WO2011046520A1

WO2011046520A1 - Polypeptide material with adjustable pore properties

Info

Publication number: WO2011046520A1
Application number: PCT/SI2009/000048
Authority: WO
Inventors: Roman Jerala; Ota Fekonja; Jelka Pohar; Helena Gradisar; Mojca Bencina; Iva Hafner Bratkovic; Robert Bremsak; Spela Miklavic; Urska Jelercic; Anja Lukan; Tibor Doles; Sabina Bozic; Marko Verce; Nika Debeljak
Original assignee: Kemijski Institut
Priority date: 2009-10-12
Filing date: 2009-10-12
Publication date: 2011-04-21
Also published as: AT511130A2; AT511130A5

Abstract

Invention represents a polypeptide material with adjustable pore properties composed of two or three dimensional assembly of fusion proteins of at least two protein domains, where at least one domain is a coiled-coil segment and at least one domain is protein domain prone to oligomerization. The invention refers to polypeptide material used for applications such as the chemical catalysis and the separation of molecules according to their properties.

Description

Polypeptide material with adjustable pore properties

Field of invention

The field of this invention is bionanomaterial, a new material composed of two or three dimensional assembly of fusion proteins composed of at least two protein domains, where at least one domain is a coiled-coil forming segment and at least one domain is a protein domain prone to oligomerization. Field of the invention is fusion protein of the polypeptide material and DNA coding protein of the polypeptide material. Field of invention is also application of the polypeptide material for the separation of molecules according to their properties and for chemical or enzyme catalysis.

Background of the invention

Membrane-based filtration systems are widely used in preparation of drinking water and are gaining importance in food industry and removal of water pollutants, pharmaceutical industry for the removal of infectious microorganisms and unwanted components and concentration of selected components, for the membrane chemical or bio-reactors.

Ultrafiltration systems for the separation of macromolecules are typically made of organic polymers, where the size of pores is defined primarily by physical principles employed for the creation of pores of the desired size, such as physical crosslinking of fibers or evaporation of solvent. Membranes composed od dense protein layers have been assembled from the bacterial S- layer. Peng et al. (Peng et al., 2009, Nature Nanotechnology, 4, 353-357), describe ultrafiltration membranes composed of protein ferritin. While the performance of membrane is varied with pH defining analyte and membrane charge, the size of the pore cannot be varied and is determined by dense packing of protein molecules. When three molecules of ferritin with diameter of 12 nm surround a pore, its diameter is about 2.2 nm, limiting the use of this system. Membrane preparation based on packing proteins clearly limits the possible range of pore size as well as geometry and their chemical properties. U.S. Patent No. 6,756,039 Bl reports on regular structures produced by self-assembly of fusion proteins composed of rigidly linked monomers capable of associating into oligomers. It discloses production of discrete cage and one dimensional fibre and suggests three dimensional lattices formed by self-assembly of fusion protein of two different monomers, which assemble into a dimer and a trimer. The selection of appropriate monomers is relatively complex since the monomers are linked by a helix extending from C-terminus of the first monomer to N-terminus of the second. This rigid linking defines orientation of monomers in fusion protein and consequently defines and limits the possible structures made by these fusion proteins and use thereof.

WO/2004/033487 and U.S. Patent Application 2008/0097080 Al disclose protein lattices and structures composed of fusion proteins called protomers comprising of monomers forming oligomers with rotational symmetry axes of order N. The lattices disclosed in WO/2004/033487 have a potential application as support for crystallizing proteins and electron microscopy, but lack the flexibility in design to enable formation of pores of different properties with minor manipulations of the building blocks. Neither 6,756,039 Bl nor WO/2004/033487 or U.S. Patent Application 2008/0097080 Al describe methods for preparation of protein filtration membranes.

Herein disclosed invention describes self-assembling protein building blocks, which can be adjusted to modify the properties of pores and thus prepare different materials with variable technological properties. Coiled-coil elements as the specifically defined part of disclosed building blocks can be selected with respect to the length, which directly results in the pore size as well as with respect to the amino acid side chain residues at surface exposed positions that do not prevent formation of coiled-coil structure. Those amino acid residues, preferably at positions b, c and f of the coiled-coil register define the chemical properties of pores and provide new technological properties to the material.

Summary of the invention

The invention refers to the polypeptide material with adjustable pore properties composed of two or three dimensional assembly of fusion proteins of at least two protein domains, where at least one domain is a coiled-coil-forming segment and at least one domain is a protein domain prone to oligomerization with a state of oligomerization at least 3.

According to invention the oligomerization state of the protein domain prone to oligomerization is between 3 and 12, typically between 3 and 6.

The invention refers to the polypeptide material where the shape and the size of the pore is defined by self-assembly of the said protein domains in the said fusion proteins, and the length of the coiled-coil-forming segment fused to protein domain prone to oligomerization defines the size of the pore.

The invention refers to the polypeptide material where the length of the coiled-coil-forming segment is from 2 to 100 heptads (14 to 700 amino acid residues).

