutic agent and a study material.
Also, control of biological activity by delivering a macromolecule, such as DNA, RNA, protein, oligonucleotide, and peptide, to a cell is very limited due to a cell membrane that constitutes a non-permeable barrier to these molecules. Biologically active macromolecules have very low delivery efficiency into tissues or cells, lack cell-specific targeting ability, and in many cases, decompose in vivo. Due to such obstacles, it is difficult to realize the efficacy of biologically active macromolecules.
Thus, many efforts such as developing various intracellular delivery platforms have been made. For example, recently developed delivery systems using short peptides have been studied. These peptides are cell-permeable and easy to introduce into cells, which characteristics have attracted the attention of researchers. However, the development of a system using delivery peptides has been at a standstill due to lack of commercial-level manufacture, in vitro and in vivo stability issues, and other such obstacles.
Accordingly, even with the availability of such conventional techniques, there remains a need for new compositions and methods for intracellular delivery of biologically active molecules.
SUMMARY
Provided are protein complexes for intracellular delivery, the protein complexes including an in vitro stabilization protein, a membrane translocation sequence domain, a biologically active molecule, and an in vivo stabilization protein.
Also, provided are methods of delivering a biologically active molecule into a cell by using the protein complex, a method for preparing a protein complex, and related methods and compositions.



BRIEF DESCRIPTION OF THE DRAWINGS
These and/or other aspects will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings in which:
FIG. 1 is a schematic view of a protein complex for intracellular delivery;
FIGS. 2A and 2B show sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results of purified products of a protein complexes as described herein. In FIG. 2A, protein complex #12-1 is Ub7KR(G76A)-MTS-NLS-SABP; protein complex #12-5 is Ub7KR(G76A)-MTS-p53-NLS-SABP; protein complex #12-6 is Ub7KR(G76A)-p53-NLS-SABP; protein complex #12-7 is Ub7KR(G76A)-MTS-p18-NLS-SABP; and protein complex #12-8 is Ub7KR(G76A)-p18-NLS-SABP. In FIG. 2B, protein complex #44 is Ub7KR(G76A)-MTS-p53-p18-NLS; protein complex #52 is Ub7KR (G76A)-MTS-p53-18-NLS-SABP; protein complex #54 is Ub7KR-MTS-p53-p18-NLS-SABP; protein complex #66 is Ub7KR(G76A)-p53-p18-NLS; protein complex #56 is Ub7KR(G76A)-p53-p18-NLS-SABP; and protein complex #57 is Ub7KR-p53-p18-NLS. M refers to a size marker;
FIG. 3 shows SDS-PAGE results of serum albumin binding of a protein complex that does not include SABP (A panel), and of a protein complex that includes SABP (B panel); and
FIGS. 4A to 4E show cell growth suppression efficacy of a p53-p18 protein evaluated by administering a protein complex described herein to colorectal cancer cell lines [HCT116 (FIG. 4A), SW48 (FIG. 4B), LoVo (FIG. 4C) and LSI174T (FIG. 4D)) and a skin carcinoma cell line (A431 (FIG. 4E), Korean Cell Line Bank].



