POLYPEPTIDES WITH PHASE TRANSITION, TRIBLOCK POLYPEPTIDES OF THE POLYPEPTIDE-CALMODULIN-POLYPEPTIDE WITH MULTI-STIMULI RESPONSIVENESS, HYDROGEL OF THE TRIBLOCK POLYPEPTIDES, AND ITS USES

Abstract

Disclosed is a polypeptide having a phase transition behavior, wherein the polypeptide consists of a Val-Pro-Gly-Xaa-Gly pentapeptide repeat or a Val-Pro-Ala-Xaa-Gly) pentapeptide repeat, and the polypeptide includes a [Val-Pro-Gly-Xaa-Gly]n or a [Val-Pro-Ala-Xaa-Gly]n (SEQ ID NO:2) pentapeptide repeat. In addition, the present invention provides a multi-stimuli polypeptide composed of polypeptide-calmodulin-polypeptide having a phase transition behavior and a hydrogel prepared using the same. A dynamic protein hydrogel according to the present invention may be used as a drug carrier, as a scaffold for tissue engineering or as a kit for tissue or organ regeneration.

Claims

1. A triblock polypeptide having multi-stimuli responsiveness, wherein the triblock polypeptide is consisted of a calmodulin block; and polypeptide blocks having a phase transition behavior linked to both ends of the calmodulin block, and is represented by Formula 4 below:
[hydrophobic EBP]m-[hydrophilic EBP]x-CalM-[hydrophilic EBP]x-[hydrophobic EBP]m, wherein  Formula 4 the [hydrophobic EBP]m refers to a polypeptide block having a phase transition behavior; the [hydrophobic EBP]m-[hydrophilic EBP]x refers to a polypeptide block having a phase transition behavior; the hydrophobic EBP is consisted of a hydrophobic polypeptide is a pentapeptide of Val-Pro-Ala-X.sub.aa-Gly (SEQ ID NO: 2); the hydrophilic EBP is consisted of a hydrophilic polypeptide is a pentapeptide of Val-Pro-Ala-X.sub.aa-Gly (SEQ ID NO: 2); wherein X.sub.aa is an amino acid other than proline; m or x, each respectively, is an integer of 2 or more, and is the number of repeats of the hydrophobic polypeptide and hydrophilic polypeptide having a phase transition behavior; and the CalM is a calmodulin block, wherein an amino acid sequence encoding the calmodulin is SEQ ID NO: 38.

2. The triblock polypeptide according to claim 1, wherein the polypeptide block is further linked a cysteine block with both end of thereof.

3. The triblock polypeptide according to claim 1, wherein the stimuli are temperature and/or a ligand, and the polypeptide block having a phase transition behavior exhibits thermal responsiveness and the calmodulin block exhibits ligand responsiveness.

4. The triblock polypeptide according to claim 2, wherein the cysteine block is consisted of an amino acid sequence in which (Gly-Ala-Cys) repeats one or more times.

5. The triblock polypeptide according to claim 2, wherein the cysteine block is consisted of an amino acid sequence, Gly-Ala-Cys-Gly-Ala-Cys-Gly-Ala-Cys-Gly-Ala-Cys [SEQ ID NO. 39].

6. The triblock polypeptide according to claim 1, wherein the [hydrophobic EBP]m, m is multiple of six, and the [hydrophobic EBP] is consisted of an amino acid sequence below: Val-Pro-Ala-X.sub.aa-Gly [SEQ ID NO. 40], wherein each X.sub.aa of the pentapeptide repeats is consisted of G (Gly), A (Ala), and F (Phe) in a ratio of 1:3:2.

7. The triblock polypeptide according to claim 6, wherein m is 6, 12, 18, 24, 30 or 36.

8. A hydrogel, wherein the hydrogel is prepared by a process comprising: applying a heat stimulus to the triblock polypeptide according to claim 1; and cross-linking hydrophobic EBPs of the triblock polypeptide by the heat stimulus and forming a hydrogel.

9. The hydrogel according to claim 8, wherein the hydrogel is prepared by a process comprising: after the hydrophobic EBPs are cross-linked, inducing a structural change of the calmodulin by specifically binding the calmodulin block, which constitutes the triblock polypeptide, to a ligand; and forming a dynamic hydrogel by a three-dimensional structural change of the hydrogel induced by the structural change of the calmodulin block due to the ligand responsiveness.

10. The hydrogel according to claim 8, wherein, in the cross-linking, the cross-linking is physical cross-linking that occurs at or above a transition temperature of the hydrophobic EBP.

11. The hydrogel according to claim 8, wherein the ligand is calcium and/or a drug.

12. A hydrogel, wherein the hydrogel is prepared by a process comprising: applying a heat stimulus to the triblock polypeptide according to claim 2; cross-linking hydrophobic EBPs constituting the triblock polypeptide by the heat stimulus and forming a hydrogel; and chemically cross-linking cysteine blocks constituting the triblock polypeptide.

13. A composition for drug delivery comprising the hydrogel according to claim 8.

14. A scaffold for tissue engineering comprising the hydrogel according to claim 8.

15. A kit for tissue or organ regeneration comprising the hydrogel according to claim 8.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0141] The above and other objectives, features and advantages of the present invention will become more apparent to those of ordinary skill in the art by describing exemplary embodiments thereof in detail with reference to the accompanying drawings, in which:

[0142] FIG. 1 is a schematic diagram showing construction of plasmids encoding EBP gene libraries with different DNA sizes and block polypeptides thereof. A modified vector was designed to include restriction sites recognized by three different restriction enzymes (REs), including XbaI (RE1), AcuI (RE2), and BseRI (RE3). For example, a gene construct encoding EBPP[G.sub.1A.sub.3F.sub.2].sub.12 was prepared by a ligation reaction, in which a plasmid backbone and an insert derived from a plasmid-borne gene vector harboring a gene encoding EBPP[G.sub.1A.sub.3F.sub.2].sub.6 were used: The plasmid-borne gene vector harboring a gene encoding EBPP[G.sub.1A.sub.3F.sub.2].sub.6 was double-digested by XbaI and AcuI to obtain an insert, i.e., a gene fragment encoding EBPP[G.sub.1A.sub.3F.sub.2].sub.6. On the other hand, the plasmid-borne gene vector for EBPP[G.sub.1A.sub.3F.sub.2].sub.6 was double-digested by XbaI and BseRI to obtain a plasmid backbone and then the plasmid backbone was dephosphorylated by treatment with alkaline phosphatase;

[0143] FIG. 2 shows agarose gel electrophoresis images of EBP gene libraries according to the present invention. (A) EBPE[A.sub.1G.sub.4I.sub.1], (B) EBPP[A.sub.1G.sub.4I.sub.1], (C) EBPE[K.sub.1G.sub.4I.sub.1], (D) EBPP[K.sub.1G.sub.4I.sub.1], (E) EBPE[D.sub.1G.sub.4I.sub.1], (F) EBPP[D.sub.1G.sub.4I.sub.1], (G) EBPE[E.sub.1G.sub.4I.sub.1], (H) EBPP[E.sub.1G.sub.4I.sub.1], (I) EBPP[G.sub.1A.sub.3F.sub.2], (J) EBPP[K.sub.1A.sub.3F.sub.2], (K) EBPP[D.sub.1A.sub.3F.sub.2], and (L) EBPP[H.sub.3A.sub.2I.sub.1]. The number of EBP repeat units is indicated below each DNA band. The bilateral lanes on all agarose gels represent different DNA size markers (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 1.0, 1.5, 2.0, and 3.0 kbp, from bottom to top);