The invention refers to the polypeptide material where its technological properties are defined among others by the introduction of positively charged, negatively charged, hydrophobic, hydrophilic residues, cysteine residues, histidine residues or other amino-acid residues imparting specific interactions to surface exposed segments, whereas the coiled-coil-forming segment retains the ability to form coiled coils, and the residues that determine the technological properties of material pore are preferentially introduced into the positions b, c or f of the said coiled-coil-forming segments.

The invention refers also to the polypeptide material where the chemical properties of pore are defined also by the properties of the said protein domains in the said fusion proteins, including but not limited to, the net charge, surface exposed hydrophobic residues and size of the protein domains.

The invention refers to the of polypeptide material with adjustable pore properties composed of at least two protein domains, where at least one domain is a coiled-coil-forming segment and at least one domain is protein domain prone to oligomerization and the segments are optionally linked to each other with a flexible linker containing from one to 20 amino acid residues, preferentially from one to 6 amino acid residues, protein optionally contains signaling sequence directing protein secretion and polypeptide tags. The invention refers to the polypeptide material with adjustable pore properties where coiled- coil-forming segment is based on the sequence of SEQ ID no. 2, SEQ ID no.4 or SEQ ID no. 6 or designed peptides with functionally similar properties.

The invention refers to the polypeptide material with adjustable pore properties where protein domain prone to oligomerization is preferentially selected among the SEQ ID no. 8, SEQ ID no. 10, SEQ ID no. 12, SEQ ID no. 14, SEQ ID no. 84 and sequences with homology above 50 % to the sequences, which retain the ability to form oligomers of the same type.

The invention refers to the polypeptide material with adjustable pore properties formed by assembly of fusion proteins selected from SEQ ID no: 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82.

The invention refers to the polypeptide material with adjustable pore properties which is prepared by mixing two fusion proteins according to claims 1 to 10, where the coiled-coil-forming segment of each of the two fusion proteins form a parallel coiled coil with coiled-coil-forming segment from the other fusion protein and in one fusion protein the oligomerization domain is positioned at the N-terminus and in the other at the C-terminus and the oligomerization state of both oligomerization domains is the same.

The invention refers to the polypeptide material with adjustable pore properties where the coiled- coil-forming segments are selected from natural parallel coiled coils or designed coiled coils or are selected among the pairs SEQ ID no: 16, 18, 20, 22, 24, 26, 28, 30, 32.

The invention refers to the polypeptide material with adjustable pore properties where the oligomeric domains are selected from tetrameric proteins, preferentially selected from pairs SEQ ID no: 10 and SEQ ID no: 12 or from trimeric proteins, preferentially selected from SEQ ID no: 8 and SEQ ID no: 84.

The invention refers DNA coding polypeptide material with adjustable pore properties where DNA is operatively linked to the regulatory elements, promoter and terminator, which drive expression of fusion proteins in the host cells. The invention refers to the application of the polypeptide material with adjustable pore properties to separate or concentrate molecules, molecular assemblies, viruses or nanoparticles according to their properties.

The invention refers to the application of the polypeptide material with adjustable pore properties for chemical catalysis.

Description of Figures

Figure 1 : A ) Schematic representation of an antiparallel coiled-coil dimer shows distribution of heptad peptide motif amino acid residues. Residues at positions b, c, and f are exposed to surface and can thus be modified in order to adjust pore properties. B) Schematic representation of polypeptide material, which is formed by assembly of fusion protein composed of antiparallel coiled-coil dimer-forming segment and protein domain prone to assemble into tetramers. C) Shematic representation of polypeptide material as in (B), demonstrating that variation of length of coiled-coil-forming segment and introduction of positive charge into exposed residues in coiled-coil dimer enables formation of pores with different physicochemical and thus technological properties.

Figure 2: A) Isolation of fusion protein dimtetra-Al. Cell lysate (lane 1) and precipitate (inclusion bodies) (lane 2) were checked for expression of dimtetra-Al. Inclusion bodies were purified, solubilized in 6M GdnHCl and dialysed against MilliQ water resulting in pure dimtetra- Al (lane 3). Lane 4 is a polypeptide standard. B) Far-UV CD spectrum of dimtetra-Al polypeptide material suspension shows alpha-helical secondary structure, confirming correct folding of dimtetra-Al in polypeptide material.

Figure 3: Filtration of Ml 3 bacteriophages (A) and dextran blue (B) through dimtetra-Al membrane. The M13 bacteriophages could not be detected in filtrate (n.d.).Concentration of dextran blue was determined from absorbance at 625 nm. Detailed description of the invention

Before further description it is to be assumed that the invention is not limited to presented embodiments since modifications of particular embodiments can still be in the scope of appended claims. Unless defined otherwise, all technical and scientific terms used herein possess the same meaning as it is commonly known to experts in the field of invention. The terminology to be used in the description of the invention has the purpose of description of a particular segment of the invention and has no intention of limiting the invention. All publications mentioned in the description of the invention are listed as references. In the description of the invention and in the claims, the description is in the singular form, but also includes the plural form, what is not specifically highlighted for the ease of understanding.

