Abstract:
Disclosed are synthetic polypeptides modeled after NspI nucleoporin which are useful for forming hydrogels characterized by selective permeability. The polypeptides and hydrogels formed from them include phenylalanine-glycine (FG) repeats, which are believed to participate in the selectivity of the nuclear pore complex. Also disclosed are filtering devices, drug delivery devices, and methods of separating or selectively filtering macromolecules using the hydrogels.

Description:
RELATED APPLICATION 
       [0001]    This application claims benefit of priority to U.S. Provisional Patent Application No. 62/015,012, filed Jun. 20, 2014. 
     
    
     GOVERNMENT SUPPORT 
       [0002]    This invention was made with Government support under Contract No. W911NF-13-D-0001 awarded by the Army Research Office. The Government has certain rights in the invention. 
     
    
     BACKGROUND OF THE INVENTION 
       [0003]    The entry and exit of large molecules from the eukaryotic cell nucleus is tightly controlled by nuclear pore complexes (NPCs). Although small molecules can enter and exit the nucleus without regulation, macromolecules such as RNA, mRNA, ribosomal proteins, DNA polymerase, lamins, carbohydrates, signaling molecules, and lipids require association with karyopherins called importins to enter the nucleus and exportins to exit. 
         [0004]    Nuclear pore complexes are large protein complexes that span the nuclear envelope, which is the double membrane surrounding the eukaryotic cell nucleus. The proteins that make up the nuclear pore complex are known as nucleoporins. 
         [0005]    Nucleoporins are only required for the transport of large hydrophilic molecules above 40 kDa, as smaller molecules pass through nuclear pores via passive diffusion. For example, nucleoporins play an important role in the transport of mRNA from the nucleus to the cytoplasm after transcription. Depending on their function, certain nucleoporins are localized to a single side of the nuclear pore complex, either cytosolic or nucleoplasmic. Other nucleoporins may be found on both faces. 
         [0006]    There are three distinct types of nucleoporins, each having a unique structure and function. These three types are structural nucleoporins, membrane nucleoporins, and FG-nucleoporins. 
         [0007]    Structural nucleoporins form the ring portion of the NPC. They span the membrane of the nuclear envelope and are often referred to as the scaffolding of a nuclear pore. Structural nucleoporins come together to form Y-complexes that are composed of seven nucleoporins. Each nuclear pore contains sixteen Y-complexes for a total of 112 structural nucleoporins. 
         [0008]    Membrane nucleoporins are localized to the curvature of a nuclear pore. These proteins are embedded within the nuclear membrane at the region where the inner and outer leaflets connect. 
         [0009]    FG-nucleoporins are so named because they contain repeats of the amino acid residues phenylalanine (F) and glycine (G). FG repeats are small hydrophobic segments that break up long stretches of hydrophilic amino acids. These FG-repeat segments are found in long random-coil portions of the protein which stretch into the channel of nuclear pores and are believed to be primarily responsible for the selective exclusivity of nuclear pore complexes. These segments of FG-nucleoporins form a mass of chains which allow smaller molecules to diffuse through but exclude large hydrophilic macromolecules. These macromolecules are only able to cross a nuclear pore if they are associated with a transport molecule (karyopherin) that temporarily interacts with a nucleoporin&#39;s FG-repeat segments. FG-nucleoporins also contain a globular portion that serves as an anchor for attachment to the nuclear pore complex. 
         [0010]    Karyopherins and their cargo are passed between FG-repeats until they diffuse down their concentration gradient and through the nuclear pore complex. The release of their cargo from karyopherins is driven by Ran, a G protein. Ran is small enough that it can diffuse through nuclear pores down its concentration gradient without interacting with nucleoporins. Ran binds to either GTP or GDP and has the ability to change a karyopherin&#39;s affinity for its cargo. Inside the nucleus, RanGTP causes an importin karyopherin to change conformation, allowing its cargo to be released. RanGTP can also bind to exportin karyopherins and pass through the nuclear pore. Once it has reached the cytosol, RanGTP can be hydrolyzed to RanGDP, allowing the exportin&#39;s cargo to be released. 
         [0011]    Adapting artificially engineered protein polymers from consensus repeats of natural proteins is an attractive approach to mimic the unprecedented performance of natural materials. Tough silk-like polypeptides, thermoresponsive elastin-like polypeptides, and resilient and elastic resilin-like polypeptides have been synthesized to mimic the functions of natural materials. Important design principles have been developed for these artificial biopolymers that enable rational control over their thermodynamic, structural, and mechanical properties. The simplified repeat allows for a detailed understanding of sequence-structure-property relationships to be developed, and these tailor-made materials open up opportunities for applications such as drug delivery, tissue engineering, photonic films and smart responsive devices. 
         [0012]    An additional natural material that has interesting engineering properties is the protein matrix which fills the nuclear pore complex (NPC) in the nuclear envelope and controls transport into the nucleus. It allows passage of less than 0.1% of all proteins while translocating over 1,000 molecules per pore per second. Ribbeck K et al.,  EMBO J  20: 1320 (2001); Yang W D et al.,  Proc Natl Acad Sci USA  101: 12887 (2004). The protein matrix is composed of nucleoporins, proteins containing Phe-Gly (FG) repeat sequences which contribute to specific binding of the nuclear transport receptors (NTRs) that facilitate transport of a specific subset of biological molecules into the matrix. 
         [0013]    Individual nucleoporins can form hydrogels in vitro that recapitulate the enhanced permeability of selectively-labeled macromolecules into the gel, similar to the intact NPC, with varying degrees of passive diffusion of inert molecules. Labokha A A et al.,  EMBO J  32: 204 (2013); Jovanovic-Talisman T et al.,  Nature  457: 1023 (2009). This selectivity is rare in synthetic polymer hydrogels, making these natural materials an intriguing model for new filtration technologies. In spite of the advanced filtering function of natural nucleoporin hydrogels, until now a fundamental understanding of the sequence-structure-property relationships needed for materials engineering has been lacking, due to the complex sequence of the proteins and the inability to synthesize them recombinantly in high yields. 
       SUMMARY OF THE INVENTION 
       [0014]    To adapt the function of nucleoporin hydrogels in a biosynthetic material, artificially engineered protein polymers were designed that can replicate the biological selective transport of the hydrogel in a synthetic mimic using a consensus repeat adapted from a well-investigated nucleoporin, Nsp1. Frey S et al.,  Science  314: 815 (2006); Ader C et al.,  Proc Natl Acad Sci USA  107: 6281 (2010); Frey S et al.,  Cell  130: 512 (2007). The polymers provide a valuable tool for material engineering and an opportunity to tune the selectivity, transport rates, and barrier function of nucleoporin-inspired materials through rational repeat sequence design. 
         [0015]    As described in detail herein, designed peptides 1NLP and 2NLP, extracted from partial NspI nucleoporin, are useful for the preparation of nucleoporin-based hydrogels characterized by selective filtering capability. 
         [0016]    As described herein, recombinant nucleoporin-like polypeptides P-1NLP-P, P-2NLP-P, and P-cNspI-P are useful for the preparation of hydrogels characterized by selective filtering capability. 
         [0017]    As described in detail herein, hydrogels of the invention are useful as selectively permeable barriers. 
         [0018]    Also as described in detail herein, hydrogels of the invention are useful for sequestration of compounds, including macromolecules. 
         [0019]    Additionally, hydrogels of the invention find use as models for the nuclear pore in assays for nuclear permeability of drugs, biomaterials, nanoparticles, and other compounds. 
         [0020]    As described in detail herein, various nuclear transport receptors, such as importin β and NTF2, can be used as carriers which can selectively bring target molecules into P-1NLP-P, P-2NLP-P, and P-cNspI-P hydrogels. 
         [0021]    Also as described in detail herein, hydrogels of the invention are useful for collecting selected target molecules into hydrogel with nuclear transport receptor associated with target molecule-specific binding tag. 
         [0022]    An aspect of the invention is a polypeptide comprising a plurality of contiguous instances of a subsequence represented by PAFSFGAKPDEKKDSDTSK (SEQ ID NO:1). 
         [0023]    In certain embodiments, the polypeptide comprises 16 contiguous instances of the subsequence represented by SEQ ID NO:1. 
         [0024]    In certain embodiments, the polypeptide consists of 16 contiguous instances of the subsequence represented by SEQ ID NO:1. 
         [0025]    In certain embodiments, the polypeptide further comprises a first leucine zipper domain endblock flanking the N-terminal end of the plurality of contiguous instances of the subsequence represented by SEQ ID NO:1; and a second leucine zipper domain endblock flanking the C-terminal end of the plurality of contiguous instances of the subsequence represented by SEQ ID NO:1. 
         [0026]    In certain embodiments, the leucine zipper domain endblock consists of a P domain. 
         [0027]    In certain embodiments, the P domain consists of the peptide represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3). 
         [0028]    In certain embodiments, the polypeptide comprises the sequence represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASPAFSFGAKPDE KKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPD EKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKP DEKKDSDTSKPAFSFGAKPDEKKDSDTSKTSPAFSFGAKPDEKKDSDTSKPAFSFGA KPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFG AKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSF GAKPDEKKDSDTSKTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMES DAS (SEQ ID NO:4). 
         [0029]    An aspect of the invention is a polypeptide comprising a plurality of contiguous instances of a subsequence represented by PAFSFGAKPDEKKDDDTSK (SEQ ID NO:2). 
         [0030]    In certain embodiments, the polypeptide comprises 16 contiguous instances of the subsequence represented by SEQ ID NO:2. 
         [0031]    In certain embodiments, the polypeptide consists of 16 contiguous instances of the subsequence represented by SEQ ID NO:2. 
         [0032]    In certain embodiments, the polypeptide further comprises a first leucine zipper domain endblock flanking the N-terminal end of the plurality of contiguous instances of the subsequence represented by SEQ ID NO:2; and a second leucine zipper domain endblock flanking the C-terminal end of the plurality of contiguous instances of the subsequence represented by SEQ ID NO:2. 
         [0033]    In certain embodiments, the leucine zipper domain endblock consists of a P domain. 
         [0034]    In certain embodiments, the P domain consists of the peptide represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3). 
         [0035]    In certain embodiments, the polypeptide comprises the sequence represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASPAFSFGAKPDE KKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPD EKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKP DEKKDDDTSKPAFSFGAKPDEKKDDDTSKTSPAFSFGAKPDEKKDDDTSKPAFSFG AKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSF GAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAF SFGAKPDEKKDDDTSKTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVM ESDAS (SEQ ID NO:5). 
         [0036]    An aspect of the invention is a polypeptide comprising a core sequence represented by PSFSFGAKSDENKAGATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTA KPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKPDENKASAT SKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNE TSKPAFSFGAKSDEKKDGDASKPAFSFGAKSDEKKDSDSSKPAFSFGTKSNEKKDS GSSKPAFSFGAKPDEKKNDEVSKPAFSFGAKANEKKESDESKSAFSFGSKPTGKEE GDGAKAAISFGAKPEEQKSSDTSK (SEQ ID NO:6); and a first leucine zipper domain endblock flanking the N-terminal end or the C-terminal end of the core sequence. 
         [0037]    In certain embodiments, the first leucine zipper domain endblock flanks the N-terminal end of the core sequence. 
         [0038]    In certain embodiments, the polypeptide further comprises a second zipper domain endblock flanking the C-terminal end of the core sequence. 
         [0039]    In certain embodiments, the leucine zipper domain endblock consists of a P domain. 
         [0040]    In certain embodiments, the P domain consists of the peptide represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3). 
         [0041]    In certain embodiments, the polypeptide comprises the sequence represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASDNKTTNTTPSF SFGAKSDENKAGATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKP AFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKPDENKASATSK PAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETS KPAFSFGAKSDEKKDGDASKPAFSFGAKSDEKKDSDSSKPAFSFGTKSNEKKDSGS SKPAFSFGAKPDEKKNDEVSKPAFSFGAKANEKKESDESKSAFSFGSKPTGKEEGD GAKAAISFGAKPEEQKSSDTSKPAFTFGTSAPQMLRELQETNAALQDVRELLRQQV KEITFLKNTVMESDAS (SEQ ID NO:7). 
         [0042]    An aspect of the invention is a nucleic acid molecule encoding a polypeptide of the invention. 
         [0043]    An aspect of the invention is an expression vector comprising a nucleic acid molecule of the invention. 
         [0044]    An aspect of the invention is a cell, comprising an expression vector of the invention. 
         [0045]    An aspect of the invention is a hydrogel, comprising a polypeptide of the invention. 
         [0046]    An aspect of the invention is a filtering device, comprising a hydrogel of the invention; and a housing or support for the hydrogel. 
         [0047]    An aspect of the invention is a drug delivery device, comprising a drug; and a hydrogel of the invention. 
         [0048]    An aspect of the invention is a method of separating or selectively filtering macromolecules, comprising contacting a source of macromolecules with a hydrogel of the invention. 
         [0049]    In certain embodiments, the macromolecule is selected from the group consisting of RNA, mRNA, DNA, proteins, glycoproteins, carbohydrates, lipids, toxins, and any combination thereof. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0050]      FIG. 1A  depicts design of synthetic protein polymers, P-cNsp1-P and P-NLPs-P, which gel by association of pentameric (P) coiled-coil endblock domains (coils). Filled circles represent Phe-Gly (FG) sequences. 
           [0051]      FIG. 1B  depicts 3D gel network formed from assembly of designed artificially engineered proteins. Black dotted circles highlight Phe-mediated interactions within synthetic hydrogels. 
           [0052]      FIG. 2A  depicts a series of three western blots SDS-PAGE of indicated lyophilized protein samples. 
           [0053]      FIG. 2B  is a graph depicting yields of designed proteins flanked by P domains. 
           [0054]      FIG. 3A  is a panel of three western blots depicting expression levels of 1NLP, 2NLP, and cNsp1*, where cNsp1* was obtained as a product from P-intein-cNsp1. 
           [0055]      FIG. 3B  is a bar graph depicting expression levels of cNsp1, 1NLP, 2NLP, and cNsp1*, where cNsp1* was obtained as a product from P-intein-cNsp1. 
           [0056]      FIG. 4A  depicts a schematic of capillary transport assay set-up. Blue (darker) and green (lighter) circles represent importin β and IBB-MBP-EGFP, respectively. 
           [0057]      FIG. 4B  depicts a time course transport measurement of 20 w/v % P-cNsp1-P hydrogel with β. 
           [0058]      FIG. 4C  is a graph depicting 20 w/v % P-cNsp1-P hydrogels in the presence (solid line) or absence (dotted line) of importin β. 
           [0059]      FIG. 4D  is a graph depicting 20 w/v % P-1NLP-P hydrogels in the presence (solid line) or absence (dotted line) of importin β. Scale bar, 900 μm. 
           [0060]      FIG. 4E  is a graph depicting 20 w/v % P-2NLP-P hydrogels in the presence (solid line) or absence (dotted line) of importin β. Scale bar, 900 μm. 
           [0061]      FIG. 4F  is a graph depicting absorption of cargo-importin β complexes by P-cNsp1-P, P-1NLP-P and P-2NLP-P hydrogels in one hour. * denotes p&lt;0.05. 
           [0062]      FIG. 4G  is a graph depicting fluorescence intensity measurements on P-2NLP-P hydrogels (20 w/v %) with 10% 1,6 hexanediol. Scale bar, 900 μm. 
           [0063]      FIG. 4H  is a graph depicting selective permeability test performed on P-1NLP-P biosynthetic hydrogels (20 w/v %) with the addition of 5 μM MBP-mCherry, a model inert molecule, into 5 μM IBB-MBP-EGFP/importin β cargo complex mixtures. Scale bar, 900 μm. 
           [0064]      FIG. 4I  is a graph depicting selective permeability test performed on P-2NLP-P biosynthetic hydrogels (20 w/v %) with the addition of 5 μM MBP-mCherry, a model inert molecule, into 5 μM IBB-MBP-EGFP/importin β cargo complex mixtures. Scale bar, 900 μm. 
           [0065]      FIG. 5A  is a graph depicting selective permeability of P-2NLP-P gel in 20 w/v %  FIG. 5B  is a graph depicting selective permeability of P-2NLP-P gel in 10 w/v %. 
           [0066]      FIG. 6  is a panel of four western blots depicting protein expression levels of Nsp1, P-cNsp1-P, P-1NLP-P, and P-2NLP-P. L, protein ladder; E, elution fraction. 
           [0067]      FIG. 7A  is a graph depicting frequency sweep, linear oscillatory shear rheology of 20 w/v % P-cNsp1-P hydrogels in the absence (blue curves) or presence (red curves) of 10% 1,6 hexanediol. The gel modulus and the crossover frequency in the absence of hexanediol are 9.3 kPa and 0.08 rad/s. 
           [0068]      FIG. 7B  is a graph depicting frequency sweep, linear oscillatory shear rheology of 20 w/v % P-1NLP-P hydrogels in the absence (blue curves) or presence (red curves) of 10% 1,6 hexanediol. The gel modulus and the crossover frequency in the absence of hexanediol are 10.7 kPa and 0.02 rad/s. 
           [0069]      FIG. 7C  is a graph depicting frequency sweep, linear oscillatory shear rheology of 20 w/v % P-2NLP-P hydrogels in the absence (blue curves) or presence (red curves) of 10% 1,6 hexanediol. The gel modulus and the crossover frequency in the absence of hexanediol are 7.5 kPa and 0.04 rad/s. 
           [0070]      FIG. 7D  is a graph depicting frequency sweep, linear oscillatory shear rheology of 20 w/v % P-C 30 -P gel, which lacks FG repeats in its midblock. C is a peptide having amino acid sequence AGAGAGPEG (SEQ ID NO:8). 
           [0071]      FIG. 7E  is a graph depicting Raman spectra of 20 w/v % cNsp1 midblocks, measured in buffer containing 50 mM Tris/HCl (pH 7.5) and 200 mM NaCl (blue curve) and with the addition of 10% hexanediol (red curve). The shaded boxes highlight Raman bands of 486, 685 and 710 cm −1  that decrease in intensity with the addition of hexanediol. 
           [0072]      FIG. 7F  is a graph depicting Raman spectra of 20 w/v % 1NLP midblocks, measured in buffer containing 50 mM Tris/HCl (pH 7.5) and 200 mM NaCl (blue curve) and with the addition of 10% hexanediol (red curve). The shaded boxes highlight Raman bands of 486, 685 and 710 cm −1  that decrease in intensity with the addition of hexanediol. 
           [0073]      FIG. 7G  is a graph depicting Raman spectra of 20 w/v % 2NLP midblocks, measured in buffer containing 50 mM Tris/HCl (pH 7.5) and 200 mM NaCl (blue curve) and with the addition of 10% hexanediol (red curve). The shaded boxes highlight Raman bands of 486, 685 and 710 cm −1  that decrease in intensity with the addition of hexanediol. 
           [0074]      FIG. 8  is a series of four graphs depicting strain sweep oscillatory shear rheology of indicated 20 w/v % hydrogels at 100 rad/s and 25° C. C is a peptide having amino acid sequence AGAGAGPEG (SEQ ID NO:8). 
           [0075]      FIG. 9A  is a western blot depicting P-C 30 -P. C is a peptide having amino acid sequence AGAGAGPEG (SEQ ID NO:8). 
           [0076]      FIG. 9B  is a graph depicting strain sweep oscillatory shear rheology of indicated P-C 30 -P hydrogels prepared by hydrating lyophilized samples (20 w/v %) with buffer containing 50 mM Tris/HCl (pH 7.5) and 200 mM NaCl in the presence or absence of 10% 1,6-hexanediol. 
           [0077]      FIG. 10  is a graph depicting Raman spectra of cNsp1 (20 w/v %) with selected band assignments; buffer contained 50 mM Tris/HCl (pH 7.5) and 200 mM NaCl. Δ=deformation; σ=stretching. 
           [0078]      FIG. 11  is a graph depicting Raman spectra of cNsp1 (upper spectrum) and 1NLP (lower spectrum) lyophilized samples with selected band assignments. 
           [0079]      FIG. 12  is a series of three graphs depicting diffusion of various sizes of FITC-dextran through the indicated hydrogels. Gel pore radii are estimated between 2.3 nm and 4.5 nm. 
           [0080]      FIG. 13A  is a graph depicting permeability profile of the P-cNsp1-P hydrogel for various sizes of FITC-dextran (as per  FIG. 12 ). The florescence intensity profile on the gel at 1 hour is shown. Areas under the solid curves (&gt;0 μm) were calculated and compared to the passive diffusion by inert molecules (dashed curve). 
           [0081]      FIG. 13B  is a graph depicting permeability profile of the P-1NLP-P hydrogel for various sizes of FITC-dextran (as per  FIG. 12 ). The florescence intensity profile on the gel at 1 hour is shown. Areas under the solid curves (&gt;0 μm) were calculated and compared to the passive diffusion by inert molecules (dashed curve). 
           [0082]      FIG. 13C  is a graph depicting permeability profile of the P-2NLP-P hydrogel for various sizes of FITC-dextran (as per  FIG. 12 ). The florescence intensity profile on the gel at 1 hour is shown. Areas under the solid curves (&gt;0 μm) were calculated and compared to the passive diffusion by inert molecules (dashed curve). 
           [0083]      FIG. 14A  is a graph depicting indicated gel and buffer interface changes by gel swelling during time lapse measurement of the capillary assay for IBB-MBP-GFP. 
           [0084]      FIG. 14B  is a graph depicting indicated gel and buffer interface changes by gel swelling during time lapse measurement of the capillary assay for IBB-MBP-GFP+importin β. 
           [0085]      FIG. 15  is a graph depicting circular dichroism (CD) analysis of cNsp1 and indicated NLPs. 
           [0086]      FIG. 16  is a graph depicting permeability profiles of P-cNsp1-P hydrogel (20 w/v %) with 10% 1,6 hexanediol, for IBB-MBP-GFP with or without importin β. 
           [0087]      FIG. 17A  is a graph depicting time lapse measurements of fluorescence profiles measured to investigate the selective permeability of P-2NLP-P gel (20 w/v %) in the mixture of IBB-MBP-EGFP, MBP-mCherry, and β. Solid curves represent green fluorescence intensity observed in the gel. 
           [0088]      FIG. 17B  is a graph depicting movement of the gel/buffer boundary over the course of the selective permeability tests of P-2NLP-P gel (20 w/v %) in the mixture of IBB-MBP-EGFP, MBP-mCherry, and β. For the control experiment, only MBP-mCherry was tested. 
           [0089]      FIG. 18  is a schematic depicting a method for selective capture of a target molecule by a hydrogel of the invention. 
           [0090]      FIG. 19A  is a photographic image depicting NTF2-GFP in P-2NLP-P. 
           [0091]      FIG. 19B  is a photographic image depicting NTF2-GFP in P-1NLP-P. 
           [0092]      FIG. 19C  is a photographic image depicting NTF2-GFP in P-cNsp1-P. 
           [0093]      FIG. 19D  is a photographic image depicting 40 kg/mol FITC-dextran in P-2NLP-P. 
           [0094]      FIG. 19E  is a photographic image depicting 40 kg/mol FITC-dextran in P-1NLP-P. 
           [0095]      FIG. 19F  is a photographic image depicting 40 kg/mol FITC-dextran in P-cNsp1-P. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0096]    Recent in vitro results indicate that the recombinant Nsp1 2-601  can be divided into an N-terminal sequence Nsp1 2-277  and a C-terminal sequence Nsp1 274-601 . Ader C et al.,  Proc Natl Acad Sci USA  107: 6281 (2010). In Nsp1, the C-terminal sequence contributes to selective transport of NTR-cargo complexes and less non-specific binding of inert molecules, core functions for selective transport. However, the C-terminal sequence alone forms a liquid that cannot restrict the passage of inert molecules. The N-terminal sequence is critical for gelation, suggesting that network formation is required for a fully functional selective transport system. 
         [0097]    To prepare synthetic gels, the N-terminal sequence of Nsp1, which gels slowly over a period of hours, was replaced with well-investigated pentameric (P) coiled-coil domains flanking the C-terminal sequence (cNsp1). Petka W A et al.,  Science  281: 389 (1998); Shen W et al.,  Nat Mater  5: 153 (2006); Olsen B D et al.,  Macromolecules  43: 9094 (2010). This triblock protein construct, P-cNsp1-P, gels in minutes, and the transient interactions of the P domains allow network relaxation that is thought to be critical to transport. Ribbeck K et al.,  EMBO J  20: 1320 (2001). 
         [0098]    Analysis of the cNsp1 consensus sequence (Nsp1 282-585 ) allows reduction of the protein to a polymer of short repeating segments. Nsp1 282-585  is composed of 16 repeats of a 19-amino acid Phe-Gly (FG)-containing sequence, with a high consensus at each position except position 15, where equal numbers of Asp (D) and Ser (S) are observed. To elucidate the 16 consecutive 19-amino acid segments, the 304-amino acid sequence of Nsp1 282-585  (SEQ ID NO:9) can be written thus: 
         [0000]                            PSFS FG AKSDENKAGATSK                       PAFS FG AKPEEKKD D NSSK                       PAFS FG AKSNEDKQ D GTAK                       PAFS FG AKPAEKNNNETSK                       PAFS FG AKSDEKKDGDASK                       PAFS FG AKPDENKA S ATSK                       PAFS FG AKPEEKKD D NSSK                       PAFS FG AKSNEDKQ D GTAK                       PAFS FG AKPAEKNNNETSK                       PAFS FG AKSDEKKDGDASK                       PAFS FG AKSDEKKD S DSSK                       PAFS FG TKSNEKKD S GSSK                       PAFS FG AKPDEKKN D EVSK                       PAFS FG AKANEKKE S DESK                       SAFS FG SKPTGKEEGDGAK                       AAIS FG AKPEEQKS S DTSK            
where the repeating FG sequences and the D and S residues at position 15 are shown in bold.
 