DETAILED DESCRIPTION
Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description. Expressions such as “at least one of,” when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.
An aspect of the present invention provides a protein complex for cell delivery, including an in vitro stabilization protein, a membrane translocation sequence domain, a biologically active molecule, and an in vivo stabilization protein.
The protein complex for cell delivery may include an in vitro stabilization protein.
The term “in vitro stabilization protein” used herein refers to a protein for increasing solubility and stability of the protein complex when the protein complex is experimentally purified in vitro. In other words, the in vitro stabilization protein can be any protein that, when complexed with or otherwise attached to a cargo molecule (e.g., any of the biologically active molecules described herein) fused with a hydrophobic cell penetrating peptide (CPP) or membrane translocation sequence domain (MTS), provides a complex with increased water solubility as compared to the cargo molecule fused to the CPP/MTS without the in vitro stabilization protein. The in vitro stabilization protein is part of the protein complex and does not induce immunogenicity in vivo.
According to an embodiment of the present invention, the in vitro stabilization protein may be a ubiquitin (Ub), a Ub-like protein, an ankyrin repeat motif-including protein, or a transferrin protein.
Ub is the most conserved protein in nature that consists of 76 amino acids (SEQ ID No. 19), shows complete homogeny among evolutionarily various species including insects, trout, and humans, and is water-soluble. Also, Ub is known as a protein that has stability with respect to pH change, is not easily denatured even at high temperature, and has stability with respect to a protease. An ankyrin repeat motif is a 33-residue motif in proteins consisting of two alpha helices separated by loops. The ankyrin repeat motif mediates protein-protein interactions and is among the most common structural motif in known proteins. Transferrins are iron-binding glycoproteins that control the level of free iron in biological fluids. The Ub, Ub-like protein, the ankyrin repeat motif-including protein, or the transferrin protein may improve insolubility of the protein complex.
The Ub or Ub-like protein may be at least one selected from the group consisting of wild-type Ub, wild-type Ub-like protein, mutant Ub, and mutant Ub-like protein. In an embodiment, the wild-type Ub may have an amino acid sequence with SEQ ID No. 19. The mutant Ub refers to that an amino acid sequence of the wild-type Ub is substituted with another amino acid sequence. Examples of the mutant Ub are Ub formed by substituting Lys of a wild-type Ub with Arg, and Ub formed by substituting RGG at the C-terminus of a wild-type Ub with RGA. According to an embodiment of the present invention, in a mutant-type Ub formed by substituting Lys of a wild-type Ub with Arg, the substitution may occur with at least one Lys present at locations 6, 11, 27, 29, 33, 48 and/or 63 of the wild-type Ub with SEQ ID No. 19, and the substitution may occur in the Lys positions independently or in combination. According to an embodiment of the present invention, the mutant-type Ub may be a mutant Ub obtained by substituting Gly at location 76 in a wild-type Ub polypeptide with another amino acid, for example, with alanine (Ala).
According to an embodiment of the present invention, the Ub-like protein is a protein that has characteristics (e.g., stability characteristics) similar to those of Ub, and, for example, may be Nedd8 (e.g., UniProtKB/Swiss-Prot: Q15843), SUMO-1 (e.g., UniProtKB/Swiss-Prot: P63165), SUMO-2 (e.g., UniProtKB/Swiss-Prot: P61956), NUB1 (e.g., UniProtKB/Swiss-Prot: Q9Y5A7), PIC1, UBL3 (e.g., UniProtKB/Swiss-Prot: O95164), UBL5 (e.g., UniProtKB/Swiss-Prot: Q9BZL1), or ISG15 (e.g., UniProtKB/Swiss-Prot: P05161).
According to an embodiment of the present invention, an ankyrin repeat motif-including protein or a transferrin protein may be wild type or mutant type thereof. For example, mutant type of an ankyrin repeat motif-including protein or a transferrin protein may be wild type may be genetically engineered.
According to an embodiment of the present invention, the Ub, Ub-like protein, ankyrin repeat motif-including protein, or transferrin protein may include at its C-terminus an amino acid sequence that is cleavable by a protease, or an amino acid sequence that is not cleavable by a protease. The amino acid sequence that is cleavable by a protease may be obtained from data base known in the art. For example, proteases and amino acid sequences that are cleavable by the proteases can be determined using available tools, such as the PEPTIDECUTTERTM database maintained by the Swiss Institute of Bioinformatics, Quartier Sorge—Batiment Genopode, 1015 Lausanne, Switzerland. When the cleavable amino acid sequence is included, once the protein complex is transferred into a cell, the Ub, Ub-like protein, ankyrin repeat motif-including protein, or transferring protein is cleaved by an intracellular protease, thereby enabling the biologically active molecule to perform its intracellular function. Even after the cleavage, the biologically active molecule still includes a membrane translocation sequence domain. Since the length of a polypeptide of the membrane translocation sequence domain is very short, the membrane translocation sequence domain does not affect the function of the biologically active molecule. In embodiments where the Ub or Ub-like protein is not cleaved by the intracellular protease, the biologically active molecule is selected such that it will still perform its intracellular function.