Polypeptide material

The basis of the invention is the discovery that two or three dimensional polypeptide materials with adjustable pore properties are assembled from fusion proteins comprising coiled-coil- forming segments and protein domains prone to oligomerization. These materials have broad applicability from separation science to chemical catalysis.

The term 'polypeptide material with adjustable pore properties' refers to material with pores which have particular shape, size and physicochemical properties. The present invention is about polypeptide material composed of fusion proteins consisting of at least two domains, which are chemically linked or genetically encoded as a fusion gene, whereas at least one domain is composed of a coiled-coil-forming segment and at least one domain is composed of a protein domain prone to oligomerization. The protein domains can originate from naturally occurring proteins or can be designed based on the knowledge on the properties of coiled-coil proteins available to the expert in the field (patent US 7,045,537 Bl) to have specific desired characteristics. Coiled-coil-forming segment

At least one of the domains in the said fusion proteins forming polypeptide material consists of a coiled-coil-forming segment. The term "coiled-coil-forming segment" refers to a heptad repeat polypeptide motif, designated as abcdefg, where amino-acid residues a and d are preferentially hydrophobic and e and g are preferentially charged. Two or more of such polypeptide chains (coiled-coil-forming segments) interact to form a "coiled coil", referring to helices that coil around each other, with heptad motif repeating approximately every 2 turns. When two chains form a coiled-coil dimer, amino acids a and d participate in hydrophobic core while the nature of e and g Interaction is electrostatic (Figure 1A). In such complexes, residues at positions b, c and / do not participate significantly in intermolecular interactions in the formed dimer and could in such a way harbor amino-acid residues with different properties such as charged, hydrophobic, cysteine and others allowing for chemical modification, metal chelation, specific interactions etc. that the inventors discovered to be used to modify the chemical properties of the pores.

Coiled-coil sequences could be designed or taken from naturally existing coiled-coil peptides or coiled-coil domains of proteins. They are present in many of the naturally occurring proteins e.g. transcription factors, oncoproteins, tropomyosin, viral proteins etc.

The basis of this invention is that the physicochemical properties of the said pores (surrounded by by coiled-coils fused to oligomerizing proteins) could be defined and adjusted by variations of the size of coiled-coil segment and variations and modifications of residues at positions b, c and / These residues could be positively or negatively charged, hydrophobic, hydrophilic, allowing for chemical modification (Asn, Gin, Cys), they could chelate metal ions or other amino acid residues imparting specific interactions, the only limitation being that these amino acid residues or their modifications do not disable coiled coil formation.

The basis of this invention is also that size of the said pore could be defined and adjusted by variations in the length of the coiled-coil-forming segments. By "the size of the pore" it is meant the dimensions of the pore surrounded by the subject fusion proteins. The length of the coiled- coil-forming segment could be from 2 to more than 150 heptads (14 to 1050 amino-acid residues), usually from 2 to 100 heptads (14 to 700 amino-acid residues). The subject coiled coil (formed by assembly of said coiled-coil-forming segments) could be either made from identical coiled-coil-forming segments (homologous) or from different coiled- coil-forming segments (heterologous). By "identical coiled-coil-forming segments" it is understood proteins having the same primary structure. By "different coiled-coil-forming segments" it is understood proteins having different primary structure that can form a coiled-coil composed of at least two different coiled-coil-forming segments. The orientation of chains (coiled-coil-forming segments) in the coiled coil could be either parallel (all protein chains in the same direction) or antiparallel (chains run in opposite directions). Principles determining the identification and properties of coiled-coil segments are well known to the researchers skilled in this subject.

Some of the naturally occurring coiled-coil-forming segments and sequences designed by inventors are listed in Table 1.

Table 1 : Coiled-coil-forming segments

Protein domain prone to oligomerization

The basis of this invention is also that at least one domain of the fusion protein is an oligomerization-prone protein domain. The term "protein domain prone to oligomerization" refers to typically but not exclusively to naturally occurring protein or fragment of naturally occurring protein. These proteins or their domains tend to associate with the same or other proteins (or domains thereof) to form homologous or heterologous protein dimeric or higher oligomeric species. Homodimer (homologous dimer) is formed when two identical protein domains associate. Heterodimer (heterologous dimer) is formed by association of two different protein domains. By "identical protein domains" it is meant proteins having the same primary structure. By "different protein domains" it is meant proteins having different primary structure. Higher oligomeric species are composed of at least three protein domains, which could be of the same primary structure (homologous oligomers), or containing protein domains of different primary structure (heterologous oligomers). The structural information about selected naturally occurring proteins prone to oligomerization is well known to experts from the field. The subject protein domain has typically from 5 to 400 amino-acid residues, more usually from 15 to 200 amino-acid residues. The term "oligomerization state" referring to the number of subject protein domains in protein oligomers is from 3 and 12, typically between 3 and 6. Examples of protein domains prone to oligomerization are listed in Table 2.