         [0099]    To capture the highest frequency of occurrence in all positions of cNsp1, two separate repeat units were designed: one where position 15 was Asp (D) (PAFSFGAKPDEKKDDDTSK; SEQ ID NO:1), and another where position 15 was Ser (S) (PAFSFGAKPDEKKDSDTSK; SEQ ID NO:2). These sequences were cloned to form an artificial protein polymer of 16 such units, producing two nucleoporin-like polypeptides (NLPs) denoted 1NLP and 2NLP, respectively. Both NLPs were genetically fused with P domain endblocks (P-1NLP-P and P-2NLP-P,  FIG. 1A ) to construct polymers that form gels due to coiled-coil physical association ( FIG. 1B ). Since these simplified NLP polymers can mimic the properties of natural cNsp1, the polymers represent a valuable tool for material engineering and an opportunity to tune the selectivity, transport rates and barrier function of nucleoporin-inspired materials through rational repeat sequence design. 
         [0100]    Compounds of the Invention 
         [0101]    2NLP 
         [0102]    An aspect of the invention is a polypeptide comprising a plurality of contiguous instances of a subsequence represented by PAFSFGAKPDEKKDSDTSK (SEQ ID NO:1). 
         [0103]    In certain embodiments, the polypeptide consists of a plurality of contiguous instances of a subsequence represented by SEQ ID NO:1. 
         [0104]    In certain embodiments, the polypeptide comprises 16 contiguous instances of the subsequence represented by SEQ ID NO:1. 
         [0105]    In certain embodiments, the polypeptide consists of 16 contiguous instances of the subsequence represented by SEQ ID NO:1. 
         [0106]    In certain embodiments, the polypeptide further comprises a first leucine zipper domain endblock flanking the N-terminal end of the plurality of contiguous instances of the subsequence represented by SEQ ID NO:1. 
         [0107]    In certain embodiments, the polypeptide further comprises a first leucine zipper domain endblock flanking the C-terminal end of the plurality of contiguous instances of the subsequence represented by SEQ ID NO:1. 
         [0108]    In certain embodiments, the first leucine zipper domain endblock comprises a pentameric coiled-coil domain (P domain). 
         [0109]    In certain embodiments, the first leucine zipper domain endblock consists of a pentameric coiled-coil domain (P domain). 
         [0110]    In certain embodiments, the P domain comprises the peptide represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3). 
         [0111]    In certain embodiments, the P domain consists of the peptide represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3). 
         [0112]    In certain embodiments, the polypeptide further comprises a first leucine zipper domain endblock flanking the N-terminal end of the plurality of contiguous instances of the subsequence represented by SEQ ID NO:1; and a second leucine zipper domain endblock flanking the C-terminal end of the plurality of contiguous instances of the subsequence represented by SEQ ID NO:1. 
         [0113]    In certain embodiments, the first leucine zipper domain endblock comprises a P domain. 
         [0114]    In certain embodiments, the first leucine zipper domain endblock consists of a P domain. 
         [0115]    In certain embodiments, the first leucine zipper domain endblock P domain comprises the peptide represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3). 
         [0116]    In certain embodiments, the first leucine zipper domain endblock P domain consists of the peptide represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3). 
         [0117]    In certain embodiments, the second leucine zipper domain endblock comprises a P domain. 
         [0118]    In certain embodiments, the second leucine zipper domain endblock consists of a P domain. 
         [0119]    In certain embodiments, the second leucine zipper domain endblock P domain comprises the peptide represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3). 
         [0120]    In certain embodiments, the second leucine zipper domain endblock P domain consists of the peptide represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3). 
         [0121]    In certain embodiments, the first leucine zipper domain endblock comprises a P domain; and the second leucine zipper domain endblock comprises a P domain. 
         [0122]    In certain embodiments, the first leucine zipper domain endblock comprises a P domain; and the second leucine zipper domain endblock consists of a P domain. 
         [0123]    In certain embodiments, the first leucine zipper domain endblock consists of a P domain; and the second leucine zipper domain endblock comprises a P domain. 
         [0124]    In certain embodiments, the first leucine zipper domain endblock consists of a P domain; and the second leucine zipper domain endblock consists of a P domain. 
         [0125]    In certain embodiments, the first leucine zipper domain endblock P domain comprises the peptide represented by SEQ ID NO:3; and the second leucine zipper domain endblock P domain comprises the peptide represented by SEQ ID NO:3. 
         [0126]    In certain embodiments, the first leucine zipper domain endblock P domain comprises the peptide represented by SEQ ID NO:3; and the second leucine zipper domain endblock P domain consists of the peptide represented by SEQ ID NO:3. 
         [0127]    In certain embodiments, the first leucine zipper domain endblock P domain consists of the peptide represented by SEQ ID NO:3; and the second leucine zipper domain endblock P domain comprises the peptide represented by SEQ ID NO:3. 
         [0128]    In certain embodiments, the first leucine zipper domain endblock P domain consists of the peptide represented by SEQ ID NO:3; and the second leucine zipper domain endblock P domain consists of the peptide represented by SEQ ID NO:3. 
         [0129]    In certain embodiments, the polypeptide comprises the sequence represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASPAFSFGAKPDE KKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPD EKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKP DEKKDSDTSKPAFSFGAKPDEKKDSDTSKTSPAFSFGAKPDEKKDSDTSKPAFSFGA KPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFG AKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSF GAKPDEKKDSDTSKTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMES DAS (SEQ ID NO:4). 
         [0130]    In certain embodiments, the polypeptide consists of the sequence represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASPAFSFGAKPDE KKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPD EKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKP DEKKDSDTSKPAFSFGAKPDEKKDSDTSKTSPAFSFGAKPDEKKDSDTSKPAFSFGA KPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFG AKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSF GAKPDEKKDSDTSKTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMES DAS (SEQ ID NO:4). 
         [0131]    1NLP 
         [0132]    An aspect of the invention is a polypeptide comprising a plurality of contiguous instances of a subsequence represented by PAFSFGAKPDEKKDDDTSK (SEQ ID NO:2). 
         [0133]    In certain embodiments, the polypeptide consists of a plurality of contiguous instances of a subsequence represented by SEQ ID NO:2. 
         [0134]    In certain embodiments, the polypeptide comprises 16 contiguous instances of the subsequence represented by SEQ ID NO:2. 
         [0135]    In certain embodiments, the polypeptide consists of 16 contiguous instances of the subsequence represented by SEQ ID NO:2. 
         [0136]    In certain embodiments, the polypeptide further comprises a first leucine zipper domain endblock flanking the N-terminal end of the plurality of contiguous instances of the subsequence represented by SEQ ID NO:2. 
         [0137]    In certain embodiments, the polypeptide further comprises a first leucine zipper domain endblock flanking the C-terminal end of the plurality of contiguous instances of the subsequence represented by SEQ ID NO:2. 
         [0138]    In certain embodiments, the first leucine zipper domain endblock comprises a pentameric coiled-coil domain (P domain). 
         [0139]    In certain embodiments, the first leucine zipper domain endblock consists of a pentameric coiled-coil domain (P domain). 
         [0140]    In certain embodiments, the P domain comprises the peptide represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3). 
         [0141]    In certain embodiments, the P domain consists of the peptide represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3). 
         [0142]    In certain embodiments, the polypeptide further comprises a first leucine zipper domain endblock flanking the N-terminal end of the plurality of contiguous instances of the subsequence represented by SEQ ID NO:2; and a second leucine zipper domain endblock flanking the C-terminal end of the plurality of contiguous instances of the subsequence represented by SEQ ID NO:2. 
         [0143]    In certain embodiments, the first leucine zipper domain endblock comprises a P domain. 
         [0144]    In certain embodiments, the first leucine zipper domain endblock consists of a P domain. 
         [0145]    In certain embodiments, the first leucine zipper domain endblock P domain comprises the peptide represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3). 
         [0146]    In certain embodiments, the first leucine zipper domain endblock P domain consists of the peptide represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3). 
         [0147]    In certain embodiments, the second leucine zipper domain endblock comprises a P domain. 
         [0148]    In certain embodiments, the second leucine zipper domain endblock consists of a P domain. 
         [0149]    In certain embodiments, the second leucine zipper domain endblock P domain comprises the peptide represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3). 
         [0150]    In certain embodiments, the second leucine zipper domain endblock P domain consists of the peptide represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3). 
         [0151]    In certain embodiments, the first leucine zipper domain endblock comprises a P domain; and the second leucine zipper domain endblock comprises a P domain. 
         [0152]    In certain embodiments, the first leucine zipper domain endblock comprises a P domain; and the second leucine zipper domain endblock consists of a P domain. 
         [0153]    In certain embodiments, the first leucine zipper domain endblock consists of a P domain; and the second leucine zipper domain endblock comprises a P domain. 
         [0154]    In certain embodiments, the first leucine zipper domain endblock consists of a P domain; and the second leucine zipper domain endblock consists of a P domain. 
         [0155]    In certain embodiments, the first leucine zipper domain endblock P domain comprises the peptide represented by SEQ ID NO:3; and the second leucine zipper domain endblock P domain comprises the peptide represented by SEQ ID NO:3. 
         [0156]    In certain embodiments, the first leucine zipper domain endblock P domain comprises the peptide represented by SEQ ID NO:3; and the second leucine zipper domain endblock P domain consists of the peptide represented by SEQ ID NO:3. 
         [0157]    In certain embodiments, the first leucine zipper domain endblock P domain consists of the peptide represented by SEQ ID NO:3; and the second leucine zipper domain endblock P domain comprises the peptide represented by SEQ ID NO:3. 
         [0158]    In certain embodiments, the first leucine zipper domain endblock P domain consists of the peptide represented by SEQ ID NO:3; and the second leucine zipper domain endblock P domain consists of the peptide represented by SEQ ID NO:3. 
         [0159]    In certain embodiments, the polypeptide comprises the sequence represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASPAFSFGAKPDE KKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPD EKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKP DEKKDDDTSKPAFSFGAKPDEKKDDDTSKTSPAFSFGAKPDEKKDDDTSKPAFSFG AKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSF GAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAF SFGAKPDEKKDDDTSKTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVM ESDAS (SEQ ID NO:5). 
         [0160]    In certain embodiments, the polypeptide consists of the sequence represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASPAFSFGAKPDE KKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPD EKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKP DEKKDDDTSKPAFSFGAKPDEKKDDDTSKTSPAFSFGAKPDEKKDDDTSKPAFSFG AKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSF GAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAF SFGAKPDEKKDDDTSKTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVM ESDAS (SEQ ID NO:5). 
         [0161]    cNsp1 
         [0162]    An aspect of the invention is a polypeptide comprising a core sequence represented by PSFSFGAKSDENKAGATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTA KPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKPDENKASAT SKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNE TSKPAFSFGAKSDEKKDGDASKPAFSFGAKSDEKKDSDSSKPAFSFGTKSNEKKDS GSSKPAFSFGAKPDEKKNDEVSKPAFSFGAKANEKKESDESKSAFSFGSKPTGKEE GDGAKAAISFGAKPEEQKSSDTSK (SEQ ID NO:6); and a first leucine zipper domain endblock flanking the N-terminal end or the C-terminal end of the core sequence. 
         [0163]    In certain embodiments, the polypeptide consists of a core sequence represented by PSFSFGAKSDENKAGATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTA KPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKPDENKASAT SKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNE TSKPAFSFGAKSDEKKDGDASKPAFSFGAKSDEKKDSDSSKPAFSFGTKSNEKKDS GSSKPAFSFGAKPDEKKNDEVSKPAFSFGAKANEKKESDESKSAFSFGSKPTGKEE GDGAKAAISFGAKPEEQKSSDTSK (SEQ ID NO:6); and a first leucine zipper domain endblock flanking the N-terminal end or the C-terminal end of the core sequence. 
         [0164]    In certain embodiments, the first leucine zipper domain endblock flanks the N-terminal end of the core sequence. 
         [0165]    In certain embodiments, the first leucine zipper domain endblock flanks the C-terminal end of the core sequence. 
         [0166]    In certain embodiments, the first leucine zipper domain endblock comprises a pentameric coiled-coil domain (P domain). 
         [0167]    In certain embodiments, the first leucine zipper domain endblock consists of a pentameric coiled-coil domain (P domain). 
         [0168]    In certain embodiments, the first leucine zipper domain endblock P domain comprises the peptide represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3). 
         [0169]    In certain embodiments, the first leucine zipper domain endblock P domain consists of the peptide represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO:3). 
         [0170]    In certain embodiments, the polypeptide comprises the core domain, the first leucine zipper domain endblock flanking the N-terminal end of the core sequence, and a second leucine zipper domain endblock flanking the C-terminal end of the core sequence. 
         [0171]    In certain embodiments, the polypeptide consists of the core domain, the first leucine zipper domain endblock flanking the N-terminal end of the core sequence, and a second leucine zipper domain endblock flanking the C-terminal end of the core sequence. 
         [0172]    In certain embodiments, the first leucine zipper domain endblock P domain comprises the peptide represented by SEQ ID NO:3; and the second leucine zipper domain endblock P domain comprises the peptide represented by SEQ ID NO:3. 
         [0173]    In certain embodiments, the first leucine zipper domain endblock P domain comprises the peptide represented by SEQ ID NO:3; and the second leucine zipper domain endblock P domain consists of the peptide represented by SEQ ID NO:3. 
         [0174]    In certain embodiments, the first leucine zipper domain endblock P domain consists of the peptide represented by SEQ ID NO:3; and the second leucine zipper domain endblock P domain comprises the peptide represented by SEQ ID NO:3. 
         [0175]    In certain embodiments, the first leucine zipper domain endblock P domain consists of the peptide represented by SEQ ID NO:3; and the second leucine zipper domain endblock P domain consists of the peptide represented by SEQ ID NO:3. 
         [0176]    In certain embodiments, the polypeptide comprises the sequence represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASDNKTTNTTPSF SFGAKSDENKAGATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKP AFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKPDENKASATSK PAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETS KPAFSFGAKSDEKKDGDASKPAFSFGAKSDEKKDSDSSKPAFSFGTKSNEKKDSGS SKPAFSFGAKPDEKKNDEVSKPAFSFGAKANEKKESDESKSAFSFGSKPTGKEEGD GAKAAISFGAKPEEQKSSDTSKPAFTFGTSAPQMLRELQETNAALQDVRELLRQQV KEITFLKNTVMESDAS (SEQ ID NO:7). 
         [0177]    In certain embodiments, the polypeptide consists of the sequence represented by APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGASDNKTTNTTPSF SFGAKSDENKAGATSKPAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKP AFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDASKPAFSFGAKPDENKASATSK PAFSFGAKPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETS KPAFSFGAKSDEKKDGDASKPAFSFGAKSDEKKDSDSSKPAFSFGTKSNEKKDSGS SKPAFSFGAKPDEKKNDEVSKPAFSFGAKANEKKESDESKSAFSFGSKPTGKEEGD GAKAAISFGAKPEEQKSSDTSKPAFTFGTSAPQMLRELQETNAALQDVRELLRQQV KEITFLKNTVMESDAS (SEQ ID NO:7). 
         [0178]    An aspect of the invention is a nucleic acid molecule encoding a polypeptide of the invention. 
         [0179]    In one embodiment, the nucleic acid molecule encodes a polypeptide represented by SEQ ID NO:4. For example, in one embodiment, a DNA sequence encoding SEQ ID NO:4 is: 
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 18) 
               