The term “membrane transfer” used herein refers to a capability of delivering a biologically active molecule of a subject to be carried into a cell or nucleus in vitro and/or in vivo. Also, the term “membrane translocation sequence domain (MTS)” or “cell-penetrating peptide” used herein refers to a polypeptide having an amino acid sequence that is permeable through a cell membrane of a phospholipid bilayer (i.e., a polypeptide that will permeate a cell membrane, regardless of mechanism). In an embodiment, the membrane translocation sequence domain may have a single hydrophobic region at its N-terminal, form a helical structure, show flexibility, and/or have a relatively short amino acid sequence (e.g., about 7 to 17 amino acids). The membrane translocation sequence domain may have hydrophobic properties. According to an embodiment of the present invention, the membrane translocation sequence domain may be any one of various polypeptides that have amino acid sequences that are permeable through a cell membrane of a phospholipid bilayer. For example, the membrane translocation sequence domain may be selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 15, or SEQ ID NOs: 98-103, or full length VP22 protein or N-terminal truncated VP22 comprising amino acids 159-301 of the full length protein.
The protein complex for intracellular delivery may include a biologically active molecule.
The term “biologically active molecule” used herein refers to a molecule that is linked to the membrane translocation sequence domain to be delivered into a cell and shows activity in vivo.
According to an embodiment of the present invention, the biologically active molecule may be selected from the group consisting of a nucleic acid molecule (for example, shRNA, siRNA, DNA, or the like), a chemical compound (for example, an anticancer drug, such as Taxol or the like), and a polypeptide.
According to an embodiment of the present invention, the biologically active molecule may be a polymer of amino acids that consist of two or more residuals and examples thereof are a polypeptide and a protein. The polypeptide may be, for example, a protein, such as SV40 large T antigen and telomerase, that is engaged in cell immortalization, an anti-apoptotic protein, such as mutant p53 and Bcl[−]xL, an antibody, an oncogene, such as ras, myc, HPV E6/E7, or adenovirus Ela, a cell cycle control protein, such as cyclin and cyclin-dependent kinase, a green fluorescence protein, or an enzyme, such as beta-galactosidase and chloramphenicol acetyl transferase.
The biologically active molecule is not limited to any particular physiochemical properties. For instance, the biologically active molecule may comprise a hydrophobic or a hydrophilic protein. Also, examples of the biologically active molecule are a hormone, a hormone analogue, an enzyme, an enzyme inhibitor, a signal transduction protein or a part thereof, an antibody or a part thereof, a single-chain antibody, a binding protein or a binding domain thereof, an antigen, an attachment protein, a structure protein, a regulatory protein, a toxoprotein, cytokine, a transcription factor, or a blood coagulation factor.
Also, the biologically active molecule may be p53, p18, a protein complex formed by linking p53 and p18 (p53-p18), p21, p25, p57, p16, p15, NIP71, neuroregulin 1, PTEN tumor suppressor, ARF tumor suppressor, APC, CD95, Folliculin, MEN1, BRCA1, Von Hippel-Lindau tumor suppressor, RKIP, nm23, endostatin, insulin, insulin-like growth factor 1 (IGF-1), growth hormone, erythropoietin, granulocyte-colony stimulating factors (G-CSFs), granulocyte/macrophage-colony stimulating factors (GM-CSFs), interferon alpha, interferon beta, interferon gamma, interleukin-1 alpha and beta, interleukin-3, interleukin-4, interleukin-6, interleukin-2, epidermal growth factors (EGFs), calcitonin, adrenocorticotropic hormone (ACTH), tumor necrosis factor (TNF), atobisban, buserelin, cetrorelix, deslorelin, desmopressin, dynorphin A (1-13), elcatonin, eleidosin, eptifibatide, growth hormone releasing hormone-II (GHRH-II), gonadorelin, goserelin, histrelin, leuprorelin, lypressin, octreotide, oxytocin, pitressin, secretin, sincalide, terlipressin, thymopentin, thymosine α1, triptorelin, bivalirudin, carbetocin, cyclosporine, exedine, lanreotide, luteinizing hormone-releasing hormone (LHRH), nafarelin, parathyroid hormone, pramlintide, enfuvirtide (T-20), thymalfasin, or ziconotide.
The protein complex for intracellular delivery may include an in vivo stabilization protein.