The basis of this invention is also that the properties of protein domain prone to oligomerization, such as the isoelectric point and the size also define pore properties and that oligomerization state of the formed oligomers from the said protein domains influences properties of the said pore.

Table 2: Protein domains prone to oligomerization

Linker

In contrast to fusion proteins in U.S. Patent No. 6,756,039 where protein domains are rigidly linked (by a rigid linker such as alpha helix), fusion proteins of the said invention contain flexibly linked protein domains. The term "linker" refers to shorter amino acid sequences, whose role could be only to separate the individual domains or segments of the protein. The role of the linker peptide in the fusion protein, may also be the introduction of flexibility or interruption of the regular secondary structure or for posttranslational modifications, including the introduction of sites for improved processing. The length of the linker peptide is not restricted and is defined by the desired technological properties of the material, however, it is usually up to 30 amino acid residues long, preferentially from one to 20 amino acid residues, more preferentially from one to six amino acid residues. Any amino acid could be included in a linker, preferentially amino acid residues are selected among small or hydrophilic amino acids or amino acids that introduce special conformational or functional properties, such as serine, glycine, treonine, proline, valine, alanine, cysteine or others.

Fusion proteins and self-assembling polypeptide materials thereof

Fusion proteins of the present invention comprise said protein domains connected by a short peptide linker described above. Protein domains in said fusion protein can occur in any order and the technological properties of the material may vary depending on the order. The number of protein domains (coiled-coil-forming segments and protein domains prone to oligomerization) in fusion protein could vary up to 8, but is typically from 2 to 4.

The basis of this invention is that selected protein domains of the said fusion proteins tend to associate under appropriate conditions to form two dimensional or three dimensional polypeptide materials. By term "two-dimensional material" it is meant a planar assembly of fusion proteins (the size of the assembled material is much greater in two dimensions than in the third dimension). The term "three dimensional" polypeptide materials refers to materials that have fusion proteins assembling in three dimensions. To facilitate two dimensional or three- dimensional protein assembly at least one of the protein domains (either coiled-coil segment or oligomerization-prone protein domain) has to form protein assemblies of the oligomerization state 3 or higher.

The major features of polypeptide materials are:

a) the shape and the size of the pore is defined by self-assembly of the said domains in the said fusion proteins b) the length of the coiled-coil-forming segment fused to protein domain prone to oligomerization defines the size of the pore

c) the pore properties are defined by the physicochemical properties of amino acids introduced into the positions b, c or/ of the coiled-coil-forming segment

d) the pore properties are defined also by the properties of the said monomeric proteins in the said fusion proteins

There are several possible implementations of polypeptide materials with adjustable pore properties composed of single fusion proteins varying in selection of protein domains that are defined by the said design of the fusion protein. However, when protein domains tend to form heterologous assemblies (such as heterologous coiled coils), more than one fusion protein is needed to form the said polypeptide material. The number of the said fusion proteins to prepare the polypeptide material is between 1 and 10, typically 1 to 3.

Assembly of polypeptide pores from one or two different fusion proteins

Polypeptide material according to the invention can be assembled either from one single type of fusion proteins of coiled-coil-forming segment and protein domain prone to oligomerization or from two or more fusion proteins of coiled-coil-forming segment and protein domain prone to oligomerization. Inventors discovered that in case of one type of fusion protein the coiled-coil segment needs to be a homodimeric antiparallel coiled coil. In case of using two different types of materials the coiled coil can be either antiparallel heterodimer or a parallel heterodimer. In case of using a parallel heterodimer the two protein domains prone to oligomerization need to be fused to the N-terminal segment of the heterodimeric coiled-coil segment in one fusion protein and to the C-terminal end of the coiled-coil segment in the second fusion protein. For the regular assembly of protein membranes the state of aggregation of both oligomerization segments need to have the same state of oligomerization, for example trimer, represented by example by trimerization domains of CutAl SEQ ID no: 84 or foldon, SEQ ID no: 8 or tetramer, represented by example of tetramer ization domain of p53 SEQ ID no: 10 or tetramerization domain of shaker channel, SEQ ID no: 12. If the stability of the coiled-coil segment is higher than the stability of protein oligomerization domain both oligomerization domains of both fusion proteins at the N- or C-terminal end can be identical.

Signal peptide, peptide tag sequences

Additional amino acid sequences at either end or within the segments of the fusion protein, which are not necessary for formation of polypeptide material, could be appended to fusion protein e.g. to simplify purification, allow detection or endow the material with additional functional properties.

The term "signal sequence" or "signal peptide" refers to the amino-acid sequence, which is important for directing the protein to a certain location in the cell. Signaling sequences also vary depending on the host organism in which the fusion protein is expressed. Amino-acid sequences of the signaling sequences are well known to experts, as well as which signal sequence is functional in a certain organism.

The term "tag sequence" refers to the sequence of amino acids, which is added to the protein to facilitate purification/isolation/detection of the protein.