               
                 gcgccgcagatgctgcgcgaactgcaggaaaccaacgcggcgctgcagga 
               
               
                   
               
               
                 tgtgcgcgaactgctgcgccagcaggtgaaagaaattacctttctgaaaa 
               
               
                   
               
               
                 acaccgtgatggaaagcgatgcgagcggcgcgagcccggcgtttagcttt 
               
               
                   
               
               
                 ggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtt 
               
               
                   
               
               
                 tagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaac 
               
               
                   
               
               
                 cggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgatacc 
               
               
                   
               
               
                 agcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatag 
               
               
                   
               
               
                 cgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaa 
               
               
                   
               
               
                 aagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggat 
               
               
                   
               
               
                 gaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaa 
               
               
                   
               
               
                 accggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttg 
               
               
                   
               
               
                 gcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaaccagcccg 
               
               
                   
               
               
                 gcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccag 
               
               
                   
               
               
                 caaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcg 
               
               
                   
               
               
                 ataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaa 
               
               
                   
               
               
                 gatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatga 
               
               
                   
               
               
                 aaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaac 
               
               
                   
               
               
                 cggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggc 
               
               
                   
               
               
                 gcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttag 
               
               
                   
               
               
                 ctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccgg 
               
               
                   
               
               
                 cgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagc 
               
               
                   
               
               
                 aaaaccagcgcgccgcagatgctgcgcgaactgcaggaaaccaacgcggc 
               
               
                   
               
               
                 gctgcaggatgtgcgcgaactgctgcgccagcaggtgaaagaaattacct 
               
               
                   
               
               
                 ttctgaaaaacaccgtgatggaaagcgatgcgagc. 
               
             
          
         
       
     
         [0180]    In one embodiment, the nucleic acid molecule encodes a polypeptide represented by SEQ ID NO:5. For example, in one embodiment, a DNA sequence encoding SEQ ID NO:5 is: 
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 19) 
               
               
                 gcgccgcagatgctgcgcgaactgcaggaaaccaacgcggcgctgcagga 
               
               
                   
               
               
                 tgtgcgcgaactgctgcgccagcaggtgaaagaaattacctttctgaaaa 
               
               
                   
               
               
                 acaccgtgatggaaagcgatgcgagcggcgcgagcccggcgtttagcttt 
               
               
                   
               
               
                 ggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtt 
               
               
                   
               
               
                 tagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaac 
               
               
                   
               
               
                 cggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgatacc 
               
               
                   
               
               
                 agcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatga 
               
               
                   
               
               
                 tgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaa 
               
               
                   
               
               
                 aagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggat 
               
               
                   
               
               
                 gaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaa 
               
               
                   
               
               
                 accggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttg 
               
               
                   
               
               
                 gcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaaccagcccg 
               
               
                   
               
               
                 gcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccag 
               
               
                   
               
               
                 caaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatg 
               
               
                   
               
               
                 ataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaa 
               
               
                   
               
               
                 gatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatga 
               
               
                   
               
               
                 aaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaac 
               
               
                   
               
               
                 cggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggc 
               
               
                   
               
               
                 gcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttag 
               
               
                   
               
               
                 ctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccgg 
               
               
                   
               
               
                 cgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagc 
               
               
                   
               
               
                 aaaaccagcgcgccgcagatgctgcgcgaactgcaggaaaccaacgcggc 
               
               
                   
               
               
                 gctgcaggatgtgcgcgaactgctgcgccagcaggtgaaagaaattacct 
               
               
                   
               
               
                 ttctgaaaaacaccgtgatggaaagcgatgcgagc. 
               