[0144] FIG. 3 shows copper-stained SDS-PAGE gel (4 to 20% gradient) images of EBPs according to the present invention. (A) EBPE[A.sub.1G.sub.4I.sub.1], (B) EBPP[A.sub.1G.sub.4I.sub.1], (C) EBPE[K.sub.1G.sub.4I.sub.1], (D) EBPP[K.sub.1G.sub.4I.sub.1], (E) EBPE[D.sub.1G.sub.4I.sub.1] and (F) EBPP[D.sub.1G.sub.4I.sub.1]. Two-sided lanes on the SDS-PAGE gels contain standard protein size markers (7, 15, 24, 35, 40, 50, 65, 90, 110, and 150 kDa, from bottom to top);

[0145] FIGS. 4A to 4F show the thermal profiles of EBPs according to the present invention, with FIG. 4A showing EBPE[A.sub.1G.sub.4I.sub.1].sub.n, FIG. 4B showing EBPP[A.sub.1G.sub.4I.sub.1].sub.n, FIG. 4C showing EBPE[K.sub.1G.sub.4I.sub.1].sub.n, FIG. 4D showing EBPP[K.sub.1G.sub.4I.sub.1].sub.n, FIG. 4E showing EBPP[D.sub.1G.sub.4I.sub.1].sub.n and FIG. 4F showing EBPP[G.sub.1A.sub.3F.sub.2].sub.n. To obtain thermal profiles, 25 μM EBP solutions were prepared in PBS buffer or PBS buffer supplemented with 1 to 3 M NaCl, and optical absorbance of the EBP solutions was measured at 350 nm while heating the solutions at a heating rate of 1° C./min;

[0146] FIG. 5 shows schematic diagrams of dynamic protein hydrogels composed of genetically engineered ABA-type EBP-CalM-EBP triblock polypeptides. Two different types of EBP blocks, such as a hydrophobic EBP having a low T.sub.t and a hydrophilic EBP having a high T.sub.t, were introduced. (A) hydrophobic EBP-CalM-hydrophobic EBP, (B) hydrophobic EBP-hydrophilic EBP-CalM-hydrophilic EBP-hydrophobic EBP, (C) Cys block-hydrophobic EBP-CalM-hydrophobic EBP-Cys block, and (D) Cys block-hydrophobic EBP-hydrophilic EBP-CalM-hydrophilic EBP-hydrophobic EBP-Cys block;

[0147] FIG. 6 shows the DNA agarose gel electrophoresis (0.8%) images (left) and the copper-stained SDS-PAGE (4-20% gradient) gel images (right) of a series of constructed Cys-EBPP[G.sub.1A.sub.3F.sub.2].sub.6n-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.6n-Cys (n: integer). Each lane of the gel on either side represents (1) Cys block-EBPP[G.sub.1A.sub.3F.sub.2].sub.6-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.6-Cys block, (2) Cys block-EBPP[G.sub.1A.sub.3F.sub.2].sub.12-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.12-Cys block, (3) Cys block-EBPP[G.sub.1A.sub.3F.sub.2].sub.18-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.18-Cys block, (4) Cys block-EBPP[G.sub.1A.sub.3F.sub.2].sub.24-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.24-Cys block, (5) Cys block-EBPP[G.sub.1A.sub.3F.sub.2].sub.30-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.30-Cys block, and (6) Cys block-EBPP[G.sub.1A.sub.3F.sub.2].sub.36-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.36-Cys block;

[0148] FIG. 7 shows the thermal profiles of Cys block-EBPP[G.sub.1A.sub.3F.sub.2].sub.6n-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.6n-Cys block in 10 mM HEPES buffer at pH 7.4 (A) and in 10 mM HEPES buffer supplemented with 10 mM CaCl.sub.2 (B), when characterized by turbidity profiling as a function of temperature at a heating rate of 1° C./min. Each number in (A) and (B) represents (1) Cys block-EBPP[G.sub.1A.sub.3F.sub.2].sub.6-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.6-Cys block, (2) Cys block-EBPP[G.sub.1A.sub.3F.sub.2].sub.12-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.12-Cys block, (3) Cys block-EBPP[G.sub.1A.sub.3F.sub.2].sub.18-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.18-Cys block, (4) Cys block-EBPP[G.sub.1A.sub.3F.sub.2].sub.24-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.24-Cys block, (5) Cys block-EBPP[G.sub.1A.sub.3F.sub.2].sub.30-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.30-Cys block, and (6) Cys block-EBPP[G.sub.1A.sub.3F.sub.2].sub.36-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.36-Cys block;

[0149] FIG. 8 shows the images of the reversible sol-gel transition behaviors of Cys block-EBPP[G.sub.1A.sub.3F.sub.2].sub.12-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.12-Cys block in 10 mM HEPES buffer at pH 7.4, as a function of temperature, when sequentially incubated at 4° C. (A), 37° C. (B and C), and 4° C. (D);

[0150] FIG. 9 shows temperature-dependent rheological behaviors of Cys block-EBPP[G.sub.1A.sub.3F.sub.2].sub.12-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.12-Cys block in 10 mM HEPES buffer at pH 7.4 as a function of polypeptide concentration (1 rad/s, 2% shear strain): (A) 16.6 wt. %, (B) 23.1 wt. %, (C) 27.0 wt. %, and (D) 28.6 wt. %. Storage modulus (G′), loss modulus (G″), and loss angle (δ) obtained by measuring each oscillatory shear were plotted as a function of temperature at a heating rate of 1° C./min; and

[0151] FIG. 10 shows oscillatory rheological profiles of 27.0 wt. % Cys block-EBPP[G.sub.1A.sub.3F.sub.2].sub.12-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.12-Cys block in 10 mM HEPES buffer (A and C) or in 10 mM HEPES buffer containing 168 mM CaCl.sub.2 (B and D), wherein A and B are profiles as a function of temperature at 1 rad/s and 2% shear strain, and C and D are profiles as a function of frequency at 10 or 40° C. and 2% shear strain.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Materials

[0152] A pET-21a vector and BL21 (DE3) E. coli cells were obtained from Novagen Inc. (Madison, Wis., U.S.). Top10 competent cells were obtained from Invitrogen (Carlsbad, Calif., U.S.). Oligonucleotides were chemically synthesized in Cosmo Gene Tech (Seoul, South Korea). FastAP, a thermosensitive alkaline phosphatase, and restriction endonucleases including BamHI and XbaI were purchased from Fermentas (Ontario, Canada). Other restriction endonucleases including BseRI, AcuI and other restriction enzymes were obtained from New England Biolabs (Ipswich, Mass., U.S.). T4 DNA ligase was obtained from Elpis Bio-tech (Taejeon, South Korea). All kits for DNA mini-preparation, gel extraction, and PCR purification were obtained from Geneall Biotechnology (Seoul, South Korea). Dyne Agarose High was obtained from DYNE BIO (Seongnam, South Korea). All Top10 cells were grown in TB DRY media (MO BIO Laboratories, Carlsbad, Calif., U.S.) and super optimal broth with catabolite repression (SOC) media (Formedium, UK) was supplemented with 20 mM glucose. All BL21 (DE3) cells were grown in Circlegrow media obtained from MP Biomedicals (Solon, Ohio, U.S.). Ready Gel (Tris-HCl, 2-20%), a precast gel, was obtained from Bio-Rad (Hercules, Calif., U.S.). Phosphate buffered saline (PBS, pH 7.4), ampicillin, polyethyleneimine (PEI), calcium chloride, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N, N, N′, N′-tetraacetic acid (EGTA), phenothiazine (PTZ), chlorpromazine (CPZ), and trifluoperazine (TFP) were obtained from Sigma-Aldrich (St Louis, Mo.).