The position of the signaling sequence and marker sequence are optional but it must allow functional expression of the protein and maintain the function for which these amino acid sequences were selected, which is known to experts in the field.

Nucleic acids encoding fusion proteins and host organisms

The invention refers also to fusion protein of polypeptide material and DNA.

The term "DNA/nucleic acid" refers to polynucleotide molecules such as DNA and RNA, including cDNA, genomic DNA, synthetic DNA, chimeric DNA and RNA. Nucleic acids may be either double-stranded or single-stranded. Nucleic acids may contain nucleic analogues or derivatives.

The subject fusion protein, encoded by the DNA, can be synthesized in the host organism that expresses the heterologous nucleic acid, encoding the fusion protein. The term "homologous protein" refers to proteins with preserved functional properties and well preserved amino-acid sequences, preferably with at least 50% conservation, with a minimum of 20 % conservation, determined by protein alignment techniques, known to experts in the field.

In general, the heterologous nucleic acid is inserted into an expression vector. An expression vector generally contains operationally linked control elements, which are operationally linked to the DNA coding for the fusion protein according to the invention. It is understood that the control elements are selected according to desired expression quantity. The promoter may be either constitutive or inducible depending on the desired pattern of expression. The promoter may be of either native origin or foreign origin (not presented in the cells, where it is applicable), and may be natural or synthetic. The promoter is chosen in such a way that it functions in the target cells of the host organism. In addition, initiation signals for the efficient translation of the fusion protein, including the ATG and corresponding sequences, are included.

Suitable vectors include, but are not limited to: plasmids, viral vectors, and others. An expression vector may be prepared for expression in prokaryotic and eukaryotic cells. For example, prokaryotic cells are bacteria, primarily Escherichia coli. According to the invention the use of prokaryotic cells is intended for preparation of a sufficient quantity of nucleic acids and to produce fusion proteins.

The invention includes also host cells and organisms that contain nucleic acid according to the invention (transient or stable), which codes for the subject fusion protein. The term "host organism" refers to the organism, in which the DNA coding for protein, has been introduced in order to be expressed. Appropriate host cells are known in the state of the art and include bacterial and eukaryotic cells. The transfer, of vectors into host cells is carried out by conventional methods, known in the state of the art, and the methods refer to transformation, transfection, including: chemical transfer, electroporation, microinjection, DNA lipofection, cell sonication, particle bombardment, viral DNA transfer, and more. In the context of the invention, the introduction of DNA is by electroporation and viral transfer into cells of vertebrates or vertebrate cell lines. 009 000048

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Transient expression refers to the introduction of the vector DNA, which according to the invention is not incorporated into the genome of cells. Stable intake is achieved by incorporating DNA of the invention into the host genome. The transfer of the DNA according to the invention, especially for the preparation of the host organism, which has a stable DNA integrated, may according to the invention be controlled by the presence of markers. DNA coding for markers refers to resistance to drugs, for example antibiotics, and may be included in the vector with DNA according to the invention, or on a separate vector.

The host organism may be prokaryotic or eukaryotic. Eukaryotic cells, suitable for the fusion protein expression, are not limited as long as cell lines are compatible with propagation methods of the expression vector and with the expression of the fusion protein. The preferred eukaryotic cells include, but are not limited to, yeast, fungi, plant cells and mammalian cells, such as: mouse, rat, monkey or human fibroblasts.

For the DNA expression any bacterial host can be used according to the invention. For the expression of proteins of the present invention, the use of bacteria or yeast is preffered. The protein can be expressed in bacteria E. coli or B. subtilis or in yeast S. cerevisiae or P. pastoris. The preferred bacterium for protein expression is E. coli. Invention refers to the expression of the protein in bacteria or yeast. Invention refers to bacteria or yeast, that express protein, preferably to the bacteria E. coli or to yeast P. pastoris.

Preparation and application of polypeptide material for the separation of molecules according to their properties

The necessary feature of subject fusion proteins is that they assemble into polypeptide material under appropriate conditions. When polypeptide material is formed from single fusion protein appropriate conditions are found by screenings of solubility, macromolecular assembly and secondary protein structure of soluble part but especially of precipitate in buffers of various pH (usually from pH 3 to pH 9), ionic strength, organic solvents (e.g. DMSO, acetonitrile, trifluoroethanol, hexafluoroisopropanol).

For the formation of filtration device for the separation of molecules it is important to assemble the material with the least amount of defects in the connectivity. This is achieved by a gradual refolding from the conditions which favor nonassembled and soluble form of structures into the conditions which favor formation of intermolecular interactions through coiled-coil-forming segments and oligomerization protein domains. Important factors for self-assembly were also the concentration of fusion protein and temperature. The polypeptide material for filtration is formed when concentration of protein is between 0.1 μg/mL and 20 mg/mL, typically between 0.05 and 10 mg/mL. Self-assembly of the polypeptide material can also be triggered by slow cooling of the fusion protein from the temperature above the melting point to the temperature below the folding transition, typically to the room temperature.