             
          
         
       
     
         [0181]    In one embodiment, the nucleic acid molecule encodes a polypeptide represented by SEQ ID NO:6. For example, in one embodiment, a DNA sequence encoding SEQ ID NO:6 is: 
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 20) 
               
               
                 ccgagctttagctttggcgcgaaaagcgatgaaaacaaagcgggcgcgac 
               
               
                   
               
               
                 cagcaaaccggcgtttagctttggcgcgaaaccggaagaaaaaaaagatg 
               
               
                   
               
               
                 ataacagcagcaaaccggcgtttagctttggcgcgaaaagcaacgaagat 
               
               
                   
               
               
                 aaacaggatggcaccgcgaaaccggcgtttagctttggcgcgaaaccggc 
               
               
                   
               
               
                 ggaaaaaaacaacaacgaaaccagcaaaccggcgtttagctttggcgcga 
               
               
                   
               
               
                 aaagcgatgaaaaaaaagatggcgatgcgagcaaaccggcgtttagcttt 
               
               
                   
               
               
                 ggcgcgaaaccggatgaaaacaaagcgagcgcgaccagcaaaccggcgtt 
               
               
                   
               
               
                 tagctttggcgcgaaaccggaagaaaaaaaagatgataacagcagcaaac 
               
               
                   
               
               
                 cggcgtttagctttggcgcgaaaagcaacgaagataaacaggatggcacc 
               
               
                   
               
               
                 gcgaaaccggcgtttagctttggcgcgaaaccggcggaaaaaaacaacaa 
               
               
                   
               
               
                 cgaaaccagcaaaccggcgtttagctttggcgcgaaaagcgatgaaaaaa 
               
               
                   
               
               
                 aagatggcgatgcgagcaaaccggcgtttagctttggcgcgaaaagcgat 
               
               
                   
               
               
                 gaaaaaaaagatagcgatagcagcaaaccggcgtttagctttggcaccaa 
               
               
                   
               
               
                 aagcaacgaaaaaaaagatagcggcagcagcaaaccggcgtttagctttg 
               
               
                   
               
               
                 gcgcgaaaccggatgaaaaaaaaaacgatgaagtgagcaaaccggcgttt 
               
               
                   
               
               
                 agctttggcgcgaaagcgaacgaaaaaaaagaaagcgatgaaagcaaaag 
               
               
                   
               
               
                 cgcgtttagctttggcagcaaaccgaccggcaaagaagaaggcgatggcg 
               
               
                   
               
               
                 cgaaagcggcgattagctttggcgcgaaaccggaagaacagaaaagcagc 
               
               
                   
               
               
                 gataccagcaaa. 
               
             
          
         
       
     
         [0182]    In one embodiment, the nucleic acid molecule encodes a polypeptide represented by SEQ ID NO:7. For example, in one embodiment, a DNA sequence encoding SEQ ID NO: 7 is: 
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 21) 
               
               
                 gcgccgcagatgctgcgcgaactgcaggaaaccaacgcggcgctgcagga 
               
               
                   
               
               
                 tgtgcgcgaactgctgcgccagcaggtgaaagaaattacctttctgaaaa 
               
               
                   
               
               
                 acaccgtgatggaaagcgatgcgagcggcgcgagcgataacaaaaccacc 
               
               
                   
               
               
                 aacaccaccccgagctttagctttggcgcgaaaagcgatgaaaacaaagc 
               
               
                   
               
               
                 gggcgcgaccagcaaaccggcgtttagctttggcgcgaaaccggaagaaa 
               
               
                   
               
               
                 aaaaagatgataacagcagcaaaccggcgtttagctttggcgcgaaaagc 
               
               
                   
               
               
                 aacgaagataaacaggatggcaccgcgaaaccggcgtttagctttggcgc 
               
               
                   
               
               
                 gaaaccggcggaaaaaaacaacaacgaaaccagcaaaccggcgtttagct 
               
               
                   
               
               
                 ttggcgcgaaaagcgatgaaaaaaaagatggcgatgcgagcaaaccggcg 
               
               
                   
               
               
                 tttagctttggcgcgaaaccggatgaaaacaaagcgagcgcgaccagcaa 
               
               
                   
               
               
                 accggcgtttagctttggcgcgaaaccggaagaaaaaaaagatgataaca 
               
               
                   
               
               
                 gcagcaaaccggcgtttagctttggcgcgaaaagcaacgaagataaacag 
               
               
                   
               
               
                 gatggcaccgcgaaaccggcgtttagctttggcgcgaaaccggcggaaaa 
               
               
                   
               
               
                 aaacaacaacgaaaccagcaaaccggcgtttagctttggcgcgaaaagcg 
               
               
                   
               
               
                 atgaaaaaaaagatggcgatgcgagcaaaccggcgtttagctttggcgcg 
               
               
                   
               
               
                 aaaagcgatgaaaaaaaagatagcgatagcagcaaaccggcgtttagctt 
               
               
                   
               
               
                 tggcaccaaaagcaacgaaaaaaaagatagcggcagcagcaaaccggcgt 
               
               
                   
               
               
                 ttagctttggcgcgaaaccggatgaaaaaaaaaacgatgaagtgagcaaa 
               
               
                   
               
               
                 ccggcgtttagctttggcgcgaaagcgaacgaaaaaaaagaaagcgatga 
               
               
                   
               
               
                 aagcaaaagcgcgtttagctttggcagcaaaccgaccggcaaagaagaag 
               
               
                   
               
               
                 gcgatggcgcgaaagcggcgattagctttggcgcgaaaccggaagaacag 
               
               
                   
               
               
                 aaaagcagcgataccagcaaaccggcgtttacctttggcaccagcgcgcc 
               
               
                   
               
               
                 gcagatgctgcgcgaactgcaggaaaccaacgcggcgctgcaggatgtgc 
               
               
                   
               
               
                 gcgaactgctgcgccagcaggtgaaagaaattacctttctgaaaaacacc 
               
               
                   
               
               
                 gtgatggaaagcgatgcgagc. 
               
             
          
         
       
     
         [0183]    In one embodiment, the nucleic acid molecule encodes a polypeptide represented by SEQ ID NO:10 (see below). For example, in one embodiment, a DNA sequence encoding SEQ ID NO:10 is: 
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 22) 
               
               
                 atggatattggcattaacagcgatccgagcaccggcgcgggcgcgtttgg 
               
               
                   
               
               
                 caccggccagagcacctttggctttaacaacagcgcgccgaacaacacca 
               
               
                   
               
               
                 acaacgcgaacagcagcattaccccggcgtttggcagcaacaacaccggc 
               
               
                   
               
               
                 aacaccgcgtttggcaacagcaacccgaccagcaacgtgtttggcagcaa 
               
               
                   
               
               
                 caacagcaccaccaacacctttggcagcaacagcgcgggcaccagcctgt 
               
               
                   
               
               
                 ttggcagcagcagcgcgcagcagaccaaaagcaacggcaccgcgggcggc 
               
               
                   
               
               
                 aacacctttggcagcagcagcctgtttaacaacagcaccaacagcaacac 
               
               
                   
               
               
                 caccaaaccggcgtttggcggcctgaactttggcggcggcaacaacacca 
               
               
                   
               
               
                 ccccgagcagcaccggcaacgcgaacaccagcaacaacctgtttggcgcg 
               
               
                   
               
               
                 accgcgaacgcgaacaaaccggcgtttagctttggcgcgaccaccaacga 
               
               
                   
               
               
                 tgataaaaaaaccgaaccggataaaccggcgtttagctttaacagcagcg 
               
               
                   
               
               
                 tgggcaacaaaaccgatgcgcaggcgccgaccaccggctttagctttggc 
               
               
                   
               
               
                 agccagctgggcggcaacaaaaccgtgaacgaagcggcgaaaccgagcct 
               
               
                   
               
               
                 gagctttggcagcggcagcgcgggcgcgaacccggcgggcgcgagccagc 
               
               
                   
               
               
                 cggaaccgaccaccaacgaaccggcgaaaccggcgctgagctttggcacc 
               
               
                   
               
               
                 gcgaccagcgataacaaaaccaccaacaccaccccgagctttagctttgg 
               
               
                   
               
               
                 cgcgaaaagcgatgaaaacaaagcgggcgcgaccagcaaaccggcgttta 
               
               
                   
               
               
                 gctttggcgcgaaaccggaagaaaaaaaagatgataacagcagcaaaccg 
               
               
                   
               
               
                 gcgtttagctttggcgcgaaaagcaacgaagataaacaggatggcaccgc 
               
               
                   
               
               
                 gaaaccggcgtttagctttggcgcgaaaccggcggaaaaaaacaacaacg 
               
               
                   
               
               
                 aaaccagcaaaccggcgtttagctttggcgcgaaaagcgatgaaaaaaaa 
               
               
                   
               
               
                 gatggcgatgcgagcaaaccggcgtttagctttggcgcgaaaccggatga 
               
               
                   
               
               
                 aaacaaagcgagcgcgaccagcaaaccggcgtttagctttggcgcgaaac 
               
               
                   
               
               
                 cggaagaaaaaaaagatgataacagcagcaaaccggcgtttagctttggc 
               
               
                   
               
               
                 gcgaaaagcaacgaagataaacaggatggcaccgcgaaaccggcgtttag 
               
               
                   
               
               
                 ctttggcgcgaaaccggcggaaaaaaacaacaacgaaaccagcaaaccgg 
               
               
                   
               
               
                 cgtttagctttggcgcgaaaagcgatgaaaaaaaagatggcgatgcgagc 
               
               
                   
               
               
                 aaaccggcgtttagctttggcgcgaaaagcgatgaaaaaaaagatagcga 
               
               
                   
               
               
                 tagcagcaaaccggcgtttagctttggcaccaaaagcaacgaaaaaaaag 
               
               
                   
               
               
                 atagcggcagcagcaaaccggcgtttagctttggcgcgaaaccggatgaa 
               
               
                   
               
               
                 aaaaaaaacgatgaagtgagcaaaccggcgtttagctttggcgcgaaagc 
               
               
                   
               
               
                 gaacgaaaaaaaagaaagcgatgaaagcaaaagcgcgtttagctttggca 
               
               
                   
               
               
                 gcaaaccgaccggcaaagaagaaggcgatggcgcgaaagcggcgattagc 
               
               
                   
               
               
                 tttggcgcgaaaccggaagaacagaaaagcagcgataccagcaaaccggc 
               
               
                   
               
               
                 gtttacctttggcaaactggcggcggcgctggaacatcatcatcatcatc 
               
               
                   
               
               
                 at. 
               
             
          
         
       
     
         [0184]    In one embodiment, the nucleic acid molecule encodes a polypeptide represented by SEQ ID NO:11 (see below). For example, in one embodiment, a DNA sequence encoding SEQ ID NO:11 is: 
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 23) 
               
               
                 atggatattggcattaacagcgatccgggcagcggcagcggcgcgagcg 
               
               
                   
               
               
                 ataacaaaaccaccaacaccaccccgagctttagctttggcgcgaaaag 
               
               
                   
               
               
                 cgatgaaaacaaagcgggcgcgaccagcaaaccggcgtttagctttggc 
               
               
                   
               
               
                 gcgaaaccggaagaaaaaaaagatgataacagcagcaaaccggcgttta 
               
               
                   
               
               
                 gctttggcgcgaaaagcaacgaagataaacaggatggcaccgcgaaacc 
               
               
                   
               
               
                 ggcgtttagctttggcgcgaaaccggcggaaaaaaacaacaacgaaacc 
               
               
                   
               
               
                 agcaaaccggcgtttagctttggcgcgaaaagcgatgaaaaaaaagatg 
               
               
                   
               
               
                 gcgatgcgagcaaaccggcgtttagctttggcgcgaaaccggatgaaaa 
               
               
                   
               
               
                 caaagcgagcgcgaccagcaaaccggcgtttagctttggcgcgaaaccg 
               
               
                   
               
               
                 gaagaaaaaaaagatgataacagcagcaaaccggcgtttagctttggcg 
               
               
                   
               
               
                 cgaaaagcaacgaagataaacaggatggcaccgcgaaaccggcgtttag 
               
               
                   
               
               
                 ctttggcgcgaaaccggcggaaaaaaacaacaacgaaaccagcaaaccg 
               
               
                   
               
               
                 gcgtttagattggcgcgaaaagcgatgaaaaaaaagatggcgatgcgag 
               
               
                   
               
               
                 caaaccggcgtttagattggcgcgaaaagcgatgaaaaaaaagatagcg 
               
               
                   
               
               
                 atagcagcaaaccggcgtttagattggcaccaaaagcaacgaaaaaaaa 
               
               
                   
               
               
                 gatagcggcagcagcaaaccggcgtttagctttggcgcgaaaccggatg 
               
               
                   
               
               
                 aaaaaaaaaacgatgaagtgagcaaaccggcgtttagctttggcgcgaa 
               
               
                   
               
               
                 agcgaacgaaaaaaaagaaagcgatgaaagcaaaagcgcgtttagcttt 
               
               
                   
               
               
                 ggcagcaaaccgaccggcaaagaagaaggcgatggcgcgaaagcggcga 
               
               
                   
               
               
                 ttagattggcgcgaaaccggaagaacagaaaagcagcgataccagcaaa 
               
               
                   
               
               
                 ccggcgtttacctttggcaccagcggcagcggcaaactggcggcggcgc 
               
               
                   
               
               
                 tggaacatcatcatcatcatcat. 
               
             
          
         
       
     
         [0185]    In one embodiment, the nucleic acid molecule encodes a polypeptide represented by SEQ ID NO:12 (see below). For example, in one embodiment, a DNA sequence encoding SEQ ID NO:12 is: 
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 24) 
               
               
                 atggatattggcattaacagcgatccggcgccgcagatgctgcgcgaact 
               
               
                   
               
               
                 gcaggaaaccaacgcggcgctgcaggatgtgcgcgaactgctgcgccagc 
               
               
                   
               
               
                 aggtgaaagaaattacctttctgaaaaacaccgtgatggaaagcgatgcg 
               
               
                   
               
               
                 agcggcgcgagcgcgattagcggcgatagcctgattagcctggcgagcac 
               
               
                   
               
               
                 cggcaaacgcgtgagcattaaagatctgctggatgaaaaagattttgaaa 
               
               
                   
               
               
                 tttgggcgattaacgaacagaccatgaaactggaaagcgcgaaagtgagc 
               
               
                   
               
               
                 cgcgtgttttgcaccggcaaaaaactggtgtatattctgaaaacccgcct 
               
               
                   
               
               
                 gggccgcaccattaaagcgaccgcgaaccatcgctttctgaccattgatg 
               
               
                   
               
               
                 gctggaaacgcctggatgaactgagcctgaaagaacatattgcgctgccg 
               
               
                   
               
               
                 cgcaaactggaaagcagcagcctgcagctgagcccggaaattgaaaaact 
               
               
                   
               
               
                 gagccagagcgatatttattgggatagcattgtgagcattaccgaaaccg 
               
               
                   
               
               
                 gcgtggaagaagtgtttgatctgaccgtgccgggcccgcataactttgtg 
               
               
                   
               
               
                 gcgaacgatattattgtgcataacgcgagcgataacaaaaccaccaacac 
               
               
                   
               
               
                 caccccgagctttagctttggcgcgaaaagcgatgaaaacaaagcgggcg 
               
               
                   
               
               
                 cgaccagcaaaccggcgtttagctttggcgcgaaaccggaagaaaaaaaa 
               
               
                   
               
               
                 gatgataacagcagcaaaccggcgtttagctttggcgcgaaaagcaacga 
               
               
                   
               
               
                 agataaacaggatggcaccgcgaaaccggcgtttagctttggcgcgaaac 
               
               
                   
               
               
                 cggcggaaaaaaacaacaacgaaaccagcaaaccggcgtttagctttggc 
               
               
                   
               
               
                 gcgaaaagcgatgaaaaaaaagatggcgatgcgagcaaaccggcgtttag 
               
               
                   
               
               
                 ctttggcgcgaaaccggatgaaaacaaagcgagcgcgaccagcaaaccgg 
               
               
                   
               
               
                 cgtttagctttggcgcgaaaccggaagaaaaaaaagatgataacagcagc 
               
               
                   
               
               
                 aaaccggcgtttagctttggcgcgaaaagcaacgaagataaacaggatgg 
               
               
                   
               
               
                 caccgcgaaaccggcgtttagctttggcgcgaaaccggcggaaaaaaaca 
               
               
                   
               
               
                 acaacgaaaccagcaaaccggcgtttagctttggcgcgaaaagcgatgaa 
               
               
                   
               
               
                 aaaaaagatggcgatgcgagcaaaccggcgtttagctttggcgcgaaaag 
               
               
                   
               
               
                 cgatgaaaaaaaagatagcgatagcagcaaaccggcgtttagctttggca 
               
               
                   
               
               
                 ccaaaagcaacgaaaaaaaagatagcggcagcagcaaaccggcgtttagc 
               
               
                   
               
               
                 tttggcgcgaaaccggatgaaaaaaaaaacgatgaagtgagcaaaccggc 
               
               
                   
               
               
                 gtttagctttggcgcgaaagcgaacgaaaaaaaagaaagcgatgaaagca 
               
               
                   
               
               
                 aaagcgcgtttagctttggcagcaaaccgaccggcaaagaagaaggcgat 
               
               
                   
               
               
                 ggcgcgaaagcggcgattagctttggcgcgaaaccggaagaacagaaaag 
               
               
                   
               
               
                 cagcgataccagcaaaccggcgtttacctttggcaccagcggcagcggca 
               
               
                   
               
               
                 aactggcggcggcgctggaacatcatcatcatcatcat. 
               