The term “in vivo stabilization protein” used herein refers to a protein that provides stability to the protein complex so that the protein complex stably exists in vivo where the protein complex substantially acts. The in vivo stabilization protein is part of the protein complex, and does not cause immunogenicity in vivo. The in vivo stabilization protein may be any one of various proteins that, when administered to a subject, confer stability to the complex when in the blood of a subject or other physiological environment, as appropriate for the desired end use. In other words, the in vivo stabilization protein can be any protein that, when complexed with, or otherwise attached to, a cargo molecule (e.g., any of the biologically active molecules described herein) fused with a hydrophobic cell penetrating peptide (CPP) or membrane translocation sequence domain (MTS), provides a complex with increased half life under physiological conditions (e.g., in blood or in the bloodstream of a subject) or increased resistance to renal clearance as compared to the cargo molecule fused to the CPP/MTS without the in vivo stabilization protein. According to an embodiment of the present invention, the in vivo stabilization protein may be a serum albumin binding peptide, a Fc region of antibody, or alpha antitrypsin (AAT) (e.g., GenBank Accession ABV21360.1 GI:157086955) or a fragment thereof sufficient to provide in vivo stabilization.
The protein complex for intracellular delivery may further include a nucleus-cytoplasm signal domain.
The term “nucleus-cytoplasm signal domain” used herein refers to a polypeptide sequence included in the protein complex for intracellular delivery which is to transfer the biologically active molecule into or from a nucleus. According to an embodiment of the present invention, the nucleus-cytoplasm signal domain may be a nucleus location sequence (NLS) domain or a nucleus export sequence (NES) domain. That is, NLS may be included in the protein complex for intracellular delivery to transfer the protein complex into a nucleus, and NES may be included in the protein complex for intracellular delivery to retain the biologically active molecule present in the cytoplasm.
An NLS domain is included in proteins that are transferred from cytoplasm to a nucleus, and an NES domain is included in proteins that are transferred from a nucleus to cytoplasm. The NES domain refers to a polypeptide having an amino acid sequence that is permeable through a nuclear membrane, and the amino acid sequence is not particularly limited. Non-limiting examples of the amino acid sequence of the NES domain are KKKRK (SEQ ID NO: 16), PKKKRKV (SEQ ID NO: 17), and KRPAATKKAGQAKKKK (SEQ ID NO: 18). Also, the nucleus-cytoplasm location domain may be located between the biologically active molecule and the in vivo stabilization protein in the protein complex for intracellular delivery. In this structure, the protein complex may have the maximum intracellular delivery efficiency.
In the protein complex, the nucleus-cytoplasm location domain may increase solubility of the protein complex. The closer the nucleus-cytoplasm location domain is located to the C-terminus of the protein complex, the solubility of the protein complex is further increased. When the nucleus-cytoplasm location domain has a basic property, it may be susceptible to a C-terminus peptidase. However, if the in vivo stabilization protein (for example, serum albumin or the like) is present, the in vivo stabilization protein may protect the nucleus-cytoplasm location domain from the C-terminus peptidase, thereby enabling the nucleus-cytoplasm location domain to retain its function.
The protein complex for intracellular delivery may include an in vitro stabilization protein, a membrane translocation sequence domain, a biologically active molecule, and an in vivo stabilization protein in any order. In some embodiments, the protein complex for intracellular delivery may include an in vitro stabilization protein, a membrane translocation sequence domain, a biologically active molecule, and an in vivo stabilization protein in sequence from an N-terminus thereof. The protein complex for intracellular delivery may include an in vitro stabilization protein, a membrane translocation sequence domain, a biologically active molecule, a nucleus-cytoplasm signal domain, and an in vivo stabilization protein in sequence from an N-terminus thereof.
The protein complex can comprise more than one in vitro stabilization protein, membrane translocation sequence domain, biologically active molecule, in vivo stabilization protein, and/or nucleus-cytoplasm signal domain, or the protein complex can comprise only one of each of these elements.
The elements of the protein complex may be part of a single amino acid polymer or protein. Furthermore, the elements of the protein complex may be joined by linker moieties. The linker moieties may comprise or consist of linking amino acid sequences. Any linker may be used, provided that it does not substantially interfere with the activity of the protein complex.
The protein complex may further include other elements needed or desired for the production, recovery, or use of the protein complex. For instance, the protein complex may comprise one or more tags to facilitate detection, isolation, and/or immobilization of the protein (e.g., fluorescent tags or affinity tags, such as a polyhistidine sequence). The protein complex may also include cleavable moieties or sequences to remove any such tags.