When polypeptide material is designed to assemble from several fusion proteins, the assembly takes place after mixing the fusion proteins in the appropriate molar ratio under the conditions favoring self-assembly.

Formation of filtration devices

After the polypeptide material is self-assembled, it can be additionally crosslinked by crosslinking agents such as glutaraldehyde. Depending on the size of the pore and pore properties, this polypeptide material can be used for the separation of a range of molecules from about 1 nm to about 100 nm. Examples of potential applications include filtration of pharmaceutical preparations, removal of contaminants in medical preparations, removal of pathogenic agents such as viruses, prions etc., treatment of waste waters, size separation of nanoparticles etc. Additional important feature of polypeptide material with adjustable pore properties is that not only the size but also other pore properties e.g. charge, immobilized specific reagents etc. enable separation of molecules of similar size but differing in other characteristics, such as polarity, charge, shape, specific surface properties etc.

Application of polypeptide material with adjustable pore properties for catalysis

Introduction of selected amino-acid residues in positions b, c and f of the coiled-coil-forming segment such as e.g. histidine, cysteine enables binding of metals to the pore acting as catalysts. Polypeptide material with larger pores can also be enriched by an enzyme thus providing support for enzymatic catalysis. Artificial enzymes can be formed in pores by introduction of active sites (e.g. catalytic triads) into the desired geometry into the pores of the said material.

Examples of implementation, designed to illustrate the invention, are shown below. The descriptions of examples of implementation have no intention of limiting the invention and should be understood as a demonstration of the invention.

Examples of implementation

Example 1. Preparation of DNA constructs

DNA sequences for protein domains described above were designed from amino-acid sequences of the selected protein domains using tool Designer from DNA2.0 Inc. that enables the user to design DNA fragments and optimize expression for the desired hosts (e.g. E .coli) using codon optimization. Genes were then ordered from MR.Gene GmbH (Im Gewerbepark B32, D-93059 Regensburg), cut out with restriction endonucleases (restriction enzymes) and cloned into the appropriate vector containing necessary regulatory sequences known to the experts in the field. Vectors used include commercial vectors ET, pRSETA and pSBlA2 (pSBlA2 http://partsregistry.Org/Part:pSBlA2), carrying all necessary features such as antibiotic resistance, origin of replication and multiple cloning site.

Molecular biology methods (DNA fragmentation with restriction endonucleases, DNA amplification using polymerase chain reaction-PCR, PCR ligation, DNA concentration detection, agarose gel electrophoresis, purification of DNA fragments from agarose gels, ligation of DNA fragments into a vector, transformation of chemically competent cells E.coli DH5cc, isolation of plasmid DNA with commercially available kits, screening and selection) were used for preparation of DNA constructs. All procedures were preformed under sterile conditions (aseptic technique). DNA segments were characterized by restriction analysis and sequencing.

Molecular cloning procedures are well known to the experts in the field and are described in details in molecular biology handbook (Sambrook J., Fritsch E.F., Maniatis T. 1989. Molecular cloning: A laboratory manual. 2nd ed. New York, Cold Spring Harbor Laboratory Press: 1659 p.).

Example 2. Production of fusion proteins composed of an antiparallel coiled-coil homodimerization domain and tetramerization domain

Several DNA constructs were prepared to demonstrate the feasibility of production of fusion proteins composed of an antiparallel coiled-coil homodimerization domain and tetramerization domain (Table 3).

Table 3: Fusion proteins composed of antiparallel coiled-coil homodimerization domain and p53 tetramerization domain or tetramerization domain of Shaker channel protein

Additionally to varying orientation of protein monomers in fusion protein, linker between proteins could be varied from 2 to 20 amino acids and His₆-tag could be positioned either on N- terminus or C-terminus.

Plasmids encoding open reading frames of fusion proteins from Table 3 were transformed into chemically competent E. coli BL21 (DE3) pLysS cells. Selected bacterial colonies grown on LB plates supplemented with selected antibiotic (ampicillin, kanamycin) were inoculated into 100 mL of LB growth media supplemented with antibiotic and grown overnight at 37°C. Next day the overnight culture was diluted 20-50-times reaching OD₆₀o of diluted culture between 0.1 and 0.2. The bacterial culture was grown until OD₆₀₀ reached 0.6 - 0.8 when protein expression was induced by addition of inducer IPTG (from 0.4 mM to 1 mM). In some cases the culturing temperature was lowered (25°C-30°C) about 0.5 h prior to induction. Two to five hours after induction culture broth was centrifuged and bacterial cells were resuspended in lysis buffer (Tris pH 8.0, 0.1% deoxycholate supplemented with protease inhibitor cocktail) and frozen at -80°C. Thawed cell suspension was further lysed by sonication and centrifuged. Precipitate (residual non-lysed cells, inclusion bodies) and supernatant were checked for expression of constructs by SDS-PAGE and when necessary by Western blot using anti-His-tag antibodies as primary antibodies. All constructs with p53T were mainly present in insoluble part (inclusion bodies), which was composed of >80% of the overexpressed protein. Inclusion bodies were washed several times with lysis buffer, twice with 2 M urea in 10 mM Tris pH 8.0 and once with MiliQ water. Usually this treatment resulted in >95% of protein purity. In cases where the precipitated material still contained impurities, inclusion bodies were dissolved in 6 M GdnHCl pH 8.0 and loaded on Ni²⁺-NTA columns (Quiagen, GE). Purification under denaturing conditions was followed according to the manufacturer's instructions. After elution with 250 mM imidazole pH 5.8 fractions containing protein were combined and dialysed twice against 10 mM Hepes pH 7.5 or other appropriate buffer.