             
          
         
       
     
         [0186]    In one embodiment, the nucleic acid molecule encodes a polypeptide represented by SEQ ID NO:13 (see below). For example, in one embodiment, a DNA sequence encoding SEQ ID NO:13 is: 
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 25) 
               
               
                 atggatattggcattaacagcgatccgggcagcggcgcgagcccggcgtt 
               
               
                   
               
               
                 tagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaac 
               
               
                   
               
               
                 cggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgatacc 
               
               
                   
               
               
                 agcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatga 
               
               
                   
               
               
                 tgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaa 
               
               
                   
               
               
                 aagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggat 
               
               
                   
               
               
                 gaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaa 
               
               
                   
               
               
                 accggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttg 
               
               
                   
               
               
                 gcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgttt 
               
               
                   
               
               
                 agctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaac 
               
               
                   
               
               
                 cagcccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatg 
               
               
                   
               
               
                 ataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaa 
               
               
                   
               
               
                 gatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatga 
               
               
                   
               
               
                 aaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaac 
               
               
                   
               
               
                 cggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggc 
               
               
                   
               
               
                 gcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttag 
               
               
                   
               
               
                 ctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccgg 
               
               
                   
               
               
                 cgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagc 
               
               
                   
               
               
                 aaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatga 
               
               
                   
               
               
                 taccagcaaaaccagcggcagcggcaaactggcggcggcgctggaacatc 
               
               
                   
               
               
                 atcatcatcatcat. 
               
             
          
         
       
     
         [0187]    In one embodiment, the nucleic acid molecule encodes a polypeptide represented by SEQ ID NO:14 (see below). For example, in one embodiment, a DNA sequence encoding SEQ ID NO:14 is: 
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 26) 
               
               
                 atggatattggcattaacagcgatccgggcagcggcgcgagcccggcgtt 
               
               
                   
               
               
                 tagctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaac 
               
               
                   
               
               
                 cggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgatacc 
               
               
                   
               
               
                 agcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatag 
               
               
                   
               
               
                 cgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaa 
               
               
                   
               
               
                 aagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggat 
               
               
                   
               
               
                 gaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaa 
               
               
                   
               
               
                 accggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttg 
               
               
                   
               
               
                 gcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgttt 
               
               
                   
               
               
                 agctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaac 
               
               
                   
               
               
                 cagcccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcg 
               
               
                   
               
               
                 ataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaa 
               
               
                   
               
               
                 gatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatga 
               
               
                   
               
               
                 aaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaac 
               
               
                   
               
               
                 cggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggc 
               
               
                   
               
               
                 gcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttag 
               
               
                   
               
               
                 ctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccgg 
               
               
                   
               
               
                 cgtttagattggcgcgaaaccggatgaaaaaaaagatagcgataccagca 
               
               
                   
               
               
                 aaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgat 
               
               
                   
               
               
                 accagcaaaaccagcaaaaccagcggcagcggcaaactggcggcggcgct 
               
               
                   
               
               
                 ggaacatcatcatcatcatcat. 
               
             
          
         
       
     
         [0188]    In one embodiment, the nucleic acid molecule encodes a polypeptide represented by SEQ ID NO:15 (see below). For example, in one embodiment, a DNA sequence encoding SEQ ID NO:15 is: 
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 27) 
               
               
                 atggatattggcattaacagcgatccggcgccgcagatgctgcgcgaact 
               
               
                   
               
               
                 gcaggaaaccaacgcggcgctgcaggatgtgcgcgaactgctgcgccagc 
               
               
                   
               
               
                 aggtgaaagaaattacctttctgaaaaacaccgtgatggaaagcgatgcg 
               
               
                   
               
               
                 agcggcgcgagcgataacaaaaccaccaacaccaccccgagctttagctt 
               
               
                   
               
               
                 tggcgcgaaaagcgatgaaaacaaagcgggcgcgaccagcaaaccggcgt 
               
               
                   
               
               
                 ttagctttggcgcgaaaccggaagaaaaaaaagatgataacagcagcaaa 
               
               
                   
               
               
                 ccggcgtttagctttggcgcgaaaagcaacgaagataaacaggatggcac 
               
               
                   
               
               
                 cgcgaaaccggcgtttagctttggcgcgaaaccggcggaaaaaaacaaca 
               
               
                   
               
               
                 acgaaaccagcaaaccggcgtttagctttggcgcgaaaagcgatgaaaaa 
               
               
                   
               
               
                 aaagatggcgatgcgagcaaaccggcgtttagctttggcgcgaaaccgga 
               
               
                   
               
               
                 tgaaaacaaagcgagcgcgaccagcaaaccggcgtttagctttggcgcga 
               
               
                   
               
               
                 aaccggaagaaaaaaaagatgataacagcagcaaaccggcgtttagattg 
               
               
                   
               
               
                 gcgcgaaaagcaacgaagataaacaggatggcaccgcgaaaccggcgttt 
               
               
                   
               
               
                 agctttggcgcgaaaccggcggaaaaaaacaacaacgaaaccagcaaacc 
               
               
                   
               
               
                 ggcgtttagctttggcgcgaaaagcgatgaaaaaaaagatggcgatgcga 
               
               
                   
               
               
                 gcaaaccggcgtttagctttggcgcgaaaagcgatgaaaaaaaagatagc 
               
               
                   
               
               
                 gatagcagcaaaccggcgtttagctttggcaccaaaagcaacgaaaaaaa 
               
               
                   
               
               
                 agatagcggcagcagcaaaccggcgtttagctttggcgcgaaaccggatg 
               
               
                   
               
               
                 aaaaaaaaaacgatgaagtgagcaaaccggcgtttagctttggcgcgaaa 
               
               
                   
               
               
                 gcgaacgaaaaaaaagaaagcgatgaaagcaaaagcgcgtttagctttgg 
               
               
                   
               
               
                 cagcaaaccgaccggcaaagaagaaggcgatggcgcgaaagcggcgatta 
               
               
                   
               
               
                 gctttggcgcgaaaccggaagaacagaaaagcagcgataccagcaaaccg 
               
               
                   
               
               
                 gcgtttacctttggcaccagcgcgccgcagatgctgcgcgaactgcagga 
               
               
                   
               
               
                 aaccaacgcggcgctgcaggatgtgcgcgaactgctgcgccagcaggtga 
               
               
                   
               
               
                 aagaaattacctttctgaaaaacaccgtgatggaaagcgatgcgagcggc 
               
               
                   
               
               
                 aaactggcggcggcgctggaacatcatcatcatcatcat. 
               
             
          
         
       
     
         [0189]    In one embodiment, the nucleic acid molecule encodes a polypeptide represented by SEQ ID NO:16 (see below). For example, in one embodiment, a DNA sequence encoding SEQ ID NO:16 is: 
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 28) 
               
               
                 atggatattggcattaacagcgatccggcgccgcagatgctgcgcgaact 
               
               
                   
               
               
                 gcaggaaaccaacgcggcgctgcaggatgtgcgcgaactgctgcgccagc 
               
               
                   
               
               
                 aggtgaaagaaattacctttctgaaaaacaccgtgatggaaagcgatgcg 
               
               
                   
               
               
                 agcggcgcgagcccggcgtttagctttggcgcgaaaccggatgaaaaaaa 
               
               
                   
               
               
                 agatgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatg 
               
               
                   
               
               
                 aaaaaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaa 
               
               
                   
               
               
                 ccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttgg 
               
               
                   
               
               
                 cgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgttta 
               
               
                   
               
               
                 gctttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccg 
               
               
                   
               
               
                 gcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccag 
               
               
                   
               
               
                 caaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatg 
               
               
                   
               
               
                 ataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaa 
               
               
                   
               
               
                 gatgatgataccagcaaaaccagcccggcgtttagctttggcgcgaaacc 
               
               
                   
               
               
                 ggatgaaaaaaaagatgatgataccagcaaaccggcgtttagctttggcg 
               
               
                   
               
               
                 cgaaaccggatgaaaaaaaagatgatgataccagcaaaccggcgtttagc 
               
               
                   
               
               
                 tttggcgcgaaaccggatgaaaaaaaagatgatgataccagcaaaccggc 
               
               
                   
               
               
                 gtttagctttggcgcgaaaccggatgaaaaaaaagatgatgataccagca 
               
               
                   
               
               
                 aaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatgatgat 
               
               
                   
               
               
                 accagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaaga 
               
               
                   
               
               
                 tgatgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaa 
               
               
                   
               
               
                 aaaaagatgatgataccagcaaaccggcgtttagctttggcgcgaaaccg 
               
               
                   
               
               
                 gatgaaaaaaaagatgatgataccagcaaaaccagcgcgccgcagatgct 
               
               
                   
               
               
                 gcgcgaactgcaggaaaccaacgcggcgctgcaggatgtgcgcgaactgc 
               
               
                   
               
               
                 tgcgccagcaggtgaaagaaattacctttctgaaaaacaccgtgatggaa 
               
               
                   
               
               
                 agcgatgcgagcggcggcaaactggcggcggcgctggaacatcatcatca 
               
               
                   
               
               
                 tcatcat. 
               
             
          
         
       
     
         [0190]    In one embodiment, the nucleic acid molecule encodes a polypeptide represented by SEQ ID NO:17 (see below). For example, in one embodiment, a DNA sequence encoding SEQ ID NO:17 is: 
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 29) 
               
               
                 atggatattggcattaacagcgatccggcgccgcagatgctgcgcgaact 
               
               
                   
               
               
                 gcaggaaaccaacgcggcgctgcaggatgtgcgcgaactgctgcgccagc 
               
               
                   
               
               
                 aggtgaaagaaattacctttctgaaaaacaccgtgatggaaagcgatgcg 
               
               
                   
               
               
                 agcggcgcgagcccggcgtttagctttggcgcgaaaccggatgaaaaaaa 
               
               
                   
               
               
                 agatagcgataccagcaaaccggcgtttagctttggcgcgaaaccggatg 
               
               
                   
               
               
                 aaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgcgaaa 
               
               
                   
               
               
                 ccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttgg 
               
               
                   
               
               
                 cgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgttta 
               
               
                   
               
               
                 gctttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccg 
               
               
                   
               
               
                 gcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgataccag 
               
               
                   
               
               
                 caaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcg 
               
               
                   
               
               
                 ataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaa 
               
               
                   
               
               
                 gatagcgataccagcaaaaccagcccggcgtttagattggcgcgaaaccg 
               
               
                   
               
               
                 gatgaaaaaaaagatagcgataccagcaaaccggcgtttagctttggcgc 
               
               
                   
               
               
                 gaaaccggatgaaaaaaaagatagcgataccagcaaaccggcgtttagct 
               
               
                   
               
               
                 ttggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaaccggcg 
               
               
                   
               
               
                 tttagattggcgcgaaaccggatgaaaaaaaagatagcgataccagcaaa 
               
               
                   
               
               
                 ccggcgtttagctttggcgcgaaaccggatgaaaaaaaagatagcgatac 
               
               
                   
               
               
                 cagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaaaaagata 
               
               
                   
               
               
                 gcgataccagcaaaccggcgtttagctttggcgcgaaaccggatgaaaaa 
               
               
                   
               
               
                 aaagatagcgataccagcaaaccggcgtttagctttggcgcgaaaccgga 
               
               
                   
               
               
                 tgaaaaaaaagatagcgataccagcaaaaccagcgcgccgcagatgctgc 
               
               
                   
               
               
                 gcgaactgcaggaaaccaacgcggcgctgcaggatgtgcgcgaactgctg 
               
               
                   
               
               
                 cgccagcaggtgaaagaaattacctttctgaaaaacaccgtgatggaaag 
               
               
                   
               
               
                 cgatgcgagcggcggcaaactggcggcggcgctggaacatcatcatcatc 
               
               
                   
               
               
                 atcat. 
               
             
          
         
       