Also provided is a nucleic acid encoding a protein complex as described herein. The nucleic acid may comprise (i) a nucleotide sequence encoding an in vitro stabilization protein; (ii) a nucleotide sequence encoding a membrane translocation sequence domain; (iii) a nucleotide sequence encoding a biologically active molecule; and (iv) a nucleotide sequence encoding an in vivo stabilization protein. The nucleic acid may further comprising nucleic acid sequence(s) encoding one or more additional elements described herein with respect to the protein complex (e.g., a nucleus-cytoplasm signal domain).
The nucleic acid may be part of a vector (e.g., an expression vector). Any suitable expression vector may be used.
The nucleic acid and/or vector may further comprise and/or one or more non-coding (e.g., regulatory sequences, tags, restriction sites, etc.) as may be needed or desired for the expression or recovery of the protein complex, or other purpose.
The protein complex can be produced by any suitable method, for instance, by transforming a host cell with a nucleic acid encoding the protein complex. If the host cell is in vivo, the protein complex can be recovered (isolated) from the host cell and, optionally, purified to any degree necessary depending upon the intended end use. If the host cell is in vivo, the protein complex may be produced and have a biological effect in vivo. Alternatively, or in addition, the protein complex may be synthetically produced in whole or in part. An example of a method for producing a protein complex is described in the Examples provided herein.
Also provided herein is a composition comprising the protein complex or nucleic acid encoding the protein complex and a carrier. Any carrier suitable for storing or delivering the protein complex or nucleic acid, as applicable, may be used. The carrier can be any of those conventionally used and is limited only by physio-chemical considerations, such as solubility and lack of reactivity with the nucleic acid or protein complex, and by the intended end use or route of administration. The carriers may include any of a variety of well know vehicles, adjuvants, excipients, and diluents, particularly those which are pharmaceutically acceptable.
Another aspect of the present invention provides a method of intracellular protein delivery, the method including a protein complex-cell contact.
The method may allow a protein complex to come into contact with a cell, wherein the protein complex includes an in vitro stabilization protein; a membrane translocation sequence; a biologically active molecule; and an in vivo stabilization protein. The protein complex for cell delivery is as described above.
According to an embodiment of the present invention, the method may comprise administering the protein complex to a cell by contacting the cell with the protein complex or a nucleic acid encoding the protein complex, optionally in a vector, such that the cell expresses the protein complex. The contacting or other administration may be performed in vitro or in vivo. When administration or contacting is performed in vivo, the method may include administering the protein complex or nucleic acid encoding same to a subject (e.g., animal, particularly mammal, or human).
Furthermore, whether in vivo or in vitro, the cell may be in or part of a tissue, an organ, or other system. Also, for the administering, the protein complex or nucleic acid may be dissolved in an appropriate buffer solution and then the protein complex solution is directly brought into contact with a cell, a tissue, or an organ. Alternatively, the protein complex may be non-orally administered to a subject together with a pharmaceutically acceptable support. If the administering is a non-oral administration, intravenous infusion, subcutaneous infusion, intramuscular infusion, intraperitoneal infusion, endothelium administration, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration, or the like may be used. Because the administered protein complex includes the membrane translocation sequence domain, the biologically active molecule may be delivered to a cell corresponding to a body site where the administration methods are applied.
Hereinafter, one or more embodiments of the present invention will be described in detail. However, these examples are presented herein for illustrative purpose only and the present invention is not limited thereto.
EXAMPLE 1
Preparation of Expression Vector for Protein Complex
This experiment was performed to manufacture expression vectors for protein complexes for intracellular delivery and manufacture the protein complexes, wherein the protein complexes each included a Ub wild-type protein or Ub mutant-type protein, which are hydrophilic polypeptides, a membrane translocation sequence domain (MTS), p53-p18, a nucleus localization signal domain (NLS), and a serum albumin binding peptide (SABP) were linked in a combination as listed in the following table. Hereinafter, the wild-type Ub will be referred to as Ub7KR and a mutant-type Ub formed substituting Lys of the wild-type Ub with Arg and substituting RGG of the wild-type Ub C-terminus with RGA will be referred to as Ub7KR(G76A).
A total of 11 kinds of expression vectors were manufactured by Genotech Research Co., Ltd, and pET-21b(+)(Novagen) was used as a vector for over-expression of the protein complex. The general configuration of the protein complexes are provided in Table 1.











TABLE 1









Protein complex

Amino

















Biologically


DNA
Acid


No.
Ub
MTS
active molecule
NLS
SABP
(SEQ ID)
(SEQ ID)





#12-1
Ub7KR(G76A)
◯
X
◯
◯
20
54


#12-5
Ub7KR(G76A)
◯
P53
◯
◯
21
58


#12-6
Ub7KR(G76A)
X
P53
◯
◯
22
59


#12-7
Ub7KR(G76A)
◯
P18
◯
◯
23
60


#12-8
Ub7KR(G76A)
X
P18
◯
◯
24
61


#44
Ub7KR(G76A)
◯
p53-p18
◯
X
25, 83
82


#52
Ub7KR(G76A)
◯
p53-p18
◯
◯
26, 85
84


#53
Ub7KR
◯
p53-p18
◯
X
87
86


#54
Ub7KR
◯
p53-p18
◯
◯
27, 89
88


#56
Ub7KR(G76A)
X
p53-p18
◯
◯
28, 93
92


#57
Ub7KR
X
p53-p18
◯
X
29, 95
94


#58
Ub7KR
X
p53-p18
◯
◯
97
96


#66
Ub7KR(G76A)
X
p53-p18
◯
X
30, 91
90





“X” indicates moiety not present; “◯” indicates moiety present.






Also, the respective insert DNA fragments may include a nucleotide sequence that is cleavable by Ndel at their 5′ end and a nucleotide sequence that is cleavable by XhoI at their 3′ end, and thus may be inserted into a NdeI-XhoI cleavage sequence of the pET21b(+) vector.
FIG. 1 shows a schematic view illustrating a primary structure of a protein complex for intracellular delivery (#52) according to an embodiment of the present invention.
EXAMPLE 2
Expression and Purification of Protein Complex
To over-express protein complex using each of 11 vectors prepared in Example 1, E. coli BL21 (DE3) transformed with each of vectors was used to express each of protein complexes. In this case, YT medium was used as a culture solution, and when an O.D. value was 0.5 at an absorption wavelength of 600 nm, 0.5 mM IPTG was added thereto and incubation was further performed at 18° C. for 16 hours. Cells obtained by the incubation were lysed by ultrasonic waves in buffer supplemented with 50 mM Tris-HCl pH8.0, 5% glycerol, 5 mM β-mercaptoethanol, 0.2% TRITONTM X-100, and 0.2 M NaCl, and then centrifuged using an centrifuger (10,000×g) to clarify the cell lysate. The supernatant solution was applied to an Ni2+-NTA superflow column (Qiagen) equilibrated in buffer, and then the result column was washed with five column volume (CV) of a washing buffer solution (50 mM Tris-HCl, pH 8.0, 5% glycerol, 5 mM β-mercaptoethanol, 0.2% TRITONTM X-100 and 1 M NaCl), followed by elution of bound protein with a buffer supplemented with 50 mM Tris-HCl, pH 8.0, 5% glycerol, 5 mM β-mercaptoethanol, 0.2% TRITONTM X-100 and 0.2 M NaCl. Fractions including the protein complexes were collected and a salt was removed therefrom using AMICONTM Ultra-15 Centrifugal Filter (Milipore), followed by concentration. The purified protein concentration was measured using bovine serum albumin (BSA) as a reference material.
FIG. 2 shows SDS-PAGE results of the purified protein complexes expressed from the vectors prepared according to Example 1. In FIG. 2A, a protein complex #12-1 is a protein complex having a sequence of Ub7KR(G76A)-MTS-NLS-SABP, a protein complex #12-5 is a protein complex having a sequence of Ub7KR(G76A)-MTS-p53-NLS-SABP, a protein complex #12-6 is a protein complex having a sequence of Ub7KR(G76A)-p53-NLS-SABP, a protein complex #12-7 is a protein complex having a sequence of Ub7KR(G76A)-MTS-p18-NLS-SABP, and a protein complex #12-8 is a protein complex having a sequence of Ub7KR(G76A)-p18-NLS-SABP. In FIG. 2B, a protein complex #44 is a protein complex having a sequence of Ub7KR(G76A)-MTS-p53-p18-NLS, a protein complex #52 is a protein complex having a sequence of Ub7KR(G76A)-MTS-p53-p18-NLS-SABP, a protein complex #54 is a protein complex having a sequence of Ub7KR-MTS-p53-p18-NLS-SABP, a protein complex #66 is a protein complex having a sequence of Ub7KR(G76A)-p53-p18-NLS, a protein complex #56 is a protein complex having a sequence of Ub7KR(G76A)-p53-p18-NLS-SABP, and a protein complex #57 is a protein complex having a sequence of Ub7KR-p53-p18-NLS. M means a size marker.
EXAMPLE 3
Confirmation of in vivo Stability of Protein Complex including in vivo Stabilization Protein
Whether the protein complexes prepared according to Example 2 stably exist in vivo was confirmed by identifying a binding between the protein complexes and serum albumin as a protein that exists in vivo.
First, from among the protein complexes purified according to Example 2, 500 ug of each of the #56 protein complex that includes SABP and the protein complex #66 that does not include SABP and 1500 ug of BSA were left in 1000 ul of a PBS buffer solution (1×PBS, 0.2% TWEENTM 20, 100 mM arginine, 0.2% reduced glutathione) at 18° C. for 120 minutes to provide a sufficient binding time, and then, the reaction product was loaded to an HILOADTM SUPERDEXTM S-75 16/60 column (GE healthcare), followed by elution. In this regard, elution conditions were a flow rate of 1 ml/min and a PBS buffer solution (1×PBS, 0.2% TWEENTM 20, 100 mM arginine, 0.2% reduced glutathione). The elution was performed using 2 ml of each fraction for 120 minutes. Thereafter, the eluent was applied to an Ni2+-NTA Superflow column (Qiagen) that had been equilibrated with a 50 mM Tris buffer solution (50 mM Tris-HCl, pH 8.0, 5% glycerol, 5 mM β-mercaptoethanol, 0.2% TRITONTM X-100 and 1 M NaCl), followed by elution using the same method as in Example 2 to complete the purification of the protein complexes.
FIG. 3 shows SDS-PAGE results of the eluted protein complexes as described above. Referring to FIG. 3, it was confirmed that only the protein complex #56 including a serum albumin binding peptide binds to BSA. If the size of a protein complex is small, renal clearance may occur in vivo or the protein complex may be degraded by a protease. However, serum albumin is stable in blood and is not subjected to renal clearance. Thus, a protein complex that includes a serum albumin binding peptide is stable in vivo.
EXAMPLE 4
Confirmation on Anticancer Effect of Protein Complex by using Various Cancer Cell Lines
The intracellular delivery and cancer cell therapeutic efficacy of the protein complexes prepared according to Example 2 were confirmed as follows: an anticancer effect of p53-p18 protein was confirmed by administering the protein complexes to colorectal cancer cell lines (HCT116, SW48, LoVo and LSI174T, Korean Cell Line Bank) and a skin carcinoma cell line (A431, Korean Cell Line Bank).
The respective cells were added to 10% FBS-including RPMI medium (Gibco BL) such that each well included 1×104 cells, and then treated with 0, 0.032, 0.16, 0.8, 4, 20, or 100 ug/ml of the protein complexes #56, #52 and #44 and incubated in a CO2 incubator for 96 hours at 37° C. under a humidity of 85% and CO2 of 5%. As a comparative example, ERBITUXTM, an anti-cancer therapeutic antibody, was used instead of the protein complexes at the same concentrations and the treated cells were incubated under the same conditions. Also, as a control, cell lines that were not treated with the protein complexes or ERBITUXTM were used.
As a result, as shown in FIGS. 4A to 4E, it was confirmed that a protein complex including both the NLS and the in vivo SABP substantially suppressed cell growth of all the cell lines at a concentration of 20 ug/ml, and at 100 ug/ml, all of the cell lines were completely suppressed.
As described above, a protein complex for cell delivery and a method of delivering a biologically active molecule into a cell by using the protein complex, according to the one or more of the above embodiments of the present invention, enable an effective delivery of a biologically active molecule.
EXAMPLE 5
Illustrative Further Embodiments
Additional examples of protein complex constructs are provided in Tables 2 and 3.











TABLE 2









Protein complex

Amino

















Biologically


DNA
Acid


No.
Ub
MTS
active molecule
NLS
SABP
(SEQ ID)
(SEQ ID)





#12-2
Ub7KR(G76A)
X
◯
◯
◯
31
55


#12-3
Ub7KR(G76A)
◯
p53-p18
◯
◯
32
56


#12-4
Ub7KR(G76A)
X
p53-p18
◯
◯
33
57


#12-9
Ub7KR(G76A)
◯
pDIQ-p18
◯
◯
34
62


#12-10
Ub7KR(G76A)
◯
pDIQ
◯
◯
35
63


#12-11
Ub7KR(G76A)
◯
pMI-p18
◯
◯
36
64


#12-12
Ub7KR(G76A)
◯
pMI
◯
◯
37
65


#12-13
Ub7KR(G76A)
◯
p53m-p18
◯
◯
38
66


#12-14
Ub7KR(G76A)
◯
p53m
◯
◯
39
67




















TABLE 3









Protein complex

Amino
















Biologically



DNA
Acid


No.
Ub
active molecule
AAT
MTS
NLS
(SEQ ID)
(SEQ ID)





#12-21
Ub7KR(G76A)
X
◯
◯
◯
40
68


#12-22
Ub7KR(G76A)
X
◯
X
◯
41
69


#12-23
Ub7KR(G76A)
p53
◯
◯
◯
42
70


#12-24
Ub7KR(G76A)
p53
◯
X
◯
43
71


#12-25
Ub7KR(G76A)
p18
◯
◯
◯
44
72


#12-26
Ub7KR(G76A)
p18
◯
X
◯
45
73


#12-27
Ub7KR(G76A)
p53-p18
◯
◯
◯
46
74


#12-28
Ub7KR(G76A)
p53-p18
◯
X
◯
47
75


#12-29
Ub7KR(G76A)
pDIQ-p18
◯
◯
◯
48
76


#12-30
Ub7KR(G76A)
pDIQ
◯
◯
◯
49
77


#12-31
Ub7KR(G76A)
pMI-p18
◯
◯
◯
50
78


#12-32
Ub7KR(G76A)
pMI
◯
◯
◯
51
79


#12-33
Ub7KR(G76A)
p53m-p18
◯
◯
◯
52
80


#12-34
Ub7KR(G76A)
p53m
◯
◯
◯
53
81





“◯” indicates the presence of a moiety, and “X” indicates that no moiety was used.






The Examples of protein complexes described herein may contain elements other than those listed in the table, such as polyhistidine tags, TEV cleavage sites, and intervening linking sequences between the various elements.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

## Claims

1. A protein comprising an in vitro stabilization protein, a membrane translocation sequence domain, a biologically active molecule, and an in vivo stabilization protein,
wherein the protein comprises the amino acid sequence of SEQ ID NO: 56, 58, 62, 63, 64, 65, 66, 67, 70, 72, 74, 76-82, 84, or 88.

2. A composition comprising the protein of claim 1 and a pharmaceutically acceptable carrier.

3. A method of delivering a biologically active molecule to a cell, the method comprising administering the protein of claim 1 to the cell.

4. The method of claim 3, wherein the protein is administered to the cell by contacting the cell with the protein.

5. The method of claim 3, wherein the cell is in vitro.

6. The method of claim 4, wherein the cell is in vivo in a subject, and the protein is administered to the cell by administering the protein to the subject.

7. A method of producing the protein of claim 1 comprising administering a nucleic acid encoding the protein to a cell, wherein the cell expresses the protein.