When fusion protein was present in supernatant, supernatant was loaded on Ni²⁺-NTA column and purified under native conditions. After elution with 250 mM imidazole pH 5.8 or 500 mM imidazole pH 8.0 fractions containing protein were combined and dialysed twice against 10 mM Hepes pH 7.5 or other appropriate buffer.

Example 3. Production of fusion proteins composed of an antiparallel coiled-coil homodimerization domain and trimerization domain

Several DNA constructs have been prepared to demonstrate the feasibility of production of fusion proteins composed of an antiparallel coiled-coil homodimerization domain and trimerization domain (Table 4). Table 4: Fusion proteins composed of antiparallel coiled-coil dimerization domain and trimerization domain

In addition to varying the orientation of protein monomers in fusion protein, linker between proteins could be varied from 2 to 20 amino acids and His₆-tag could be positioned either or N- terminus or C-terminus.

Production and isolation of proteins were performed as described in Example 2.

Example 4. Preparation of material selfassembly by refolding based on dilution, dialysis or temperature annealing

First screening to find conditions where the fusion proteins folded and were able to self-assemble were made by diluting protein denatured in 6M GdnHCl at ratio 1 : 100 into the buffer of various pH (citrate buffer pH 2 and pH 3, acetate buffers pH 4, pH 5, phosphate buffers pH 5, pH 6, pH 7, Hepes buffer pH 7.5, Tris buffer pH 8, carbonate buffer pH 9, pH 10), different ionic strength (100 mM, 300 mM, 1 M, 2M salts) and organic solvent up to 20% of acetonitrile, DMSO, methanol or up to 50% in case of trifluoroethanol. Visual inspection revealed the presence of macroscopic protein assemblies and their morphology, such as the fine precipitate, or more favorably gel-like polypeptide aggregate. Protein absorption spectra were measured to determine the solubility of the assembled fusion protein. Samples (soluble fraction and precipitates) were analyzed by CD spectroscopy to determine the protein secondary structure and thermal stability. When appropriate conditions for the formation of self-assembled material with pores were found, larger amount of individual polypeptide material, such as 1 mg was assembled by slow removal of denaturing agent using dialysis of denatured protein against the selected buffer.

When APH-1 was a coiled-coil part of fusion protein temperature was also an important factor, since APH-1 coil-coiled homodimer is labile at higher temperatures. These type of fusion proteins were relatively soluble at room temperature. Their CD spectra and melting temperature correspond to the properties measured for the fusion partner tetramerizatiin domain p53T. In this case annealing was done by slow decrease of temperature from 80°C to 4°C over 10 hours at protein concentration of 0.5 mg/ml.

Example 5. Refolding of fusion proteins and preparation of membrane

Membranes of fusion proteins were prepared by different methods of self-assembly by refolding starting with initial solubilization of fusion proteins in: a) 6 M GdnHCl , b) 9M LiBr, c) hexafluoroisopropanol.

Sample of the isolated protein SEQ ID: 34 was dissolved in 6 M LiBr at concentration of 0.1 mg/ml. At this concentration of LiCl the secondary structure of protein was disrupted and protein was soluble at more than 10 mg/ml. Protein assembly was initiated by dilution of protein solution in 9M LiBr into the buffer, which favors native conformation of protein and formation of coiled- coil and tetramerization of p53 domain (20 mM HEPES buffer at pH 7.5). During the refolding visible protein associate was formed. Protein suspension was slowly deposited on a 0.22 μητ, membrane PVDF filter (Millipore) with a diameter of 13 mm as a support for the assembled protein membrane. After deposition of assembled protein to the support filter the solution was washed with 10 ml of 20 mM HEPES buffer at pH 7.5 mechanical strength of the protein membrane was increased by covalent crosslinking of the self-assembled polypeptide material with 10 % glutaraldehyde. Glutaraldehyde was left to react for 1 hour with a subsequent washing step using 10 ml 1 M Tris pH 8.0 as a blocking reagent to remove any remains of nonreacted glutaraldehyde and 10 ml of deionized water. Protein membrane was left soaked in water and prevented to dry before using it as a filter. Alternatively, membrane was prepared by incubating the supporting fiter with polypeptide solution at 80°C and annealed by decreasing the temperature to 4°C as described in Example 4.

To the sample dissolved in hexafluoroisopropanol water was added to 5% and hexafluoroisopropanol was evaporated leaving protein assembled in small amount of water.

Example 6. Use of the assembled polypeptide material for the separation of large molecules and particles (bacteriophages)

To test the filtering properties of formed polypeptide material (membranes) Ml 3 bacteriophages and dextran blue were used. Ml 3 bacteriophage suspension was filtered through filter formed from polypeptide material on a solid support. Phage titer of the initial suspension and filtrate was determined according to the procedures well known to the experts in the field. Although the starting material had a phage titer of 3x 10¹⁰ pfu/ml the phages could not be detected in the filtrate, estimating the decrease in filter titer by more than 6 orders of magnitude (less than 10⁴ pfu/ml) (Figure 3A). Dextran blue solution (0.5 mg/ml) was also filtered and the filtration efficiency was followed by measuring absorbance at 625 nm (Figure 3B).

Claims

67 Claims

1. Polypeptide material with adjustable pore properties composed of two or three dimensional assembly of fusion proteins of at least two protein domains, where at least one domain is a coiled-coil-forming segment and at least one domain is a protein domain prone to

oligomerization with a state of oligomerization at least 3.

2. Polypeptide material according to claim 1, where the oligomerization state of the protein

domain prone to oligomerization is between 3 and 12, typically between 3 and 6.

3. Polypeptide material according to claim 1 or 2, where the shape and the size of the pore is defined by self-assembly of the said protein domains in the said fusion proteins, and the length of the coiled-coil-forming segment fused to protein domain prone to oligomerization defines the size of the pore.

4. Polypeptide material according to any claim from 1 to 3, where the length of the coiled-coil- forming segment is from 2 to 100 heptads (14 to 700 amino acid residues).

5. Polypeptide material according to any claim from 1 to 4, where its technological properties are defined among others by the introduction of positively charged, negatively charged, hydrophobic, hydrophilic residues, cysteine residues, histidine residues or other amino-acid residues imparting specific interactions to surface exposed segments, whereas the coiled- coil-forming segment retains the ability to form coiled coils, and the residues that determine the technological properties of material pore are preferentially introduced into the positions b, c or f of the said coiled-coil-forming segments.

6. Polypeptide material according to any claim from 1 to 5, where the chemical properties of pore are defined also by the properties of the said protein domains in the said fusion proteins, including but not limited to, the net charge, surface exposed hydrophobic residues and size of the protein domains.

7. Fusion protein of polypeptide material according to any claim from 1 to 6 composed of at least two protein domains, where at least one domain is a coiled-coil-forming segment and at least one domain is protein domain prone to oligomerization and the segments are optionally 68 linked to each other with a flexible linker containing from one to 20 amino acid residues, preferentially from one to 6 amino acid residues, protein optionally contains signaling sequence directing protein secretion and polypeptide tags.

8. Polypeptide material according to any claim from 1 to 7, where coiled-coil-forming segment is based on the sequence of SEQ ID no. 2, SEQ ID no.4 or SEQ ID no. 6 or designed peptides with functionally similar properties.

9. Polypeptide material according to any claim from 1 to 8, where protein domain prone to

oligomerization is preferentially selected among the SEQ ID no. 8, SEQ ID no. 10, SEQ ID no. 12, SEQ ID no. 14, SEQ ID no. 84 and sequences with homology above 50 % to the sequences, which retain the ability to form oligomers of the same type.

10. Polypeptide material according to any claim from 1 to 9 formed by assembly of fusion

proteins selected from SEQ ID no: 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82

11. Polypeptide material according to any claim 1 to 10 prepared by mixing two fusion proteins according to claims 1 to 10, where the coiled-coil-forming segment of each of the two fusion proteins form a parallel coiled coil with coiled-coil-forming segment from the other fusion protein and in one fusion protein the oligomerization domain is positioned at the N-terminus and in the other at the C-terminus and the oligomerization state of both oligomerization domains is the same.

12. Polypeptide material according to claim 11, where the coiled-coil-forming segments are selected from natural parallel coiled coils or designed coiled coils or are selected among the pairs SEQ ID no: 16, 18, 20, 22, 24, 26, 28, 30, 32.

13. Polypeptide material according to claim 11 or 12, where the oligomeric domains are selected from tetrameric proteins, preferentially selected from pairs SEQ ID no: 10 and SEQ ID no: 12 or from trimeric proteins, preferentially selected from SEQ ID no: 8 and SEQ ID no: 84. 69

14. DNA coding fusion protein according to claims 1 to 13 where DNA is operatively linked to the regulatory elements, promoter and terminator, which drive expression of fusion proteins in the host cells.

15. Application of the polypeptide material according to any claim from 1 to 13 to separate or concentrate molecules, molecular assemblies, viruses or nanoparticles according to their properties.

16. Application of the polypeptide material according to any claim from 1 to 13 for chemical catalysis.