     
         [0191]    An aspect of the invention is an expression vector comprising a nucleic acid molecule of the invention. 
         [0192]    As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a plasmid, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors, namely expression vectors, are capable of directing the expression of genes to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, the present invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. 
         [0193]    The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell. This means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel,  Methods in Enzymology: Gene Expression Technology  vol. 185, Academic Press, San Diego, Calif. (1991). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including proteins or polypeptides, encoded by nucleic acids as described herein. 
         [0194]    The recombinant expression vectors for use in the invention can be designed for expression of a polypeptide of the invention in prokaryotic (e.g.,  E. coli ) or eukaryotic cells (e.g., insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells). Suitable host cells are discussed further in Goeddel, supra. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase. 
         [0195]    Expression of proteins in prokaryotes is most often carried out in  E. coli  with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988 , Gene  67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. 
         [0196]    Examples of suitable inducible non-fusion  E. coli  expression vectors include pTrc (Amann et al., 1988 , Gene  69:301-315) and pET 11d (Studier et al., p. 60-89, In  Gene Expression Technology: Methods in Enzymology  vol. 185, Academic Press, San Diego, Calif., 1991). Target biomarker nucleic acid expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target biomarker nucleic acid expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a co-expressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21 (DE3) or HMS174(DE3) from a resident prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter. 
         [0197]    One strategy to maximize recombinant protein expression in  E. coli  is to express the protein in a host bacterium with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, p. 119-128, In  Gene Expression Technology: Methods in Enzymology  vol. 185, Academic Press, San Diego, Calif., 1990). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in  E. coli  (Wada et al., 1992 , Nucleic Acids Res.  20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques. 
         [0198]    In another embodiment, the expression vector is a yeast expression vector. Examples of vectors for expression in yeast  S. cerevisiae  include pYepSec1 (Baldari et al., 1987 , EMBO J.  6:229-234), pMFa (Kurjan and Herskowitz, 1982 , Cell  30:933-943), pJRY88 (Schultz et al., 1987 , Gene  54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and pPicZ (Invitrogen Corp, San Diego, Calif.). 
         [0199]    Alternatively, the expression vector is a baculovirus expression vector. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al., 1983 , Mol. Cell Biol.  3:2156-2165) and the pVL series (Lucklow and Summers, 1989 , Virology  170:31-39). 
         [0200]    In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987 , Nature  329:840) and pMT2PC (Kaufman et al., 1987 , EMBO J.  6:187-195). When used in mammalian cells, the expression vector&#39;s control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus 40 (SV40). For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook et al., ed.,  Molecular Cloning: A Laboratory Manual,  2 nd ed ., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. 
         [0201]    In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al., 1987 , Genes Dev.  1:268-277), lymphoid-specific promoters (Calame and Eaton, 1988 , Adv. Immunol.  43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989 , EMBO J.  8:729-733) and immunoglobulins (Banerji et al., 1983 , Cell  33:729-740; Queen and Baltimore, 1983 , Cell  33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989 , Proc. Natl. Acad. Sci. USA  86:5473-5477), pancreas-specific promoters (Edlund et al., 1985 , Science  230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss, 1990 , Science  249:374-379) and the α-fetoprotein promoter (Camper and Tilghman, 1989 , Genes Dev.  3:537-546). 
         [0202]    In certain embodiments, the expression vector is a prokaryotic expression vector. 
         [0203]    In certain embodiments, the expression vector is a eukaryotic expression vector. 
         [0204]    An aspect of the invention is a cell comprising an expression vector of the invention. 
         [0205]    In certain embodiments, the expression vector is a prokaryotic expression vector, and the cell is a prokaryotic cell, e.g.,  E. coli.    
         [0206]    In certain embodiments, the expression vector is a eukaryotic expression vector, and the cell is a eukaryotic cell, e.g., a yeast cell, an insect cell, or a mammalian cell. 
         [0207]    Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals. 
         [0208]    For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die). 
         [0209]    Compositions of the Invention 
         [0210]    An aspect of the invention is a hydrogel, comprising a polypeptide of the invention. 
         [0211]    An aspect of the invention is a filtering device, comprising a hydrogel of the invention; and a housing or support for the hydrogel. In certain embodiments, the hydrogel is disposed within a housing. In certain embodiments, the hydrogel is disposed within and is in contact with the housing. The housing can be configured in any suitable manner for the intended use of the filtering device, e.g., as a plate, a cone, a manifold, a cartridge, an open-ended tube, a closed-end tube (e.g., a centrifuge tube), etc. The housing optionally can include a fitting, e.g., a screw- or compression-Luer lock fitting, to facilitate connection of the filtering device to a fluid path. In certain embodiments, the hydrogel is disposed upon a support. In certain embodiments, the hydrogel is disposed upon and is in contact with the support. The support can be configured in any suitable manner for the intended use of the filtering device, e.g., as a frame, a perforated plate, a mesh, a fabric, a filter, etc. 
         [0212]    In certain embodiments, the support, together with the hydrogel, can be fitted or placed into a housing which is configured and arranged to receive the support. 
         [0213]    An aspect of the invention is a drug delivery device, comprising a drug; and a hydrogel of the invention. 
         [0214]    In certain embodiments, the drug is a macromolecule. 
         [0215]    In certain embodiments, the drug is a nucleic acid. 
         [0216]    In certain embodiments, the drug is an RNA. 
         [0217]    In certain embodiments, the drug is a DNA. 
         [0218]    In certain embodiments, the drug is a polymer. 
         [0219]    In certain embodiments, the drug is a polypeptide. 
         [0220]    In certain embodiments, the drug is a protein. 
         [0221]    In certain embodiments, the drug is a fusion protein. 
         [0222]    In certain embodiments, the drug is an antibody or an antigen-binding fragment of an antibody. 
         [0223]    In certain embodiments, the drug is a cytokine. 
         [0224]    In certain embodiments, the drug is a glycoprotein. 
         [0225]    In certain embodiments, the drug is a carbohydrate. 
         [0226]    In certain embodiments, the drug is a lipid. 
         [0227]    In certain embodiments, the drug is a toxin. 
         [0228]    In certain embodiments, the drug is a steroid. 
         [0229]    In certain embodiments, the drug is a hormone, e.g., an estrogen or a progestogen. 
         [0230]    In certain embodiments, the drug is dispersed within the hydrogel. 
         [0231]    In certain embodiments, the drug is effectively enveloped by the hydrogel. For example, the drug can be enclosed within a housing, wherein at least a portion of the housing is open to the environment but for the presence of the hydrogel. 
         [0232]    In certain embodiments, the drug is enveloped by the hydrogel. For example, the drug can be present as a core which is completely surrounded by the hydrogel; the core can include more than a single active agent, and there can be one or more additional layers present between the drug or core and the hydrogel. In certain embodiments, there can be one or more additional layers external to the hydrogel. 
         [0233]    Methods of the Invention 
         [0234]    An aspect of the invention is a method of separating or selectively filtering macromolecules, comprising contacting a source of macromolecules with a hydrogel of the invention. For example, a solution comprising a macromolecule can be contacted with a hydrogel of the invention. 
         [0235]    As used herein, the term “macromolecule” refers to any molecule having a molecular weight greater than about 1500 Da. In certain embodiments, a macromolecule has a molecular weight greater than or equal to about 10,000 Da. In certain embodiments, a macromolecule has a molecular weight greater than or equal to about 20,000 Da. In certain embodiments, a macromolecule has a molecular weight greater than or equal to about 30,000 Da. In certain embodiments, a macromolecule has a molecular weight greater than or equal to about 40,000 Da. In certain embodiments, a macromolecule has a molecular weight greater than or equal to about 50,000 Da. In certain embodiments, a macromolecule has a molecular weight greater than or equal to about 60,000 Da. In certain embodiments, a macromolecule has a molecular weight greater than or equal to about 70,000 Da. In certain embodiments, a macromolecule has a molecular weight greater than or equal to about 80,000 Da. In certain embodiments, a macromolecule has a molecular weight greater than or equal to about 90,000 Da. In certain embodiments, a macromolecule has a molecular weight greater than or equal to about 100,000 Da. 
         [0236]    In certain embodiments, the macromolecule is a naturally occurring macromolecule. In certain embodiments, the macromolecule is a synthetic or semi-synthetic macromolecule. In certain embodiments, the macromolecule is present as part of a complex or conjugate with another molecule, e.g., a karyopherin. 
         [0237]    In certain embodiments, the macromolecule is selected from the group consisting of RNA, mRNA, DNA, proteins, glycoproteins, carbohydrates, lipids, toxins, and any combination thereof. 
         [0238]    In certain embodiments, the macromolecule is RNA. 
         [0239]    In certain embodiments, the macromolecule is mRNA. 
         [0240]    In certain embodiments, the macromolecule is DNA. 
         [0241]    In certain embodiments, the macromolecule is a protein. 
         [0242]    In certain embodiments, the macromolecule is a glycoprotein. 
         [0243]    In certain embodiments, the macromolecule is a carbohydrate. 
         [0244]    In certain embodiments, the macromolecule is a lipid. 
         [0245]    In certain embodiments, the macromolecule is a toxin. 
         [0246]    Having described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention. 
       EXAMPLES 
     Materials and Methods 
       [0247]    DNA Engineering: 
         [0248]    The Nsp1 30-591  gene from the Chait group was amplified by polymerase chain reaction (PCR) to prepare BamHI (B)-NheI (N)-Nsp1 30-591 -SpeI (S)-HindIII (H) and B-N-cNsp1 (Nsp1 282-585 )-S-H. Then, both DNA fragments were subcloned into the pET22b expression plasmid (C-terminal 6×His tag) using BamHI and HindIII restriction sites. The pET22b vector was chosen due to the C-terminal 6×His tag which allows isolation of full length proteins by metal affinity chromatography. B-N-1NLP-S-H, B-N-2NLP-S-H, B-P-EcoRI-intein-N-cNsp1-S-H, B-P domain (P)-N-cNsp1-S-P-H, B-P-N-1NLP-S-P-H and B-P-N-2NLP-S-P-H were also prepared. Gene sequences of B-P domain (P)-N-1NLP 1/2 -S-P-H and B-P-N-2NLP 1/2 -S-P-H were purchased (GenScript, USA) and subcloned into the pET22b expression plasmid using BamHI and HindIII restriction sites. To prepare P-1NLP-P and P-2NLP-P, N-1NLP 1/2 -S and N-2NLP 1/2 -S were subcloned into pET22b-B-P-N-1NLP 1/2 -S-P-H and pET22b-B-P-N-2NLP 1/2 -S-P-H plasmids using the SpeI restriction enzyme site. P-cNsp1-P was prepared by subcloning N-cNsp1-S into the pET22b-B-P-N-1NLP 1/2 -S-P-H plasmid using NheI and SpeI restriction sites. 1NLP and 2NLP were prepared by subcloning N-1NLP-S and N-2NLP-S from pET22b-B-P-N-1NLP-S-P-H and pET22b-B-P-N-2NLP-S-P-H plasmids into pET22b-B-P-N-1NLP 1/2 -S-P-H and pET22b-B-P-N-2NLP 1/2 -S-P-H plasmids using NheI and SpeI restriction enzyme sites. B-P-EcoRI-intein-N sequences were purchased (Integrated DNA Technologies, USA) and subcloned into the pET22b-B-N-cNsp1-S-H plasmid using BamHI and NheI restriction enzyme sites. Sequences of all plasmids were confirmed by gene sequencing (Genewiz, USA). Prepared protein sequences are as follows. 
         [0000]    
       
         
               
             
           
               
                 Nsp1 30-591   
               
               
                 (SEQ ID NO: 10)  
               
               
                 MDIGINSDPSTGAGAFGTGQSTFGFNNSAPNNTNNANSSITPAFGSNNTG 
               
               
                   
               
               
                 NTAFGNSNPTSNVFGSNNSTTNTFGSNSAGTSLFGSSSAQQTKSNGTAGG  
               
               
                   
               
               
                 NTFGSSSLFNNSTNSNTTKPAFGGLNFGGGNNTTPSSTGNANTSNNLFGA 
               
               
                   
               
               
                 TANANKPAFSFGATTNDDKKTEPDKPAFSFNSSVGNKTDAQAPTTGFSFG 
               
               
                   
               
               
                 SQLGGNKTVNEAAKPSLSFGSGSAGANPAGASQPEPTTNEPAKPALSFGT 
               
               
                   
               
               
                 ATSDNKTTNTTPSFSFGAKSDENKAGATSKPAFSFGAKPEEKKDDNSSKP 
               
               
                   
               
               
                 AFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKK 
               
               
                   
               
               
                 DGDASKPAFSFGAKPDENKASATSKPAFSFGAKPEEKKDDNSSKPAFSFG 
               
               
                   
               
               
                 AKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDAS 
               
               
                   
               
               
                 KPAFSFGAKSDEKKDSDSSKPAFSFGTKSNEKKDSGSSKPAFSFGAKPDE 
               
               
                   
               
               
                 KKNDEVSKPAFSFGAKANEKKESDESKSAFSFGSKPTGKEEGDGAKAAIS 
               
               
                   
               
               
                 FGAKPEEQKSSDTSKPAFTFGKLAAALEHHHHHH 
               
               
                   
               
               
                 cNsp1 
               
               
                 (SEQ ID NO: 11)  
               
               
                 MDIGINSDPGSGSGASDNKTTNTTPSFSFGAKSDENKAGATSKPAFSFGA  
               
               
                   
               
               
                 KPEEKKDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSK 
               
               
                   
               
               
                 PAFSFGAKSDEKKDGDASKPAFSFGAKPDENKASATSKPAFSFGAKPEEK 
               
               
                   
               
               
                 KDDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSF 
               
               
                   
               
               
                 GAKSDEKKDGDASKPAFSFGAKSDEKKDSDSSKPAFSFGTKSNEKKDSGS 
               
               
                   
               
               
                 SKPAFSFGAKPDEKKNDEVSKPAFSFGAKANEKKESDESKSAFSFGSKPT 
               
               
                   
               
               
                 GKEEGDGAKAAISFGAKPEEQKSSDTSKPAFTFGTSGSGKLAAALEHHHH 
               
               
                   
               
               
                 HH 
               
               
                   
               
               
                 P-Intein-cNsp1 
               
               
                 (SEQ ID NO: 12)  
               
               
                 MDIGINSDPAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDA 
               
               
                   
               
               
                 SGASAISGDSLISLASTGKRVSIKDLLDEKDFEIWAINEQTMKLESAKVS  
               
               
                   
               
               
                 RVFCTGKKLVYILKTRLGRTIKATANHRFLTIDGWKRLDELSLKEHIALP 
               
               
                   
               
               
                 RKLESSSLQLSPEIEKLSQSDIYWDSIVSITETGVEEVFDLTVPGPHNFV 
               
               
                   
               
               
                 ANDIIVHNASDNKTTNTTPSFSFGAKSDENKAGATSKPAFSFGAKPEEKK 
               
               
                   
               
               
                 DDNSSKPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFG 
               
               
                   
               
               
                 AKSDEKKDGDASKPAFSFGAKPDENKASATSKPAFSFGAKPEEKKDDNSS 
               
               
                   
               
               
                 KPAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDE 
               
               
                   
               
               
                 KKDGDASKPAFSFGAKSDEKKDSDSSKPAFSFGTKSNEKKDSGSSKPAFS 
               
               
                   
               
               
                 FGAKPDEKKNDEVSKPAFSFGAKANEKKESDESKSAFSFGSKPTGKEEGD 
               
               
                   
               
               
                 GAKAAISFGAKPEEQKSSDTSKPAFTFGTSGSGKLAAALEHHHHHH 
               
               
                   
               
               
                 1NLP 
               
               
                 (SEQ ID NO: 13)  
               
               
                 MDIGINSDPGSGASPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDT 
               
               
                   
               
               
                 SKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPD  
               
               
                   
               
               
                 EKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAF  
               
               
                   
               
               
                 SFGAKPDEKKDDDTSKTSPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKK 
               
               
                   
               
               
                 DDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFG 
               
               
                   
               
               
                 AKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTS 
               
               
                   
               
               
                 KPAFSFGAKPDEKKDDDTSKTSGSGKLAAALEHHHHHH  
               
               
                   
               
               
                 2NLP 
               
               
                 (SEQ ID NO: 14)  
               
               
                 MDIGINSDPGSGASPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDT 
               
               
                   
               
               
                 SKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPD 
               
               
                   
               
               
                 EKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAF 
               
               
                   
               
               
                 SFGAKPDEKKDSDTSKTSPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKK 
               
               
                   
               
               
                 DSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFG 
               
               
                   
               
               
                 AKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTS 
               
               
                   
               
               
                 KPAFSFGAKPDEKKDSDTSKTSKTSGSGKLAAALEHHHHHH  
               
               
                   
               
               
                 P-cNsp1-P 
               
               
                 (SEQ ID NO: 15)  
               
               
                 MDIGINSDPAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDA 
               
               
                   
               
               
                 SGASDNKTTNTTPSFSFGAKSDENKAGATSKPAFSFGAKPEEKKDDNSSK 
               
               
                   
               
               
                 PAFSFGAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEK  
               
               
                   
               
               
                 KDGDASKPAFSFGAKPDENKASATSKPAFSFGAKPEEKKDDNSSKPAFSF 
               
               
                   
               
               
                 GAKSNEDKQDGTAKPAFSFGAKPAEKNNNETSKPAFSFGAKSDEKKDGDA 
               
               
                   
               
               
                 SKPAFSFGAKSDEKKDSDSSKPAFSFGTKSNEKKDSGSSKPAFSFGAKPD 
               
               
                   
               
               
                 EKKNDEVSKPAFSFGAKANEKKESDESKSAFSFGSKPTGKEEGDGAKAAI 
               
               
                   
               
               
                 SFGAKPEEQKSSDTSKPAFTFGTSAPQMLRELQETNAALQDVRELLRQQV 
               
               
                   
               
               
                 KEITFLKNTVMESDASGKLAAALEHHHHHH  
               
               
                   