Example 2: Notation for Different EBP Blocks and Block Polypeptides Thereof

[0153] Different EBPs having a pentapeptide repeat unit of Val-Pro-(Gly or Ala)-X.sub.aa-Gly[VP (G or A)XG] (SEQ ID NO: 43) are named as follows. X.sub.aa may be any amino acid except Pro. First, pentapeptide repeats of Val-Pro-Ala-X.sub.aa-Gly (VPAXG (SEQ ID NO: 2)) with plasticity are defined as an elastin-based polypeptide with plasticity (EBPP). On the other hand, pentapeptide repeats of Val-Pro-Gly-X.sub.aa-Gly (VPGXG (SEQ ID NO: 1)) are called an elastin-based polypeptide with elasticity (EBPE). Second, in [X.sub.iY.sub.jZ.sub.k].sub.n, the capital letters in the parentheses represent the single letter amino acid codes of guest residues, i.e., amino acids at the fourth position (X.sub.aa or X) of an EBP pentapeptide, and subscripts corresponding to the capital letters indicate the ratio of the guest residues in an EBP monomer gene as a repeat unit. The subscript number n of [X.sub.iY.sub.jZ.sub.k].sub.n represents the total number of repeats of an EBP. For example, EBPP[G.sub.1A.sub.3F.sub.2].sub.12 is an EBPP block including 12 repeats of the Val-Pro-Gly-X.sub.aa-Gly (SEQ ID NO: 1) pentapeptide unit, in which a ratio of Gly, Ala, and Phe at the fourth guest residue position (X.sub.aa) is 1:3:2. Finally, the EBP-CalM-EBP triblock polypeptides are named by the composition of each block in square brackets with a hyphen between blocks such as EBPP[G.sub.1A.sub.3F.sub.2].sub.12-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.12.

Example 3: Preparation of Modified pET-21a Vector for Cloning Seamless Gene

[0154] Four micrograms of a pET-21a vector was digested and dephosphorylated with 50 U of XbaI, 50 U of BamHI and 10 U of a thermosensitive alkaline phosphatase in FastDigest buffer for 20 minutes at 37° C. The restricted plasmid DNA was purified using a PCR purification kit, and then was eluted in 40 μl of distilled and deionized water. Two oligonucleotides with XbaI and BamHI compatible sticky ends were designed, i.e., SEQ ID NO. 40 (5′-ctagaaataattttgtttaactttaagaaggaggagtacatatgggctactgataatgatcttcag-3′) and SEQ ID NO. 41 (5′-gatcctgaagatcattatcagtagcccatatgtactcctccttcttaaagttaaacaaaattattt-3′). To anneal the two types of oligonucleotides, each oligonucleotide was prepared at a concentration of 2 μM in 50 μl of T4 DNA ligase buffer, heat treated at 95° C. for 2 minutes and then slowly cooled to room temperature over 3 hours. To ligate the annealed dsDNA, i.e., a DNA insert, into multiple cloning sites within the linearized pET-21a vector, 20 pmol of the annealed dsDNA and 0.1 pmol of the linearized pET-21a vector were incubated in T4 DNA ligase buffer containing 1 U of T4 DNA ligase for 30 minutes at 37° C. The modified pET-21a (mpET-21a) vector for cloning and expressing a seamless gene was transformed into Top10 competent cells, followed by plating the Top10 competent cells on a super optimal broth with catabolite repression (SOC) plate supplemented with 50 μg/ml of ampicillin. The DNA sequence of the mpET-21a vector was then verified by fluorescent dye terminator DNA sequencing (Applied Biosystems Automatic DNA Sequencer ABI 3730).

Example 4: Synthesis of EBP Monomer Gene and Oligomerization Thereof

[0155] The EBP sequences having a pentapeptide repeat unit, Val-Pro-(Gly or Ala)-X.sub.aa-Gly (SEQ ID NO: 43), in which the fourth residues were varied in different molar ratios, were designed at the DNA level to optimize T.sub.t below a physiological temperature. The DNA and amino acid sequences of EBPs with various pentapeptide repeat units for 17 EBP libraries are shown in Tables 1 and 2, respectively.

TABLE-US-00002 TABLE 1 Gene sequences corresponding to EBP libraries. Both EBPs with plasticity (EBPPs) having a pentapeptide repeat of Val-Pro-Ala-Xaa-Gly (SEQ ID NO: 2), and EBPs with elasticity (EBPEs) having a pentapeptide repeat of Val-Pro-Gly-Xaa-Gly (SEQ ID NO: 1) were all cloned to have the same guest residue composition and ratio. EBP Gene Sequence SEQ ID NO. EBPE[A.sub.1G.sub.4I.sub.1] GTC CCA GGT GGA GGT GTA CCC GGC GCG GGT GTC CCA GGT GGA GGT  3 GTA CCT GGG GGT GGG GTC CCT GGT ATT GGC GTA CCT GGA GGC GGC EBPP[A.sub.1G.sub.4I.sub.1] GTT CCA GCT GGC GGT GTA CCT GCT GCT GCT GTT CCG GCC GGT GGT  4 GTT CCG GCG GGC GGC GTG CCT GCA ATA GGA GTT CCC GCT GGT GGC EBPE[K.sub.1G.sub.4I.sub.1] GTT CCG GGT GGT GGT GTT CCG GGT AAA GGT GTT CCG GGT GGT GGT  5 GTT CCG GGT GGT GGT GGT GTT CCG GGT ATC GGT GTT CCG GGT GGC EBPP[K.sub.1G.sub.4I.sub.1] GTT CCG GCG GGT GGT GTT CCG GCG AAA GGT GTT CCG GCG GGT GGT  6 GTT CCG GCG GGT GGT GTT CCG GCG ATC GGT GTT CCG GCG GGT GGC EBPE[D.sub.1G.sub.4I.sub.1] GTT CCG GGT GGT GGT GTT CCG GGT GAT GGT GTT CCG GGT GGT GGT  7 GTT CCG GGT GGT GGT GGT GTT CCG GGT ATC GGT GTT CCG GGT GGC EBPP[D.sub.1G.sub.4I.sub.1] GTT CCG GCG GGT GGT GTT CCG GCG GAT GGT GTT CCG GCG GGT GGT  8 GTT CCG GCG GGT GGT GTT CCG GCG ATC GGT GTT CCG GCG GGT GGC EBPE[E.sub.1G.sub.4I.sub.1] GTT CCG GGT GGT GGT GTT CCG GGT GAA GGT GTT CCG GGT GGT GGT  9 GTT CCG GGT GGT GGT GGT GTT CCG GGT ATC GGT GTT CCG GGT GGC EBPP[E.sub.1G.sub.4I.sub.1] GTT CCG GCG GGT GGT GTT CCG GCG GAA GGT GTT CCG GCG GGT GGT 10 GTT CCG GCG GGT GGT GTT CCG GCG ATC GGT GTT CCG GCG GGT GGC EBPE[G.sub.1A.sub.3F.sub.2] GTC CCG GGT GCG GGC GTG CCG GGA TTT GGA GTT CCG GGT GCG GGT 11 GTT CCA GGC GGT GGT GTT CCG GGC GCG GGC GTG CCG GGC TTT GGC EBPP[G.sub.1A.sub.3F.sub.2] GTG CCG GCG GCG GGC GTT CCA GCC TTT GGT GTG CCA GCG GCG GGA 12 GTT CCG GCC GGT GGC GTG CCG GCA GCG GGC GTG CCG GCT TTT GGC EBPP[K.sub.1A.sub.3F.sub.2] GTG CCG GCG GCG GGC GTT CCA GCC TTT GGT GTG CCA GCG GCG GGA 13 GTT CCG GCC AAA GGC GTG CCG GCA GCG GGC GTG CCG GCT TT GGC EBPP[D.sub.1A.sub.3F.sub.2] GTG CCG GCG GCG GGC GTT CCA GCC TTT GGT GTG CCA GCG GCG GGA 14 GTT CCG GCC GAT GGC GTG CCG GCA GCG GGC GTG CCG GCT TTT GGC EBPP[K.sub.3F.sub.3] GTT CCA GCG TTT GGC GTG CCA GCG AAA GGT GTT CCG GCG TTT GGG 15 GTT CCC GCG AAA GGT GTG CCG GCC TTT GGT GTG CCG GCC AAA GGC EBPP[D.sub.3F.sub.3] GTT CCA GCG TTT GGC GTG CCA GCG GAT GGT GTT CCG GCG TTT GGG 16 GTT CCC GCG GAT GGT GTG CCG GCC TTT GGT GTG CCG GCC GAT GGC EBPP[H.sub.3A.sub.3I.sub.1] GTG CCG GCG CAT GGA GTT CCT GCC GCC GGT GTT CCT GCG CAT GGT 17 GTA CCG GCA ATT GGC GTT CCG GCA CAT GGT GTG CCG GCC GCC GGC EBPP[H.sub.5G.sub.1] GTT CCG GCC GGA GGT GTA CCG GCG CAT GGT GTT CCG GCA CAT GGT 18 GTG CCG GCT CAC GGT GTG CCT GCG CAT GGC GTT CCT GCG CAT GGC EBPE[G.sub.1C.sub.3F.sub.2] GTG CCG GCG TGC GGC GTT CCA GCC TTT GGT GTG CCA GCG TGC GGA 19 GTT CCG GCC GGT GGC GTG CCG GCA TGC GGC GTG CCG GCT TTT GGC

TABLE-US-00003 TABLE 2 Amino acid sequences corresponding to EBP libraries. SEQ ID EBP Amino acid Sequence NO. EBPE[A.sub.1G.sub.4I.sub.1] VPGGG VPGAG VPGGG VPGGG VPGIG VPGGG 20 EBPP[A.sub.1G.sub.4I.sub.1] VPAGG VPAAG VPAGG VPAGG VPAIG VPAGG 21 EBPE[K.sub.1G.sub.4I.sub.1] VPGGG VPGKG VPGGG VPGGG VPGIG VPGGG 22 EBPP[K.sub.1G.sub.4I.sub.1] VPAGG VPAKG VPAGG VPAGG VPAIG VPAGG 23 EBPE[D.sub.1G.sub.4I.sub.1] VPGGG VPGDG VPGGG VPGGG VPGIG VPGGG 24 EBPP[D.sub.1G.sub.4I.sub.1] VPAGG VPADG VPAGG VPAGG VPAIG VPAGG 25 EBPE[E.sub.1G.sub.4I.sub.1] VPGGG VPGEG VPGGG VPGGG VPGIG VPGGG 26 EBPP[E.sub.1G.sub.4I.sub.1] VPAGG VPAEG VPAGG VPAGG VPAIG VPAGG 27 EBPE[G.sub.1A.sub.3F.sub.2] VPGAG VPGFG VPGAG VPGGG VPGAG VPGFG 28 EBPP[G.sub.1A.sub.3F.sub.2] VPAAG VPAFG VPAAG VPAGG VPAAG VPAFG 29 EBPP[K.sub.1A.sub.3F.sub.2] VPAAG VPAFG VPAAG VPAGG VPAAG VPAFG 30 EBPP[D.sub.1A.sub.3F.sub.2] VPAAG VPAFG VPAAG VPAGG VPAAG VPAFG 31 EBPP[K.sub.3F.sub.3] VPAFG VPAKG VPAFG VPAKG VPAFG VPAKG 32 EBPP[D.sub.3F.sub.3] VPAFG VPADG VPAFG VPADG VPAFG VPADG 33 EBPP[H.sub.3A.sub.3I.sub.1] VPAHG VPAAG VPAHG VPAIG VPAHG VPAAG 34 EBPP[H.sub.5G.sub.1] VPAGG VPAHG VPAHG VPAHG VPAHG VPAHG 35 EBPP[G.sub.1C.sub.3F.sub.2] VPACG VPAFG VPACG VPAGG VPACG VPAFG 36

[0156] In Table 1, SEQ ID NO. 3 to 10 may be classified as gene sequences for hydrophilic EBP blocks, and SEQ ID NO. 11 to 19 may be classified as gene sequences for hydrophobic EBP blocks, in which Phe and His are incorporated. That is, as described above, when the LCST of an EBP is lower than the body temperature, the EBP exhibits hydrophobicity, and when the LCST of an EBP is higher than the body temperature, the EBP exhibits hydrophilicity. Due to this property of EBPs, the hydrophilic and hydrophobic properties of EBPs may be relatively defined when EBPs are applied to biotechnology.

[0157] Different EBPs having a pentapeptide repeat unit, Val-Pro-(Gly or Ala)-X.sub.aa-Gly (SEQ ID NO: 43) [where X.sub.aa may be any amino acid except Pro], which are capable of responding to unique stimuli including temperature and pH, were designed at the DNA level. EBPs with plasticity (EBPPs) having a pentapeptide repeat unit of Val-Pro-Ala-X.sub.aa-Gly (SEQ ID NO: 2) and EBPs with elasticity (EBPEs) having a pentapeptide repeat unit of Val-Pro-Gly-X.sub.aa-Gly (SEQ ID NO: 1) were all cloned to have the same guest residue composition and ratio. Tables 1 and 2 represent the gene and amino acid sequences of different EBPs having respective pentapeptide repeat units. For example, EBPE[G.sub.1A.sub.3F.sub.2].sub.12 and EBPP[G.sub.1A.sub.3F.sub.2].sub.12 not only show almost the same molar mass, but also the fourth residues of these EBP pentapeptide repeat units represent the same combination. In addition, these EBP blocks have different mechanical properties because the third amino acid residue (Ala or Gly) of the pentapeptide repeat units are different. Positively and negatively charged EBPs were constructed by introducing charged amino acids such as Lys, Asp, Glu, His and the like as guest residues.

[0158] To anneal each pair of oligonucleotides encoding various EBPs, each oligonucleotide was prepared at a concentration of 2 μM in 50 μl of T4 DNA ligase buffer, heat treated at 95° C. for 2 minutes and then slowly cooled to room temperature over 3 hours. Four micrograms of a modified pET-21a (mpET-21a) vector was digested and dephosphorylated with 15 U of BseRI and 10 U of FastAP thermosensitive alkaline phosphatase for 30 minutes at 37° C. The restricted plasmid DNA was purified using a PCR purification kit, and then was eluted in 40 μl of distilled and deionized water. To ligate the annealed dsDNA, i.e., a DNA insert, into multiple cloning sites within the linearized mpET-21a vector, 90 pmol of the annealed dsDNA and 30 pmol of the linearized mpET-21a vector were incubated in T4 DNA ligase buffer containing 1 U of T4 DNA ligase for 30 minutes at 16° C. The ligated plasmid was transformed into Top10 chemically competent cells, followed by plating the Top10 competent cells on a SOC plate supplemented with 50 μg/ml of ampicillin. DNA sequences was then verified by DNA sequencing. After all EBP monomer genes were constructed, each EBP gene was synthesized by ligating each of 36 types of repetitive genes (as an insert) into the corresponding vector containing each of the same 36 types of repetitive genes, as follows. A cloning procedure for EBP libraries and fusions thereof is illustrated in FIG. 1. Vectors harboring gene copies corresponding to EBP monomers were digested and dephosphorylated with 10 U of XbaI, 15 U of BseRI and 10 U of FastAP thermosensitive alkaline phosphatase in CutSmart buffer for 30 minutes at 37° C. The restricted plasmid DNA was purified using a PCR purification kit, and then was eluted in 40 μl of distilled and deionized water. For preparation of an insert part, a total of 4 μg of an EBP monomer gene were digested with 10 U of XbaI and 15 U of AcuI in CutSmart buffer for 30 minutes at 37° C. After digestion, the reaction product was separated by agarose gel electrophoresis and the insert was purified using a gel extraction kit. A ligation reaction was performed by incubating 90 pmol of the purified insert with 30 pmol of the linearized vector in T4 DNA ligase buffer containing 1 U of T4 DNA ligase for 30 minutes at 16° C. The product was transformed into Top10 chemically competent cells, then the cells were plated on a SOC plate supplemented with 50 μg/ml of ampicillin. Transformants were initially screened by a diagnostic restriction digest on an agarose gel and further confirmed by DNA sequencing as described above.

[0159] As described above, EBP gene libraries having different DNA sizes were synthesized using the designed plasmid vector and three different restriction endonucleases. FIG. 1 illustrates a recursive directional ligation (RDL) method, in which EBP monomer genes are ligated to form oligomerized EBP genes. For example, a gene construct encoding EBPP[G.sub.1A.sub.3F.sub.2].sub.12 was prepared by a ligation reaction, in which a plasmid backbone and an insert derived from a plasmid-borne gene vector harboring a gene encoding EBPP[G.sub.1A.sub.3F.sub.2].sub.6 were used: The plasmid-borne gene vector harboring a gene encoding EBPP[G.sub.1A.sub.3F.sub.2].sub.6 was double-digested by XbaI and AcuI to obtain an insert, i.e., a gene fragment encoding EBPP[G.sub.1A.sub.3F.sub.2].sub.6. On the other hand, the plasmid-borne gene vector for EBPP[G.sub.1A.sub.3F.sub.2].sub.6 was double-digested by XbaI and BseRI to obtain a plasmid backbone and then the plasmid backbone was dephosphorylated by treatment with an alkaline phosphatase. The RDL method using two different double restriction enzymes has several advantages. First, due to the different shapes of the protrusions of both an insert and a restricted vector, a self-ligating reaction of the restricted vector did not occur, and the insert and the restricted vector were efficiently linked in a head-tail orientation. Second, due to the mechanism of a type III restriction endonuclease, an additional DNA sequence encoding each linker between blocks is not required. Each EBP gene was oligomerized to generate 36, 72, 108, 144, 180, and 216 EBP pentapeptide repeats. Using two restriction endonucleases XbaI and BamHI, oligomerized gene sizes with 540, 1080, 1620, 2160, 2700, and 3240 base pairs (bps) were confirmed. As characterized by agarose gel electrophoresis, FIG. 2 depicts the restricted DNA bands of EBP libraries with DNA size markers on both end lanes. For example, EBPE[A.sub.1G.sub.4I.sub.1] in FIG. 2(A) clearly shows a restricted DNA band corresponding to a DNA region encoding an oligomerized pentapeptide sequence containing Ala, Gly, Ile in a ratio of 1:4:1 as a guest residue. All of the restricted DNA bands are shown as corresponding lengths as compared to the molecular size markers.

[0160] The triblock polypeptides composed of EBP genes and calmodulin were overexpressed in E. coli having a T7 promoter and purified by multiple cycles of inverse transition cycling (ITC). FIG. 3 shows copper-stained SDS-PAGE gel images of the purified EBPs. EBPs shifted at least 20% more than theoretically calculated molecular weights. Two-sided lanes on SDS-PAGE gels contain standard protein size markers (7, 15, 24, 35, 40, 50, 65, 90, 110, and 150 kDa, from bottom to top). In FIGS. 3(A) and 3(B), EBPE[A.sub.1G.sub.4I.sub.1] and EBPP[A.sub.1G.sub.4I.sub.1] represent a series of corresponding proteins with a molecular weight greater than a theoretical molecular weight (in the case of EBPE[A.sub.1G.sub.4I.sub.1], 14.0, 27.7, 41.3, 55.0, and 68.6 kDa, from left to right). In general, as shown in FIGS. 3(C) and 3(D), positively charged EBP libraries, including EBPE[K.sub.1G.sub.4I.sub.1] and EBPP[K.sub.1G.sub.4I.sub.1], showed higher molecular weights than nonpolar EBP libraries, including EBPE[A.sub.1G.sub.4I.sub.1] and EBPP[A.sub.1G.sub.4I.sub.1]. In addition, as shown in FIGS. 3(E) and 3(F), negatively charged EBP libraries, including EBPE[D.sub.1G.sub.4I.sub.1] and EBPP[D.sub.1G.sub.4I.sub.1], have differently charged characteristics, and thus exhibited higher molecular weights than positively charged EBP libraries.

[0161] The EBP libraries were characterized. FIGS. 4A to 4F show thermal transition behaviors of EBPs determined by measuring optical absorbance at 350 nm (absorbance.sub.350) at a heating rate of 1° C./min. An inverse transition temperature (T.sub.t) is defined as a temperature at which the first derivative (d (OD.sub.350)/dT) of turbidity, which is a function of temperature, was the maximum. Based on environmental conditions such as salt concentration and pH and the different third and fourth amino acids of an EBP pentapeptide repeat unit, the T.sub.t of an EBP was finely controlled in PBS and PBS was supplemented with 1 to 3 M NaCl. For example, EBPE[A.sub.1G.sub.4I.sub.1].sub.12 (FIG. 4A) with Gly at the third amino acid of an EBP pentapeptide repeat exhibited a T.sub.t about 15° C. higher than that of EBPP[A.sub.1G.sub.4I.sub.1].sub.12 (FIG. 4B) with Ala at the third amino acid of an EBP pentapeptide repeat in PBS containing 1 M NaCl, because Gly at the third amino acid of an EBP pentapeptide repeat has higher hydrophilicity than Ala. In general, charged EBP libraries have a higher T.sub.t than nonpolar EBP libraries because charged residues were introduced into the fourth amino acid of the EBP pentapeptide repeat of the charged EBPs. Negatively charged EBP libraries, such as EBPP[D.sub.1G.sub.4I.sub.1] (FIG. 4E), have different pK.sub.a values for Asp and Lys at the fourth amino acid of an EBP pentapeptide repeat, and thus have a higher T.sub.t than positively charged EBP libraries, such as EBPE[K.sub.1G.sub.4I.sub.1] (FIG. 4C) and EBPP[K.sub.1G.sub.4I.sub.1] (FIG. 4D). For reference, FIGS. 4A to 4E represent hydrophilicity, and FIG. 4F represents hydrophobicity.

Example 5: Gene Design for Block Including Calmodulin and Cys

[0162] A CalM gene was derived from Rattus norvegicus CalM 3 (GenBank: BC063187.1). Originally, the gene has two BseRI recognition sites, of which the sequence is GAGGAG. Accordingly, to construct the gene libraries of EBP-CalM-EBP triblock polypeptides by molecular cloning, GAG, a codon corresponding to Glu-83 of CalM, was modified to GAA because GAG and GAA are codons for Glu.

[0163] A rationally designed recombinant CalM gene was prepared through a gene synthesis service provided by Invitrogen (Carlsbad, Calif., U.S.), and restricted by XbaI and BseRI to perform seamless ligation for construction of the gene libraries of EBP-CalM-EBP triblock polypeptides. In addition, to perform reversible intermolecular crosslinking by oxidation and reduction as well as drug conjugation for chemotherapeutics, a Cys-incorporated block (Cys block) with multiple Cys residues was designed such that Cys was cyclically located in a repetitive gene sequence, (Gly-Ala-Cys).sub.4. A pair of oligonucleotides for the Cys-incorporated block were chemically synthesized by Cosmo Genetech (Seoul, Korea) and annealed to a dsDNA fragment including restriction sites for AcuI and BseRI.

[0164] As shown in Table 3, a CalM gene derived from Rattus norvegicus CalM 3 was prepared. Since both GAG and GAA are codons for Glu, GAG, a codon corresponding to Glu-83 of CalM, was modified to GAA (the dark colored area in Table 3). Using this modified CalM gene, seamless ligation was performed for construction of the gene libraries of EBP-CalM-EBP triblock polypeptides, which have restriction sites for restriction enzymes including XbaI and BseRI. In addition, to perform reversible intermolecular crosslinking by oxidation and reduction as well as drug conjugation for chemotherapeutics, a Cys-incorporated block (Cys block) with multiple Cys residues was designed at the DNA level such that Cys was cyclically located in a repetitive gene sequence, (Gly-Ala-Cys).sub.4.

TABLE-US-00004 TABLE 3 Gene (SEQ ID NO. 37) and amino acid (SEQ ID NO. 38) sequences of calmodulin CalM) GCT GAC CAG CTG ACC GAA GAA CAG ATT GCA GAG TTC AAG GAA GCC TTC TCC CTC TTT  A   D   Q   L   T   E   E   Q   I   A   E   F   K   E   A   F   S   L   F GAC AAG GAT GGA GAT GGC ACC ATT ACC ACC AAG GAG CTG GGG ACT GTG ATG AGA TCG  D   K   D   G   D   G   T   I   T   T   K   E   L   G   T   V   M   R   S CTG GGG CAA AAC CCC ACT GAG GCG GAA CTG CAG GAC ATG ATC AAT GAG GTG GAT GCT  L   G   Q   N   P   T   E   A   E   L   Q   D   M   I   N   E   V   D   A GAT GGC AAT GGG ACC ATT GAC TTC CCA GAG TTC CTG ACC ATG ATG GCC AGA AAG ATG  D   G   N   G   T   I   D   F   P   E   F   L   T   M   M   A   R   K   M AAG GAT ACA GAC AGC GAG GAA GAG ATA CCA GAG GCC TTC CGT GTC TTT GAC AAG GAT  K   D   T   D   S   E   E   E   I   P   E   A   F   R   V   F   D   K   D GGG AAT GGC TAC ATC AGT GCT GCT GAG CTG CGT CAC GTC ATG ACG AAC CTG GGG GAG  G   N   G   Y   I   S   A   A   E   L   R   H   V   M   T   N   L   G   E AAG CTG ACT GAG GAG GAA GTG GAT GAG ATG ATC CGA GAG GCG GAC ATT GAT GGA GGC  K   L   T   D   E   E   V   D   E   M   I   R   E   A   D   I   D   G   G GGC CAG GTC AAT TAT GAA GAG TTT GTA CAG ATG ATG ACT  G   Q   V   N   Y   E   E   F   V   Q   M   M   T ※Area marked in bold is mutated region, and underlined representation is dityrosine binding region

Example 6: Gene Construction of EBP-CalM-EBP Triblock Polypeptide

[0165] Each plasmid containing EBP, CalM, or a Cys-incorporated block was used to prepare genes for the fusion polypeptide libraries of EBP[G.sub.1A.sub.3F.sub.2].sub.n-CalM-EBP[G.sub.1A.sub.3F.sub.2].sub.n with Cys blocks at both ends. A plasmid vector harboring an EBP gene was digested and dephosphorylated with 10 U of XbaI, 10 U of BseRI and 10 U of FastAP, a thermosensitive alkaline phosphatase, in CutSmart buffer for 30 minutes at 37° C. The restricted plasmid DNA was purified using a PCR purification kit, and then was eluted in 40 μl of distilled and deionized water. A total of 4 μg of the CalM gene was digested with 10 U of XbaI and 15 U of AcuI in the CutSmart buffer for 30 minutes at 37° C. to prepare the CalM gene as an insert, followed by separation using agarose gel electrophoresis and purification using a gel extraction kit. A ligation reaction was performed by incubating 90 pmol of the purified insert with 30 pmol of the linearized vector in T4 DNA ligase buffer containing 1 U of T4 DNA ligase for 30 minutes at 16° C. The product was transformed into Top10 chemically competent cells, then the cells were plated on a SOC plate supplemented with 50 μg/ml of ampicillin. Transformants were initially screened by a diagnostic restriction digest on an agarose gel and further confirmed by DNA sequencing as described above.

Example 7: Gene Expression and Purification of EBPs and Block Polypeptides Thereof

[0166] E. coli strain BL21(DE3) cells were transformed with each vector containing an EBP or EBP block polypeptide, and then inoculated in 50 ml of CircleGrow media supplemented with 50 μg/ml ampicillin. Preculturing was performed in a shaking incubator at 200 rpm overnight at 37° C. 50 ml of CircleGrow media was then inoculated in 500 ml of CircleGrow media with 50 μg/ml ampicillin and incubated in a shaking incubator at 200 rpm for 16 hours at 37° C. When optical density at 600 nm (OD.sub.600) reached 1.0, overexpression of an EBP gene or a block polypeptide gene thereof was induced by addition of IPTG at a final concentration of 1 mM. The cells were centrifuged at 4500 rpm for 10 minutes at 4° C. The expressed EBPs and block polypeptides thereof were purified by inverse transition cycling (ITC) as reported previously (see Dong Woo Lim, Kimberly Trabbic-Carlson, J. Andrew MacKay and Ashutosh Chilkoti, “IMPROVED NON-CHROMATOGRAPHIC PURIFICATION OF A RECOMBINANT PROTEIN BY CATIONIC ELASTIN-LIKE POLYPEPTIDES”, Biomacromolecules, 8 (5), 1417-1424, 2007). The cell pellet was resuspended in 30 ml of HEPES buffer, and the cells were lysed by sonication for 10 s at 20 s intervals (VC-505, Sonics & Materials Inc, Danbury, Conn.) on an ice bath. The cell lysate was centrifuged in a 50 ml centrifuge tube at 13000 rpm for 15 min at 4° C. to precipitate the insoluble debris of the cell lysate. A supernatant containing water soluble EBPs was then transferred to a new 50 ml centrifuge tube and centrifuged with 0.5% w/v of PEI at 13000 rpm for 15 minutes at 4° C. to precipitate nucleic acid contaminants. The inverse phase transition of the EBPs were triggered by adding sodium chloride at a final concentration of 3 to 4 M, and the aggregated EBPs were separated from the lysate solution by centrifugation at 13000 rpm for 15 minutes at 37° C. The aggregated EBPs were resuspended in cold HEPES buffer, and the EBP solutions were centrifuged at 13000 rpm for 15 minutes at 4° C. to remove any aggregated protein contaminants. These aggregation and resuspension processes were repeated 5 to 10 times until the purity of EBP reached approximately 95%, as determined by SDS-PAGE and gel permeating chromatography with a high-performance liquid chromatography (HPLC) 1260 series instrument (Agilent Technologies, Palo Alto, Calif., USA) using Shodex GPC column OHpak SB-804 HQ (Showa Denko Co., Tokyo, Japan). Deionized water at 20° C. was used as an eluent at a flow rate of 1 ml/min and a GPC column was maintained at 20° C. A low-dispersity pullulan in the range of 5900 to 200000 g/mol was used as a standard. The EBPs and the block copolypeptides thereof were analyzed with a refractive index detector (RID) and a variable wavelength detector (VWD) at 280 nm. An effect of temperature on inverse phase transition of the various EBPs and the block polypeptides thereof at 25 μM concentration in HEPES buffer was determined by measuring OD.sub.350 using a Cary 100 Bio UV/Vis Spectrophotometer equipped with a multi-cell thermoelectric temperature controller (Varian Instruments, Walnut Creek, Calif.) at 10 to 85° C. at a rate of 1° C./min.

[0167] FIG. 5 shows schematic diagrams of dynamic protein hydrogels composed of genetically engineered ABA-type EBP-CalM-EBP triblock polypeptides. Two different types of EBP blocks, such as a hydrophobic EBP having a low T.sub.t and a hydrophilic EBP having a high T.sub.t, were introduced. A series of EBP-CalM-EBP triblock polypeptides in FIG. 5 were designed as follows: (A) hydrophobic EBP-CalM-hydrophobic EBP, (B) hydrophobic EBP-hydrophilic EBP-CalM-hydrophilic EBP-hydrophobic EBP, (C) Cys block-hydrophobic EBP-CalM-hydrophobic EBP-Cys block, and (D) Cys block-hydrophobic EBP-hydrophilic EBP-CalM-hydrophilic EBP-hydrophobic EBP-Cys block. EBP blocks having different molecular weights and different T.sub.ts were arranged on both sides of the CalM block, and Cys blocks were introduced at both ends of the EBP blocks for chemical cross-linking. Since EBP-CalM-EBP triblock polypeptides may reversibly exhibit thermally initiated gelation, a physically cross-linked protein hydrogel may be generated under physiological conditions at or above T.sub.t. In addition, an EBP-CalM-EBP triblock polypeptide may form a physically cross-linked protein hydrogel under aqueous solution conditions above the transition temperature of a hydrophobic EBP, and ligands such as calcium and a phenothiazine drug specifically bind to CalM, thereby inducing a structural change in CalM to dynamically change the three-dimensional network of the hydrogel. Furthermore, the thiol groups of Cys blocks at both ends may be chemically crosslinked via oxidation, and Tyr residues in the CalM block may be also chemically crosslinked into dityrosine via UV irradiation. Depending on multi-responsiveness of the EBP-CalM-EBP triblock polypeptides, physicochemical and mechanical properties thereof may be finely controlled. Consequently, EBP-CalM-EBP triblock polypeptides exhibit multi-stimuli responsiveness to temperature, calcium, and ligand molecules, forming a dynamic protein hydrogel that is triggered by the multi-stimuli.

Example 8: Rheological Measurement of Physically and Chemically Cross-Linked Hydrogels

[0168] EBP-CalM-EBP triblock polypeptide solutions were prepared in different HEPES buffers supplemented with various concentrations of oxidizing and reducing agents, CaCl.sub.2, or phenothiazine as ligands for CalM. The EBP-CalM-EBP solutions were studied in dynamic-shear rheological testing (cone-and-plate configuration, cone angle=1.58°, diameter=20 mm, TA DHR1 with Peltier plate, TA instruments, Inc., U.S.) to quantify the elastic modulus (G′), loss modulus (G″), complex shear modulus (G*), complex viscosity (η*), and loss angle (δ) as functions of temperature and frequency. The G′ characterizes the elastic behavior of a material whereas the G″ characterizes the viscous behavior of the material. The G* and η* represent the frequency-dependent stiffness, and the frequency-dependent viscous drag of a viscoelastic liquid or solid, respectively. The loss angle (δ) is a relative measure of viscous to elastic properties (Newtonian viscous fluid: δ=90°; elastic solid: δ=0°). A metal solvent trap under fully hydrating conditions was used to prevent solvent evaporation over temperatures ranging from 10 to 40° C. All samples were equilibrated for at least 5 to 10 minutes at the desired temperatures prior to each experiment. Dynamic frequency sweep measurements were performed in the linear viscoelastic regime at different temperatures, as confirmed by independent strain sweep tests (strain sweep range: 0.2 to 20%, angular frequency: 0.1 or 10 rad/s). The angular frequency ranged from 0.1 to 10 rad/s, both at 10° C. (below T.sub.t) and 40° C. (above T.sub.t) for the frequency sweep tests.

[0169] The temperature sweep tests were executed with 2% strain at 1 rad/s over a temperature range of 10 to 40° C. with one minute in duration per degree for forward heating and reverse cooling measurements to examine the reversibility of rheological and mechanical properties thereof. The storage modulus (G′), the loss modulus (G″), the complex shear modulus (G*), the complex viscosity (η*), and the loss angle (δ) were obtained from each oscillatory shear measurement. Each experiment was performed three times to ensure reproducibility.

[0170] Hydrophobic EBPP[G.sub.1A.sub.3F.sub.2] blocks with various molecular weights (M.W.) was introduced into the Cys block-hydrophobic EBP-CalM-hydrophobic EBP-Cys block at the DNA level. A DNA agarose gel electrophoresis image in FIG. 6 (left) clearly shows the different DNA sizes of a series of constructed Cys block-EBPP[G.sub.1A.sub.3F.sub.2].sub.6n-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.6n-Cys block (n: integer) in the range of 1596, 2676, 3756, 4836, 5916, and 6996 base pairs (bps) from the first to sixth lane. The triblock polypeptides were overexpressed in E. coli and purified by ITC as described above. A copper-stained SDS-PAGE gel in FIG. 6 (right) shows that these triblock polypeptides were purified to at least 95% homogeneity by five to six rounds of ITC, as characterized by GPC analysis. The theoretical molecular weights of the polypeptides shown on the right of the gel in FIG. 6 (right) are 49.0, 79.1, 109.2, 139.4, 169.5, and 199.6 kDa from the first to sixth lane. It was found that the polypeptides were partly oxidized during SDS-PAGE, indicating dimers formed via disulfide bridges.

[0171] FIG. 7 shows the thermal transition behaviors of the polypeptides in the absence (A) or presence (B) of 10 mM CaCl.sub.2 when the polypeptides were characterized by turbidity profiling as a function of temperature at a heating rate of 1° C./min. Depending on different EBPP[G.sub.1A.sub.3F.sub.2] block sizes as a hydrophobic block and calcium binding to the CalM, T.sub.ts of the polypeptides were finely controlled in the range from 29.4 to 47.3° C. As the EBPP[G.sub.1A.sub.3F.sub.2] block length increased, the T.sub.t of the polypeptides became lower irrespective of calcium binding to the CalM. In particular, when calcium ions were bound to the CalM, the CalM of the triblock polypeptides underwent a large conformational change, forming a dumbbell shaped structure. Therefore, the T.sub.t of the polypeptides in the presence of 10 mM CaCl.sub.2) were approximately 1.6 to 6.2° C. lower than those in the absence of calcium ions due to increased hydrophobicity induced by a large conformational change of the CalM. Potentially, the calcium bound CalM structure in the dumbbell shaped structure at both ends of a long central helix may make the distance between EBPP[G.sub.1A.sub.3F.sub.2] blocks within the triblock polypeptides much shorter than that without calcium.

Example 9: Viscoelastic Behavior of EBP-CalM-EBP Triblock Polypeptides

[0172] FIG. 8 shows photographic images of reversible sol-gel transition behavior of the Cys block-EBPP[G.sub.1A.sub.3F.sub.2].sub.12-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.12-Cys block in 10 mM HEPES buffer at pH 7.4 as a function of temperature. When the EBP-CalM-EBP triblock polypeptide was prepared in HEPES buffer at 27 wt. %, a viscoelastic liquid with viscous flow at 4° C. became a viscoelastic solid at 37° C. due to physical crosslinking between aggregated EBPP blocks within the triblock polypeptide at 37° C. In particular, as shown in FIG. 8(C), the physically cross-linked hydrogel at 37° C. was easily detached from a tube by simply tapping a finger, indicating rheological and mechanical properties of the physically cross-linked hydrogel at 37° C. FIGS. 8(A) to 8(D) show that transition behavior from the viscoelastic solid to the viscoelastic liquid was reversible as temperature decreased from at 37 to 4° C. because of reversible phase transition behavior of an EBPP block as shown in FIGS. 8(A) to 8(D).

[0173] FIGS. 9 and 10 show the temperature and ligand-dependent rheological and mechanical properties of the Cys block-EBPP[G.sub.1A.sub.3F.sub.2].sub.12-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.12-Cys block in 10 mM HEPES buffer at pH 7.4 as functions of polypeptide concentration and CaCl.sub.2). A series of independent strain sweep tests were performed in the range from 0.2 to 20% at various angular frequencies of 0.1, 1.0 or 10.0 rad/s. Dynamic frequency sweep measurements at 2% strain were performed in the linear viscoelastic regime at different temperatures. The angular frequency ranged from 0.1 to 10 rad/s, both at 10° C. below T.sub.t and 40° C. above T.sub.t for the dynamic frequency sweep tests. The temperature sweep tests were executed with 2% strain at 1.0 rad/s over a temperature range of 10 to 40° C. with one minute in duration per degree for forward heating and reverse cooling measurements to examine the reversibility of rheological and mechanical properties thereof. The storage modulus (G′), the loss modulus (G″), the complex shear modulus (G*), the complex viscosity (η*), and the loss angle (δ) were obtained from each oscillatory shear measurement. FIG. 9 clearly shows controlled gelation temperature (τ), which is defined as a temperature of the crossover point of G′ and G″ in the temperature sweep measurement. As shown in FIG. 9, thermally induced phase transition of the triblock polypeptide in 10 mM HEPES buffer at pH 7.4 occurred at 22 to 23° C. in the concentration range of 16.6-28.6 wt. % and the G′ and G″ values continued to increase due to intra- or intermolecular hydrophobic interactions between the aggregated EBPP blocks within the triblock polypeptide as the temperature increased. In particular, as the temperature increased above the T.sub.t, the complex shear modulus (G*) values largely increased about 3 orders of magnitude greater than that 10° C. below the T.sub.t, indicating enhanced stiffness due to the evolved viscoelasticity.

[0174] As shown in FIG. 10, an effect of calcium binding to CalM on both thermally induced phase transition and rheological properties of the triblock polypeptide was also characterized by oscillatory rheological measurements when 10 mM HEPES buffer at pH 7.4 was supplemented with 168 mM CaCl.sub.2) to have a 10-fold molar excess of CaCl.sub.2) as compared to the 4.2 mM EF-hand motif of CalM. Thermally induced phase transition of the triblock polypeptide in the presence of calcium (FIG. 10 (B)) occurred at 19° C., which is 3° C. lower than that without calcium (FIG. 10 (A)) because the CalM of the triblock polypeptides underwent a large conformational change, forming into a dumbbell shaped structure when calcium ions were bound to the CalM. Although the complex shear modulus (G*) showed similar values irrespective of calcium binding to CalM, the loss angle (δ) in the presence of calcium was 36°, which was 20° higher than that without calcium. Potentially, when a large conformational change of CalM of the triblock polypeptides was induced by calcium binding, the calcium bound CalM structure in the dumbbell shaped structure at both ends of a long central helix may make the distance between EBPP[G.sub.1A.sub.3F.sub.2] blocks within the triblock polypeptides much shorter than that without calcium, suggesting a lowered viscoelastic solid property. FIGS. 10(C) and 10(D) show the frequency-dependent rheological behavior of the triblock polypeptide without (C) and with (D) calcium binding to CalM at 10 and 40° C. In general, the elastic property at 40° C. dominated over a wide frequency range of 0.1 to 10 rad/s at 2% strain and the values of the complex viscosity (η*) are highly frequency dependent with a 10-fold difference or greater due to a intermolecular physical cross-linking of the aggregated EBPP blocks. This proved that the triblock polypeptide was a physically cross-linked hydrogel in conditions higher than the transition temperature of hydrophobic EBP, and depending on the presence or absence of calcium binding to the CalM, (C) and (D) showed different values of G′, G″ and η* depending on frequency. These indicated that rheologically and mechanically different protein hydrogels were formed.

TABLE-US-00005 TABLE 4 Transition temperature of EBP-CalM-EBP triblock polypeptides in the presence of 10 mM CaC12 as compared to free CalM without CaCl.sub.2. Free CalM CalM with EBP-CalM-EBP library (° C.) Ca.sup.2+ (° C.) A Cys-EBPP[G.sub.1A.sub.3F.sub.2].sub.6-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.6-Cys 47.3 40.1 B Cys-EBPP[G.sub.1A.sub.3F.sub.2].sub.12-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.12-Cys 43.2 36.1 C Cys-EBPP[G.sub.1A.sub.3F.sub.2].sub.18-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.18-Cys 37.4 33.5 D Cys-EBPP[G.sub.1A.sub.3F.sub.2].sub.24-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.24-Cys 35.0 31.9 E Cys-EBPP[G.sub.1A.sub.3F.sub.2].sub.30-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.30-Cys 32.4 30.8 F Cys-EBPP[G.sub.1A.sub.3F.sub.2].sub.36-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.36-Cys 29.4 30.7

TABLE-US-00006 TABLE 5 Thermal transition temperature (T.sub.t), gelation temperature (τ), complex shear modulus (|G*|) and phase angle (δ) of 27.0 wt.% Cys block-EBPP[G.sub.1A.sub.3F.sub.2].sub.12-CalM-EBPP[G.sub.1A.sub.3F.sub.2].sub.12-Cys block in HEPES and HEPES supplemented with 168 mM CaCl.sub.2 measured by oscillatory rheological profiles at 1 rad/s and 2% shear strain. Solvent T.sub.t (° C.) τ (° C.) |G*| (Pa) δ (°) HEPES 20.9 24.1 756.2 15.3 HEPES with 168mM CaC1.sub.2 17.4 24.0 404.7 33.8

[0175] The EBP-CalM-EBP triblock polypeptide as an ABA-type showed multi-responsiveness to temperature, calcium and ligand molecules at a molecular level. The physically crosslinked hydrogel of the EBP-CalM-EBP triblock polypeptide was formed by thermal and ligand stimuli, and was found to have reversible sol-gel transition behavior. These dynamic hydrogels also showed different rheological and mechanical properties via multi-responsiveness to calcium, and ligand molecules.

[0176] Potentially, the physically crosslinked hydrogels may be further chemically crosslinked via disulfide bonds of the Cys blocks via oxidation, and dityrosine of CalM via light. These multi-responsive dynamic protein hydrogels may be used in drug delivery system, tissue engineering, and regenerative medicine.

[0177] The hydrogel according to the present invention can be used in injectable drug delivery systems, functional tissue engineering and regenerative medicine.