               
               
                 P-1NLP-P 
               
               
                 (SEQ ID NO: 16)  
               
               
                 MDIGINSDPAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDA  
               
               
                   
               
               
                 SGASPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAK 
               
               
                   
               
               
                 PDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKP  
               
               
                   
               
               
                 AFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKK 
               
               
                   
               
               
                 DDDTSKTSPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFS 
               
               
                   
               
               
                 FGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDD 
               
               
                   
               
               
                 TSKPAFSFGAKPDEKKDDDTSKPAFSFGAKPDEKKDDDTSKPAFSFGAKP 
               
               
                   
               
               
                 DEKKDDDTSKTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVME 
               
               
                   
               
               
                 SDASGGKLAAALEHHHHHH  
               
               
                   
               
               
                 P-2NLP-P 
               
               
                 (SEQ ID NO: 17)  
               
               
                 MDIGINSDPAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDA 
               
               
                   
               
               
                 SGASPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAK 
               
               
                   
               
               
                 PDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKP  
               
               
                   
               
               
                 AFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKK 
               
               
                   
               
               
                 DSDTSKTSPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFS 
               
               
                   
               
               
                 FGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSD 
               
               
                   
               
               
                 TSKPAFSFGAKPDEKKDSDTSKPAFSFGAKPDEKKDSDTSKPAFSFGAKP 
               
               
                   
               
               
                 DEKKDSDTSKTSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVME 
               
               
                   
               
               
                 SDASGGKLAAALEHHHHHH  
               
             
          
         
       
     
         [0249]    Protein Expression and Purification: 
         [0250]    All prepared genes were transformed into  E. coli  OverExpress C41(DE3) cells (Lucigen, USA). For expression, a freshly-grown bacterial colony was inoculated in 10 mL LB medium with 100 μg/mL ampicillin at 37° C. overnight. 10 mL of overnight culture of all samples was inoculated into 1 L terrific broth (TB) media at 37° C. with 100 μg/mL ampicillin until OD 600 ˜1. Expression was then induced overnight at room temperature with the addition of 1 mM IPTG. The cells were harvested, lysed in 50 mM Tris (pH 8), 300 mM NaCl and 8 M urea, and frozen at −80° C. Thawed cells were sonicated, and 15 w/v % ammonium sulfate was added. Cell lysates were clarified by centrifugation (14,000 g for 30 min at 4° C.), and the proteins of interest were purified by Ni-NTA affinity chromatography under denaturing conditions with 250 mM imidazole used for protein elution. Purified samples were dialyzed against deionized (DI) water, and 20 mM Tris (pH 8) and 6 M urea were added. The proteins were further purified by ion exchange chromatography using a HiTrap Q HP column (GE Healthcare, Sweden) in an ÄKTA pure FPLC. Samples containing the desired product were dialyzed against DI water and lyophilized. P-intein-cNsp1 was prepared using the same expression condition as the other proteins, but harvested cells were resuspended in 50 mM NaH 2 PO 4 , 300 mM NaCl, and 10 mM imidazole and stored at −80° C. Thawed cells were lysed by the sonication, and cell lysates clarified by centrifugation were stored overnight at 4° C. at pH 7 for intein self-cleavage. Sun Z et al.,  Protein Expr Purif  43: 26 (2005). After adjusting the pH to 8, samples with 6×His tags were purified by Ni-NTA under native conditions and by FPLC as described above. P-C 30 -P protein was expressed and purified as reported previously (Olsen B D et al.,  Macromolecules  43: 9094 (2010)), with additional FPLC purification under denaturing conditions. Lyophilized samples were weighed to calculate yields of samples from 1 L culture ( FIG. 2B  and  FIG. 3B ). The purity of samples was determined to be greater than 95% by SDS-PAGE analysis ( FIG. 2A  and  FIG. 3A ). 
         [0251]    For transport studies, pQE80-14×His tag-TEV-IBB-MBP-EGFP, pQE80-14×His tag-TEV-MBP-mCherry and pQE30-scImp β-6×His tag from the Görlich group were transformed into SG13009 (pRep4) cells, and expression followed a previously described protocol. Frey S et al.,  EMBO J  28: 2554 (2009). After cutting off the His-tag with TEV protease (Eton Bioscience, USA) for IBB-MBP-EGFP and MBP-mCherry, additional purification was performed for all three proteins by size exclusion chromatography in buffer containing 50 mM Tris (pH 7.5) and 200 mM NaCl. The concentration of recombinant fluorescent proteins was determined using their optical absorbance. 
         [0252]    FITC-Dextran: 
         [0253]    Fluorescein isothiocyanate (FITC)-dextran with molar masses of 4, 10, 40, 70 and 150 kg/mol (catalog #46994, FD10S, FD40, 46945 and 46946), were purchased from Sigma-Aldrich (USA). Approximate Stokes&#39; radii of the FITC-dextran polymers were obtained from the supplier as follows: 1.4 nm (4 kg/mol), 2.3 nm (10 kg/mol), 4.5 nm (40 kg/mol), 6.0 nm (70 kg/mol) and 8.5 nm (150 kg/mol). 
         [0254]    Hydrogel Preparation: 
         [0255]    Lyophilized samples were dissolved at a concentration of 200 mg/mL in 50 mM Tris/HCl (pH 7.5) and 200 mM NaCl except where otherwise noted, mixed, and allowed to gel at either room temperature or 4° C. It is known that natural nucleoporins can form homogeneous hydrogels at such high protein concentration, but at lower nucleoporin concentrations, the hydrogels lose their selective permeability function. For this reason, nucleoporin hydrogels, Nsp1, are typically prepared at 150-200 mg/mL concentration throughout the literature. Since the invention concerns developing artificially engineered protein hydrogels that mimic the function of natural nucleoporin hydrogels, the nucleoporin hydrogels were benchmarked to these previous studies and used the concentration where it showed the selective transport property. Having confirmed that the designed hydrogels at 20 w/v % can show selectivity ( FIG. 4  and  FIG. 5 ), the ability of the best performing gel, P-2NLP-P, to mimic this property at a lower gel concentration, 10 w/v % ( FIG. 5 ), was checked. During the hydrogel inversion test, food coloring was added in the buffer for better visualization of the test. 
         [0256]    Rheology: 
         [0257]    Oscillatory shear rheology was performed on an Anton Paar MCR-301 in Direct Strain Oscillation mode with TruGap™ control. A Peltier heating system and environmental enclosure were employed to control sample temperature. Samples were loaded into a 25 mm cone-and-plate geometry with an angle of 1° and sealed with a mineral oil barrier to prevent dehydration. 
         [0258]    Raman Spectroscopy: 
         [0259]    A custom-built NIR confocal Raman inverted microscopy system was used for Raman measurements. 785 nm light from a continuous-wave Ti: Sapphire laser (3900S, Spectra-Physics) pumped by a frequency-doubled Nd: YAG laser (Millennia 5sJ, Spectra-Physics) was used for the excitation. A water immersion objective lens with 1.2 NA (UPLSAP060XWIR 60X/1.20, Olympus) was used both to focus the laser onto the sample, which is on top of quartz coverslip (043210-KJ, Alfa Aesar), and to collect the backscattered light. The Rayleigh light in the collected signal was removed by dichroic mirrors (LPD01-785RU-25×36×1.1, Semrock). Raman light was delivered to an imaging spectrograph (Holospec f/1.8i, Kaiser Optical Systems) and detected by a TE-cooled, back-illuminated, deep depleted CCD (PIXIS: 100BR_eXcelon, Princeton Instruments). The laser power at the sample plane was ca. 60 mW, and the signal was integrated for 5 seconds. Nine spectra were collected from each sample and averaged. Spectrum processing (cosmic ray removal, background subtraction and normalization) was performed by MATLAB (Mathworks) scripts. 
         [0260]    Capillary Transport Measurements: 
         [0261]    1.5 inch borosilicate capillaries with 0.9 mm inner diameters (Vitrocom 8290) were loaded by piercing pre-made hydrogels. 5 μM solutions of IBB-MBP-EGFP, MBP-mCherry, and/or importin β were injected into the capillary and sealed by a 1:1:1 mixture of vaseline, lanolin, and paraffin. Time lapses of cargo transport into the hydrogels were taken at 1 minute intervals for 1-3 hours on a Nikon Ti Eclipse inverted microscope using a Nikon CFI Plan UW 2× and Hamamatsu C11440-22CU camera. All fluorescence intensity profiles were obtained by averaging the fluorescence intensity within 100 μm slice width through the center of the gel and across the gel/buffer interface. The profiles were normalized by the bath concentration in the capillaries at the first time point. The interface between gel and buffer is assigned by a 20% change in the intensity compared to the bath fluorescence intensity as the zero point of the distance scale in each fluorescence intensity profile. 
         [0262]    Circular Dichroism Spectroscopy: 
         [0263]    CD spectra were obtained on an Aviv Model 202 Circular Dichroism spectrometer. CD spectra were recorded in a quartz cell of 0.1 cm path length at 25° C. between 190 and 250 nm, using a scan rate of 12 nm/min with a wavelength step of 1 nm. cNsp1 and the NLPs were dissolved in 50 mM Tris buffer with 200 mM NaCl at pH 7.5. CD band intensities, after the buffer signal subtractions, were converted into mean residue ellipticity (MRE). 
       Example 1: Preparation and Expression of Engineered Proteins 
       [0264]    To reduce protein loss during washing step in Ni-NTA chromatography denaturing purification, the washing buffer did not contain imidazole and was prepared at pH 8, causing increased impurity during elution. After the elution, eluted proteins were run on denaturing gels ( FIG. 6 ). While Nsp1 (60 kg/mol), one of nuclear pore proteins, was expressed at low levels and difficult to identify on the gel, engineered proteins (P-cNsp1-P and P-NLPs-P) were highly expressed. For better purity, anion exchange chromatography was performed, with final purified products shown in  FIG. 2A . Note that all proteins ran slower in SDS-PAGE ( FIGS. 2A, 7A, and 6 ) than their calculated and measured molar masses (Table 1), similar to previous reports for P-C 30 -P protein. Olsen B D et al.,  Macromolecules  43: 9094 (2010). 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Protein molar masses measured by MALDI 
               
             
          
           
               
                   
                 cNsp1 
                 1NLP 
                 2NLP 
                 P-cNsp1-P 
                 P-1NLP-P 
                 P-2NLP-P 
               
               
                   
               
               
                 Measured (g/mol)  
                 35,623 
                 36,756 
                 36,223 
                 46,305 
                 46,331 
                 45,780 
               
               
                 Calculated (g/mol) 
                 35,460 
                 36,461 
                 36,329 
                 45,971 
                 45,885 
                 45,437 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 DNA plasmids 
               
             
          
           
               
                 Protein name 
                 Expressed protein 
                 Vector 
               
               
                   
               
               
                 Nsp1 30-591   
                 Nsp1-His 6   
                 pET-22b 
               
               
                 cNsp1(=Nsp1 274-591 ) 
                 cNsp1-His 6   
                 pET-22b 
               
               
                 cNsp1 
                 P-Intein-cNsp1-His 6   
                 pET-22b 
               
               
                 1NLP 
                 1NLP-His 6   
                 pET-22b 
               
               
                 2NLP 
                 2NLP-His 6   
                 pET-22b 
               
               
                 P-cNsp1-P 
                 P-cNsp1-P-His 6   
                 pET-22b 
               
               
                 P-1NLP-P 
                 P-1NLP-P-His 6   
                 pET-22b 
               
               
                 P-2NLP-P 
                 P-2NLP-P-His 6   
                 pET-22b 
               
               
                 P-C 30 -P 
                 His 6 -P-C 30 -P 
                 pQE-9      
               
               
                 IBB-MBP-EGFP 
                 His 14 -TEV-IBB-MBP-mEGFP 
                 pQE-80      
               
               
                 MBP-mCherry 
                 His 14 -TEV-MBP-mCherry 
                 pQE-80      
               
               
                 scImpβ 
                 scImpβ-His 6   
                 pQE-30      
               
               
                   
               
             
          
         
       
     
         [0265]    Engineered proteins with P domain blocks—P-cNsp1-P, P-1NLP-P and P-2NLP-P—were easily synthesized in much higher yield than recombinant nucleoporin Nsp1. After protein expression and chromatographic purification, the yield of high purity protein was more than 20- to 70-fold higher than the recombinant Nsp1 protein ( FIG. 2  and  FIG. 6 ). 
         [0266]    NLPs without the coiled-coil domain were also isolated at 10 times greater yield than their parent sequence, cNsp1, after the same procedure ( FIG. 3 ). Interestingly, when the cNsp1 was fused to the P domain endblocks (P-cNsp1-P), the construct was expressed at a similar yield as the NLP constructs. 
         [0267]    Based on this observation, a single P domain together with an intein self-cleavage domain (Mathys S et al.,  Gene  231: 1 (1999)) was subcloned into the N-terminal cNsp1 gene (P-intein-cNsp1). After the self-cleavage of P-intein domains, cNsp1 was obtained at a similar yield as the NLPs ( FIG. 3 ). Significantly improved biosynthetic yields of these artificially engineered proteins enable detailed characterization of their material properties and engineering to control their performance. 
       Example 2: Preparation and Characterization of Hydrogels 
       [0268]    Engineered proteins with P domain endblocks rapidly formed hydrogels, while NLP midblocks alone failed inversion tests, indicating that structure beyond the FG repeat is necessary to give elastic mechanical properties. Consistent with a previous study on recombinant cNsp1, the NLPs without associating coiled-coil domains did not pass hydrogel inversion tests, while the designed proteins with the P domains formed hydrogels within a few minutes, mainly limited by the time required for the lyophilized sample to swell in a buffer. The engineered proteins were found to gel in number of buffer conditions commonly used for the recombinant Nsp1, demonstrating that P domain endblocks successfully replace the role of the N-terminal sequences of Nsp1 as a gel crosslinker. 
         [0269]    More particularly, while cNsp1 is known not to form hydrogels, P-cNsp1-P formed gels in all commonly used buffer conditions for Nsp1 at a protein concentration of 200 mg/mL. Buffers tested included (i) water; (ii) 0.1% TFA (in water), followed by neutralization with ¼ volume of the buffer (400 mM Tris-base, 100 mM Tris/HCl pH 7.5, 1 M NaCl); (iii) 0.1% TFA (in water) and neutralized with ⅕ volume of 200 mM Tris-HCl (pH 8.5); (iv) 0.2% TFA (in water) and neutralized with ⅕ volume of 200 mM Tris-HCl (pH 9); and (v) 100 mM phosphate buffer. 
       Example 3: Significance of Midblock FG Repeats 
       [0270]    To characterize the effect of midblock interactions on hydrogel mechanics, frequency sweep, linear oscillatory shear rheology of 20 w/v % hydrogels was performed in the absence or presence of 10% 1,6 hexanediol. Measurements were performed at 25° C. with a strain amplitude of 1%, within the linear viscoelastic range. Representative results are shown in  FIG. 8 . 
         [0271]    Rheology showed that the midblock sequence has a significant impact on the low frequency viscoelastic properties of the triblock protein gels without affecting the high frequency elastic plateau modulus. Although the midblocks cNsp1, 1NLP, and 2NLP are insufficient to cause gelation without the P domain, all three proteins with P domains formed gels with a comparable modulus (on the order of 10 kPa) with the crossover between G′ and G″ occurring below 0.1 rad/s ( FIG. 7A-C ). In all three artificially engineered hydrogels, the addition of 10% hexanediol to a 20 w/v % gel led to a decrease in the gel relaxation time, increasing the crossover frequency of the gel by approximately a factor of 5, while the stiffness of hydrogels (the plateau modulus G′) changed very little ( FIG. 7A-C ). 
         [0272]    Aliphatic alcohols, such as 1,6 hexanediol, are known to weaken FG associations, leading to a loss of selective permeability in vivo and in vitro. Comparison to a control hydrogel of similar molar mass but without FG repeats in the midblock (P-C 30 -P) showed no effect on the crossover frequency and the high frequency plateau modulus after the addition of hexanediol ( FIG. 7D  and  FIG. 9 ), indicating that the endblock P domains are unaffected by hexanediol. Therefore, the changes in crossover frequency in nucleoporin-mimetic gels, characteristic of changes in network relaxation rate, originate from differences in the state of the midblock domain. 
         [0273]    It has been shown that interchain β-sheets in some nucleoporin FG repeat hydrogels contribute to crosslinking and enhance the FG hydrogel stability, and removing these crosslinks enhances permeability and reduces selectivity. Labokha A A et al.,  EMBO J  32: 204 (2013). It is expected that the choice of crosslinking group in the hydrogel may affect biomolecular transport and mechanical properties, as crosslinking controls the mesh size of the gel and the relaxation dynamics of the junction points. These properties can influence the transport of macromolecules interacting with the network chains. 
         [0274]    Prominent changes in Raman bands upon the addition of hexanediol confirmed that these changes in gel mechanics are caused by disruption of FG repeats involved in molecular interactions within the midblocks, indicating that naturally observed FG interactions have been successfully adapted into the biosynthetic hydrogels. Upon the addition of hexanediol, a significant decrease was observed in the Raman band at 486 cm −1  corresponding to a Phe vibrational mode for cNsp1 and both consensus repeat NLP midblocks. Other Phe Raman bands (band assignments in  FIG. 10 ) are similar for all midblock polymers in the presence and absence of the hexanediol ( FIG. 7E-G ), indicating that natural cNsp1 and synthetic NLPs have a similar physicochemical environment for the Phe residues. 
         [0275]    Other common changes upon the addition of hexanediol were observed in Raman bands at 685 and 710 cm −1 . These bands do not appear in lyophilized cNsp1 or NLP ( FIG. 11 ) or in individual amino acids included in NLPs in water solution from a previous study. Zhu G et al.,  Spectrochim Acta A Mol Biomol Spectrosc  78: 1187 (2011). This suggests that the bands are a result of the association between midblocks in water. Molecular interactions between Phe and CH 3  and Pro and Lys have been suggested in cNsp1 based upon Nuclear Overhauser Exchange Spectroscopy NMR spectra by Ader et al.,  Proc Natl Acad Sci USA  107: 6281 (2010). The addition of 10% hexanediol suppressed Raman bands responsible for Phe (486 cm −1 ), Pro (856 and 1097 cm −1 ), Lys (1442 cm −1 ), and CH 3  (1452 cm −1 ) in cNsp1 ( FIG. 7E ), consistent with the NMR result, [24]  and therefore the observed Raman bands at 685 and 710 cm −1  may also relate to those residues. 
         [0276]    The similar shifts in the crossover frequency in all designed hydrogels ( FIG. 7A-C ) and large intensity differences of the 486, 685 and 710 cm −1  ( FIG. 7E-G ) by the addition of hexanediol suggest that hydrophobic interactions, including Phe-mediated associations, between the midblocks exist, similar to the natural Nsp1 hydrogel. These interactions can influence the gel relaxation without contributing significantly to the plateau modulus G′ ( FIG. 7A-C ). 
       Example 4: Transport Selectivity of Engineered Protein Hydrogels 
       [0277]    Engineered protein hydrogels containing cNsp1, 1NLP, and 2NLP midblocks selectively enhanced transport of specific biomolecules into the hydrogels, mimicking the property of natural Nsp1 gels. A fluorescence assay originally established to test recombinant nucleoporin hydrogels was performed in a capillary geometry ( FIG. 4A ) to test whether cargo-NTR complexes can permeate through the engineered biosynthetic gels with enhanced transport accumulation, while other molecules and cargo without the NTR are significantly retarded. 
         [0278]    For the assay, importin β (95 kg/mol) was chosen as a NTR due to its well-known binding to cargo with an importin β binding (IBB) domain and to the FG repeat on nucleoporin hydrogels. To reduce the passage of cargo without the NTR and easily quantify the transport of selected cargo, recombinant IBB—maltose binding protein (MBP)—enhanced green fluorescent protein (EGFP) protein fusions were prepared as a model cargo protein (75 kg/mol; Stokes&#39; radii of MBP and GFP are reported as 2.85 nm and 2.42 nm, respectively.). Based on the widely applied dextran diffusion method, it was expected that cargo diffusion into the gel would be significantly reduced since the pore size of the gels is smaller than non-interacting 40 kg/mol dextran probe (4.5 nm of Stokes&#39; radius;  FIG. 12 ). When importin β and the cargo were physically mixed and added to the capillary prepared with the engineered hydrogels, selective partitioning into the hydrogel occurred over time, while a slab diffusion profile was detected in the absence of importin β ( FIG. 4B-E  and  FIG. 13 ). The enhanced transport into the gel occurred due to a combination of diffusion and convection caused by gel swelling with buffer and cargo complexes ( FIG. 14 ). 
         [0279]    Because the length scale of the measurement was much larger than the molecular size and gel mesh size, the gel can be considered as a uniform, semi-infinite slab. The gels can also be treated as macroscopically homogeneous since they are optically clear (absence of inhomogeneity that would scatter on the length scale of visible light) and did not phase separate upon centrifugation. Under these conditions, the permeability coefficient is the product of the diffusivity and solubility coefficients. The discontinuous concentration profile at the interface during the capillary experiments suggested that the cargo-importin β complexes are more soluble in the gel phase than the cargo alone because of the physical association between importin β and FG repeats. This increase in solubility enhances the permeability of the cargo complexes. 
       Example 5: Electrostatic Effects on Transport Properties 
       [0280]    The two simplified NLP midblocks, both consensus sequences of cNsp1 but differing by a single amino acid in the repeating peptide, showed quantitative differences in transport properties. When accumulated green fluorescence intensities were calculated compared to the intensities without importin β ( FIG. 4F  and  FIG. 13 ), the P-2NLP-P gel showed almost twice the intensity of the P-1NLP-P gel. P-cNsp1-P, P-1NLP-P, and P-2NLP-P hydrogels absorbed 3.9±0.4 (mean±SD, n=3), 2.3±0.1 (n=3), and 3.8±0.3 (n=6) times more cargo-importin β complexes than inert molecules in an hour, respectively. Both NLPs have equal numbers of FG repeats, the same P domain crosslinkers for gelation, similar secondary structure as determined by circular dichroism ( FIG. 15  and Table 3), and similar passive diffusion profiles for inert molecules over time (dotted black curves in  FIG. 4D-E ). Therefore, this quantitative change in permeability is believed to originate from the change in the consensus repeat sequence. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 CD analysis of cNsp1 and NLPs a   
               
             
          
           
               
                 Protein 
                 Helix 1 
                 Helix 2 
                 Strand 1 
                 Strand 2 
                 Turns 
                 Unordered 
                 Total 
                 NRMSD 
               
               
                   
               
               
                 cNsp1 
                 0.00 
                 0.02 
                 0.18 
                 0.11 
                 0.14 
                 0.54 
                 0.99 
                 0.125 
               
               
                 1NLP 
                 0.00 
                 0.02 
                 0.19 
                 0.12 
                 0.18 
                 0.47 
                 0.98 
                 0.141 
               
               
                 2NLP 
                 0.01 
                 0.02 
                 0.18 
                 0.12 
                 0.18 
                 0.48 
                 0.99 
                 0.128 
               
               
                   
               
               
                   a By the circular dichroism secondary structure (CDSSTR) method (Kang JW et al.,  Biomed Opt Express  2: 2484 (2011)), it was found that approximately 50% structures of all proteins were disordered and others were β-strand and β-turns. 
               
             
          
         
       
     
         [0281]    Since the single amino acid change Asp in 1NLP to Ser in 2NLP occurred in the middle of the peptide between FG repeats that are known to bind importin β, the change from an anionic to neutral residue (change from a formal charge of −16 to 0 for the entire midblock of 16 repeats) suggests that electrostatic effects may affect molecular transport. It is interesting to note that the hydrogel made from P-cNsp1-P, where the midblock has a formal charge of +6, shows higher cargo-carrier accumulation on the gel interface than the P-2NLP-P hydrogel (max. fluorescence intensity: 7.6±0.7 and 5.0±0.9 for P-cNsp1-P and P-2NLP-P, respectively). However, the depth-integrated accumulation in an hour is the same for both materials ( FIG. 4F ), indicating that the P-2NLP-P gel has better permeability to the cargo-carrier than P-cNsp1-P gel. 
         [0282]    Changes in the charge of the protein based on a single substitution per repeat unit between 1NLP and 2NLP affect biomolecular transport through the designed hydrogels, despite the use of high ionic strength buffers that screen charge as under physiological conditions. Recent studies of related biological hydrogels such as mucus and cartilage have similarly observed that electrostatic effects influence molecular transport at the physiologically relevant ionic strength conditions. Selective binding can be added to synthetic systems by conjugating FG peptide onto polymer gels. The results presented here on NLP hydrogels indicate that not only FG sequences but also residues far from the FG repeat can play an important role in the performance of nuclear pore-mimetic synthetic hydrogels. 
       Example 6: Further Characterization of P-2NLP-P 
       [0283]    After identifying P-2NLP-P as the top performing construct, additional experiments were performed to explore its performance. The addition of 10% hexanediol to disrupt FG interactions eliminated selective accumulation of cargo complexes in P-2NLP-P gels ( FIG. 4G ), showing that FG repeat interactions are involved in enhanced selective transport in the biosynthetic hydrogels. This indicates that the engineered hydrogels have a filtering mechanism similar to the natural nucleoporin system. 
         [0284]    Using blends of model proteins with and without the IBB domain established the ability of the biosynthetic NLP gels to actively transport cargo proteins compared to the inert proteins. A model cargo protein incapable of binding importin β, MBP-mCherry (67 kg/mol), has smaller size than IBB-MBP-EGFP (75 kg/mol) but showed retarded transport through the biosynthetic NLP hydrogels even in the presence of importin β ( FIG. 4H-I , 3 hours assay in 10 w/v % and 20 w/v % of P-2NLP-P gel results in  FIG. 5 ). 
         [0285]      FIG. 4H-I  shows selective permeability test performed on P-1NLP-P and P-2NLP-P biosynthetic hydrogels (20 w/v %) with the addition of 5 μM MBP-mCherry, a model inert molecule, into 5 μM IBB-MBP-EGFP/importin β cargo complex mixtures. Over an hour, the cargo-carrier complexes accumulated 3.0 and 5.3 times more than MBP-mCherry (without the IBB domain) in P-1NLP-P and P-2NLP-P hydrogels, respectively. 
         [0286]    When hydrated in buffer at 20% and 10%, P-2NLP-P formed optically-clear gels that did not phase separate upon centrifugation, suggesting they formed macroscopically homogeneous networks under these conditions.  FIG. 5  shows that over three hours, the cargo-carrier complexes accumulated in the gel 10.2 and 5.2 times more than MBP-mCherry (no IBB domain), indicating that the 10 w/v % gel still showed enhanced transport of the selected biomolecules and the total accumulation depended on the number of FG sequences in the hydrogels (i.e., there are twice as many FG sequences in the 20 w/v % gel compared to the 10 w/v % gel). 
         [0287]    The results illustrate that the designed hydrogels can mimic both the selectivity and enhanced transport of natural nucleoporin hydrogels. 
       Example 7: NTR-Mediated Selective Uptake of a Target Molecule 
       [0288]    This example illustrates a generalizable method for capturing selected molecules into a hydrogel of the invention. As shown in  FIG. 18 , a peptide tag which can associate with a target molecule of interest can be genetically fused to a nuclear transport receptor (NTR). In a solution, the peptide tag fused to NTR will recognize, i.e., associate with, its target molecule and thereby spontaneously form NTR-peptide tag-target molecule complexes. Due to the interaction between NTR and the hydrogel, the NTR-peptide tag-target molecule complex is captured by and carried into the hydrogel. 
         [0289]    As a model system, incomplete green fluorescent protein (GFP) was used as a target molecule and GFP tag as a binding peptide tag. It is known that complete GFP can be constructed by mixing incomplete GFP and the GFP tag, with green fluorescence as evidence of the assembly. Cabantous S et al.,  Nat Methods  3: 845-854 (2006); Kent K P et al.,  J Am Chem Soc  130: 9664-9665 (2008). Another benefit using GFP as a model is that green fluorescence can be used to visualize selective transport of the NTR-GFP tag-incomplete GFP (i.e., NTR-GFP) complex into the hydrogel under a fluorescence microscope. 
         [0290]    GFP tag was genetically fused to the C-terminus of the NTR nuclear transport factor 2 (NTF2). Since NTF2 is homodimer, each NTF2 has two GFP tags. For protein purification, 6×Histidine tag was also fused between NTF2 and GFP tag (NTF2-His tag-GFP tag). After protein synthesis, more than 50 mg of chimeric NTF2-GFP tag was obtained. 
         [0291]    Capillary transport assay validated the method and confirmed that the hydrogel system can capture selected molecules into the hydrogel. When NTF2 with GFP tag was mixed with incomplete GFP, the solution turned from no color to light green after overnight incubation. When the solution was added to one end of a capillary where 20 wt % hydrogel filled the other end ( FIG. 19 ), the green fluorescence was accumulated onto the hydrogel over time ( FIGS. 19A-19C ). In the same experimental conditions, fluorescein-labeled dextran (40 kg/mol, hydrodynamic radius: 4.5 nm) did not get into the hydrogel ( FIGS. 19D-19F ). 
         [0292]    One or two incomplete GFP can associate with NTF2-GFP tag homodimer which has two GFP tags. The molar mass of GFP is approximately 30 kg/mol (2.42 nm hydrodynamic radius). The molar mass of NTF2-GFP tag homodimer is 36 kg/mol. Since NTF2-GFP complex (66 kg/mol with one GFP, or 96 kg/mol with two GFP) is greater than the 40 kg/mol dextran, the results clearly show that the synthetic system can mimic the natural selective filtering nuclear pore function which is carrying target molecule into the gel although the size of the NTF2-GFP complex is greater than uncomplexed inert reference molecules. 
         [0293]    The method just described opens new avenues for numerous applications for a number of applications, including for example drug delivery, food toxicology, and defense. Researchers have developed peptide library for various target molecules using phage display, and solid phase peptide synthesis techniques. By simply fusing those peptides to NTR carriers, the carriers will capture the targets and bring them into the hydrogels. More and diverse peptides will be available for specific target molecules with time; thereby, the method will be further generalized. As a specific example, Staphylococcal enterotoxin B (SEB) toxoid can be captured into the hydrogel using anti-SEB tag fused to NTF2 or importin β; the hydrogel can then be removed or destroyed for environmental decontamination. 
       INCORPORATION BY REFERENCE 
       [0294]    All patents and published patent applications mentioned in the description above are incorporated by reference herein in their entirety. 
       EQUIVALENTS 
       [0295]    Having now fully described the present invention in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims.