WO2000077196A1 - Gene et sequences de proteines du gene 35 de phage t4 - Google Patents

Gene et sequences de proteines du gene 35 de phage t4 Download PDF

Info

Publication number
WO2000077196A1
WO2000077196A1 PCT/US1999/013024 US9913024W WO0077196A1 WO 2000077196 A1 WO2000077196 A1 WO 2000077196A1 US 9913024 W US9913024 W US 9913024W WO 0077196 A1 WO0077196 A1 WO 0077196A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
ofthe
sequence
purified
nucleic acid
Prior art date
Application number
PCT/US1999/013024
Other languages
English (en)
Inventor
Edward B. Goldberg
Original Assignee
The Trustees Of Tufts College
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Trustees Of Tufts College filed Critical The Trustees Of Tufts College
Priority to AU46781/99A priority Critical patent/AU4678199A/en
Priority to PCT/US1999/013024 priority patent/WO2000077196A1/fr
Priority to CA002375998A priority patent/CA2375998A1/fr
Priority to JP2001503640A priority patent/JP2003507007A/ja
Priority to EP19990930192 priority patent/EP1185638A4/fr
Publication of WO2000077196A1 publication Critical patent/WO2000077196A1/fr
Priority to HK02104995.5A priority patent/HK1043155A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

  • the present invention relates to nucleotide sequences of bacteriophage T4 gene 35 and amino acid sequences of its encoded protein, as well as derivatives and analogs thereof and antibodies thereto.
  • the present invention further relates to the use of nucleic acids encoding bacteriophage T4 gene 35 and its encoded protein, as well as derivatives, and analogs thereof, in the construction of nanostructures, i.e., nanometer sized structures useful in the construction of microscopic and macroscopic structures.
  • Bacteriophage viruses that attack bacteria, are generally composed of a protein coat which surrounds genetic material.
  • Bacteriophage T4 a T-even phage, consists of an icosahedron shaped head which contains DNA, a tail (a hollow cylinder of contractile protein) which serves as an injection tube ofthe DNA and tail fiber appendages which emanate from the base ofthe tail.
  • the tail fibers serve to attach the phage to the bacterial surface in a process known as adsorption.
  • the gp34 homooligomer (“P34”), gp36 homooligomer (“P36”), and gp37 homooligomer (“P37”) are rod-shaped structures in which two identical ⁇ sheets, oriented in the same direction, are fused face-to-face by hydrophobic interactions between the sheets juxtaposed with a 180° rotational axis of symmetry through the long axis ofthe rod.
  • gp35 is a monomeric polypeptide that attaches specifically first to the N-terminal region ofthe P36 homooligomer and then to the C-terminus ofthe P34 homooligomer and forms a joint between these two rods having an average angle of 137° ( ⁇ 7°) or 156° ( ⁇ 12°).
  • the self assembly ofthe tail fiber is regulated by a predetermined order based on the interaction of specific protein subunits whereby structural maturation caused by formation ofthe first subassembly permits interaction with new (previously disallowed) subunits.
  • gp37 the monomeric 109 Kda translation product of gene 37
  • 2 accessory proteins gp57 and gp38
  • the N- terminus of P37 initiates the oligomerization of two gp36 molecules of 23 Kda each, in a butt-end joint to form the P36 homooligomer rod.
  • the N-terminus of P36 then attaches to the carboxy terminal region of a gp35 monomer; this interaction stabilizes P36 and forms the flexible angle joint ofthe tail fiber.
  • the amino terminal region of gp35 then attaches to the C-terminus of P34 (the homooligomerization of which requires the chaperon protein gp57).
  • these structures are created by a process of self-assembly, the instructions for which are built into the component polypeptides.
  • These natural proteins are also subject to proofreading processes that insure a high degree of quality control.
  • Advantages of using natural proteins to construct nanostructures are that the resulting structures are stiff, strong, stable in aqueous media, heat resistant, protease resistant, and can be rendered biodegradable. Additionally, large quantities of nanostructure parts and subassemblies can be easily fabricated in microorganisms and stored and used as needed.
  • Phage T4 gp35 is located between genes gp34 and gp36.
  • a sequence for gp35 is available on the NCBI database (NCBI.NIH.GON) within the sequence T4g34-t (bases 4188-5075).
  • the T4g34-t sequence reveals that gene 35 has an open reading frame, ORF35, that is predicted to encode a protein having a molecular weight of 32,334 Daltons.
  • the ⁇ CBI database also predicts an open reading frame, ORF34.1 , that extends 241 nucleotides between genes gp 34 and gp 35, and encodes a deduced protein having a molecular weight of 7,334 Daltons (in a different reading frame from ORF35).
  • the present invention relates to nucleotide sequences of bacteriophage T4 gene 35, and amino acid sequences ofthe encoded bacteriophage T4 gene 35 protein, as well as derivatives (e.g., fragments) and analogs thereof, and antibodies thereto.
  • the present invention further relates to nucleic acids hybridizable to or complementary to the foregoing nucleotide sequences, as well as equivalent nucleic acid sequences encoding a bacteriophage T4 gene 35 protein.
  • the present invention also relates to expression vectors encoding a bacteriophage T4 gene 35 protein, derivatives or analogs thereof, as well as host cells containing the expression vectors encoding the bacteriophage T4 gene 35 protein, derivative or analog thereof.
  • gene 35 (gp35)
  • gp35 the protein product of bacteriophage T4 gene 35
  • the present invention also relates to methods of production ofthe gp35 proteins, derivatives and analogs, such as, for example, by recombinant means.
  • the invention further relates to gp35 proteins, derivatives (e.g., fragments), and analogs having an angle joint domain that has been modified so as to form average angles different from the natural average angle of 137° ( ⁇ 7°) or 156° ( ⁇ 12°).
  • the invention also relates to gp35 proteins, derivatives and analogs which exhibit thermolabile interactions with tail fiber binding partners.
  • the invention further relates to gp35 derivatives and analogs which are functionally active, i.e., they are capable of displaying one or more known functional activities associated with a full-length (wild-type) gp35 protein.
  • Such functional activities include, but are not limited to, antigenicity [ability to bind (or compete with gp35 for binding) to an anti-gp35 antibody], immunogenicity (ability to generate antibody which binds to gp35), and ability to bind (or compete with gp35 for binding) to a ligand for gp35, and ability to multimerize with other phage products such as P34 and/or P36.
  • the gp35 protein, derivative or analogs thereof disclosed herein may be used for the production of anti-gp35 antibodies which antibodies may be used diagnostically in immunoassays for the detection or measurement of gp35 protein.
  • the invention also relates to fragments (and derivatives and analogs thereof) of gp35 which comprise one or more domains of a gp35 protein, e.g., the P34 or P36 binding domain, and/or retain the antigenicity of a gp35 protein (i.e., are able to be bound by an anti-gp35 antibody).
  • the present invention further relates to the use of nucleotide sequences ofgp35 and its encoded amino acid sequence in the construction of nanostructures, i.e., nanometer sized structures useful in the construction of microscopic and macroscopic structures.
  • FIGS. 1A-1B T4 bacteriophage. Schematic representation ofthe T4 bacteriophage particle ( Figure 1A), and a schematic representation ofthe bacteriophage T4 tail fiber ( Figure IB).
  • Figure 2 Sequence of bacteriophage T4 gp35.
  • the gp35 protein sequence shown in Figure 3 (encoded by nucleotides 4,127-5,011 of Figure 3) lacks amino acid numbers 1-77 of Figure 2.
  • Amino acid numbers 1-7, 18-56 and 65 of Figure 2 appear as part ofthe ORF34J sequence in Figure 3 (encoded by nucleotides 3,894-4,088 of Figure 3).
  • Figure 3 NCBI database sequence containing bacteriophage T4 gene 34, gene 35
  • gene 36 and gene 37 The nucleotide sequence containing gene 34, gene 35 and gene 36 (SEQ ID NO:3) and the amino acids encoding the gene products of gene 34 (SEQ ID NO:4; ORF 34.1, SEQ ID NO:5) gene 35 (SEQ ID NO:6), GENE 36 (SEQ ID NO:7) and gene 37 (SEQ ID NO:8).
  • the present inventor has discovered that significant errors are present in the nucleotide and amino acid sequences of gp35 disclosed in the prior art. Indeed, the inventor has discovered that the prior art predicted amino acid sequence of gp35 lacks 77 amino acid residues at the N- terminus ofthe actual protein and that 15 of the 16 amino acid residues corresponding to the N-terminal residues ofthe prior art predicted gp35 are incorrect. The invention thus provides sequences of gp35 that correct these prior art errors.
  • the present invention thus relates to nucleotide sequences of gp3 '5 and amino acid sequences of encoded gp35 proteins, as well as derivatives and analogs thereof, and antibodies thereto.
  • the present inventor has isolated and characterized the gene encoding bacteriophage T4 gp35, a tail component necessary for the formation of bacteriophage T4 tail fibers.
  • the nucleotide sequence encoding gp35 was determined to be distinct from that previously reported in the NCBI database ( Figure 3).
  • the gp35 nucleotide sequence encodes a protein that has a different N-terminus and a molecular weight that is 24% greater than that predicted by the sequence in the NCBI database (nucleotides 4,127-5,011 of Figure 3).
  • the present invention enables recombinant production and genetic manipulation ofthe gp35 protein.
  • the present invention provides a purified bacteriophage gp35 protein that is not contained in a gel (e.g., a gel suitable in which to conduct electrophoresis).
  • the invention relates to a composition comprising at least 1, 10, 50, 100 or 500 nanogram(s), 1, 10, 50, 100 or 500 microgram(s), or 1, 10, 50, 100 or 500 milligram(s), of purified non-denatured gp35 protein.
  • the gp35 gene sequence ofthe invention can be a naturally occurring sequence or in variant form, whether natural, synthetic, or recombinant.
  • the gp35 protein is not native (i.e., not naturally occurring).
  • the present invention relates to a bacteriophage T4 gp35 protein variant containing the amino acid sequence depicted in Figure 2 (SEQ ID NO:2) wherein only conservative substitutions relative to the sequence in Figure 2 are made.
  • the invention also relates to purified molecules comprising bacteriophage T4 gp35 protein fragments, which fragments consist of at least the amino acid sequence depicted in Figure 2 (SEQ ID NO:2) from amino acid numbers 1-17, 1-56, 1-78, 1-93, 8-17, 57-93, 57-64, 66-79 or 81-93, as well as derivatives thereof, e.g., in which only conservative substitutions relative to the sequence in Figure 2 are made. Nucleic acids encoding such proteins, and their complement, are also within the scope ofthe invention.
  • the invention additionally relates to proteins, derivatives, fragments or analogs containing an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85% or 90% identity to amino acids number 1 to 100 in Figure 2 over a 100 amino acid sequence.
  • amino acid sequence homology refers to amino acid sequences having identical amino acid residues or amino acid sequences containing conservative changes in amino acid residues.
  • a gp35 homologous protein is one that shares the foregoing percentages of sequences identical with the naturally occurring gp35 protein over a 100 amino acid length.
  • the invention additionally relates to proteins, derivatives, fragments or analogs containing an amino acid sequence having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% identity to amino acids number 57 to 93 in Figure 2 over a 36 amino acid sequence.
  • a gp35 homologous protein is one that shares the foregoing percentages of sequences identical with the naturally occurring gp35 protein over a 36 amino acid length.
  • the invention also relates to proteins encoded by nucleic acids hybridizable to a gp35 gene under non-stringent, moderately stringent, or stringent conditions.
  • a protein is encoded by a nucleic acid hybridizable to a DNA having a nucleotide sequence consisting ofthe coding region of SEQ ID NOJ or its complement.
  • a gp35 derivative may be a fragment or amino acid variant (e.g., an insertion, substitution and/or deletion derivative) ofthe gp35 sequence shown in Figure 2.
  • insertion, substitution and/or deletion occur outside of amino acid numbers 1-17, 1-56, 1-78, 1-93, 8-17, 57-93, 57-64, 66-79 or 81-93 depicted in Figure 2.
  • the invention also relates to gp35 analogs.
  • the gp35 fragment, amino acid variant or analog ofthe invention is capable of displaying one or more functional activities associated with a full-length native gp35 protein.
  • Such functional activities include, but are not limited to, antigenicity, t.e., the ability to bind to an anti-gp35 antibody, immunogenicity, i.e., the ability to generate an antibody which is capable of binding a gp35 protein; the ability to bind (or compete with gp35 for binding) to a ligand for gp35; and the ability to multimerize with P36 and/or P34.
  • a functional ability ofthe gp35 protein is the ability of gp35 or a gp35-P36 oligomer to bind to P34 and/or the ability of gp35 to bind to P36.
  • the invention provides gp35 fragments or variants that comprise at least a functionally active portion ofthe gp35 sequence shown in Figure 2 from amino acid numbers 1-17, 1-56, 1-78, 1-93, 8-17, 57-93, 57-64, 66-79, or 81-93.
  • the invention provides derivatives (including fragments) or analogs of a gp35 protein consisting of at least 8 contiguous amino acids, or of at least 15 contiguous amino acids, or of at least 20 contiguous amino acids, ofthe gp35 protein sequence depicted in Figure 2 (SEQ ID NO:2) from amino acids numbers 1 to 24.
  • this derivative or analog is able to be bound by an antibody directed against a gp35 protein.
  • the derivative or analog specifically binds the P34 homooligomer.
  • Nucleic acids encoding such derivatives or analogs are also within the scope ofthe invention.
  • the invention further provides derivatives, fragments or analogs of a gp35 protein consisting of at least 40, 45, 50, 60, or 70 contiguous amino acid residues ofthe gp35 protein sequence depicted in Figure 2 (SEQ ID NO:2) from amino acids numbers 1 to 100.
  • this gp35 derivative, fragment or analog lacks amino acid residues 93 to 372.
  • the invention further relates to fragments (and derivatives and analogs thereof) of gp35 which comprise one or more functional domains of a gp35 protein, e.g., the P36 or P34 binding domain, and/or retain the antigenicity of a gp35 protein (i.e., are able to be bound by an anti-gp35 antibody).
  • the fragments lack at least 10, 20, 30 or 40 contiguous amino acids ofthe sequence shown in Figure 2.
  • the invention also relates to gp35 proteins, derivatives and analogs in which internal peptide sequences are deleted without affecting the ability of gp35 to associate with its natural tail fiber partners P36 and/or P34.
  • the deletion occurs of contiguous amino acids selected from among amino acids 100-273.
  • gp35 is modified so that it interacts only with other modified, and not native, tail fiber partners; exhibit thermolabile interactions with its partners; or contains, or is conjugated to, additional functional groups that enables it to interact with heterologous binding moieties.
  • the gp35 protein, or derivatives or analogs thereof, described herein, may be used for the production of anti-gp35 antibodies, which antibodies may be used in immunoassays for the detection or measurement of gp35 protein.
  • the present invention also relates to a gp35 protein, derivative or analog that is modified in the domain that which forms an angle joint, to form an average angle that is different from the natural average angle of 137° ( ⁇ 7°) or 156° ( ⁇ 12°).
  • the present invention further relates to methods of production ofthe gp35 proteins, derivatives and analogs, such as, for example, by recombinant means.
  • the present invention additionally provides for nanostructures comprising native or modified gp35 and native or modified bacteriophage tail fiber proteins.
  • the nanostructures may be one-dimensional rods, two-dimensional polygons or open or closed sheets, or three-dimensional open cages or closed solids.
  • the gp35 protein may be modified in various ways to form novel structures with different properties for use as described in Section 5.8. 5.
  • THE ep35 CODING SEQUENCES gp35 DNA sequences and sequences complementary thereto are gp35 nucleic acids provided by the present invention. Sequences hybridizable thereto, are also provided.
  • Nucleic acids comprising gp35 DNA or RNA sequences are also provided; in various embodiments, at least 850, 880, 920, 960, or 1000 contiguous nucleotides ofthe gp35 sequence in Figure 2, are in the nucleic acid. Also included within the scope ofthe present invention are nucleic acids comprising gp 35 DNA having the sequence depicted in Figure 2 (SEQ ID NO:2), or its corresponding RNA, which do not encode other bacteriophage T4 tail fiber proteins or functionally active portions thereof. Nucleic acids can be single-stranded or double-stranded. In specific embodiments, isolated nucleic acids are provided that comprise at least 150, 175, 200, 225, 250, 275, or 285 contiguous nucleotides of nucleotides 1 to 285 in Figure 2.
  • nucleic acids ofthe invention comprise the nucleotide sequences shown in Figure 2 that encode amino acid numbers 1-17, 1-56, 1-78, 1-93, 8-17, 57-93, 57-64, 66-79, or 81-93 of Figure 2.
  • nucleic acids comprise nucleotide numbers 1 to 1,116 of Figure 2.
  • the gp35 nucleotide sequences ofthe invention preferably do not contain in contiguous linkage sequences of a bacteriophage T4 genome that are naturally in contiguous linkage flanking the gp35 sequences (i.e. , 5 ' or 3 ' to the gp35 gene).
  • the gp35 nucleotide sequences can be contiguous with non-bacteriophage T4 nucleotide sequences of at least 10 nucleotides.
  • the invention provides an isolated nucleic acid comprising a nucleotide sequence encoding a gp35 protein having the amino acid sequence depicted in Figure 2 (SEQ ID NO:2), operably linked to a heterologous promoter.
  • heterologous promoter is meant a promoter that is not the native T4 promoter that is operably linked to the gp35 sequence in the bacteriophage T4 genome. In a specific embodiment, the promoter is not a bacteriophage T4 promoter.
  • nucleotide sequence encoding the gp35 protein is that sequence depicted in Figure 2 (SEQ ID NOJ) from nucleotide numbers 1 to 1,116 contiguous to a 3' termination codon.
  • nucleic acids contain at least 850, 880, 920, 960, or 1000 contiguous nucleotides of a gp35 DNA sequence operably linked to a promoter that is not a bacteriophage promoter (i.e., a heterologous promoter).
  • the nucleic acid further comprises nucleotide sequences encoding other bacteriophage T4 proteins selected from the group consisting of gp36 and gp37, and optionally the chaperon protein gp57, operably linked to the same or a different promoter.
  • other bacteriophage T4 proteins selected from the group consisting of gp36 and gp37, and optionally the chaperon protein gp57, operably linked to the same or a different promoter.
  • native intergenic regions between the other bacteriophage T4 proteins are omitted.
  • the invention also provides single-stranded ohgonucleotides for use as primers in PCR that amplify a gp35 gene or gp 35 sequence-containing fragment, e.g., an ohgonucleotide having the sequence of a hybridizable portion (at least ⁇ 8 nucleotides) of gp 35, and another ohgonucleotide having the reverse complement of a downstream sequence in the same strand of gp35, such that each ohgonucleotide primes synthesis in a direction toward the other.
  • the 5 ' ohgonucleotide corresponds to sequence flanking nucleotides 1-280 of Figure 2.
  • the 5' primer comprises a sequence upstream of nucleotide number 1 in Figure 2 and/or also comprises a nucleotide sequence shown in Figure 2 encoding an amino-terminal portion (i.e. at least the N-terminal amino acid) of gp35.
  • the ohgonucleotide primers are preferably in the range of 10-35 nucleotides in length.
  • a kit comprising in one or more containers the foregoing primers is also provided.
  • sequence for gp35 is depicted in Figure 2 (SEQ ID NO: 1), with the coding region thereof spanning nucleotide numbers 1 to 1,116. Sequence analysis ofthe nucleotide sequence of gp35 of Figure 2 reveals an open reading frame of 1,116 nucleotides, encoding a protein of 372 amino acids (SEQ ID NO:2). in accordance with the present invention, any polynucleotide sequence which encodes the amino acid sequence of a gp35 product can be used to generate recombinant molecules which direct the expression of gp35.
  • nucleic acids consisting of at least 8 nucleotides that are useful as probes or primers (i.e., a hybridizable portion) in the detection or amplification of gp35.
  • these probes or primers have a contiguous sequence contained in nucleotides 1 to 279 of Figure 2.
  • the invention also relates to nucleic acid sequences hybridizable or complementary to the foregoing sequences or equivalent to the foregoing sequences in that the equivalent nucleic acid sequences also encode a protein product displaying gp35 functional activity.
  • nucleic acids encoding fragments and derivatives of gp35 are additionally described infra. 5
  • the invention also relates to nucleic acids hybridizable to or complementary to the above-described nucleic acids comprising gp35 sequences.
  • nucleic acids are provided which comprise a sequence absolutely complementary to at least 10, 25, 50, 100, or 200 nucleotides or the entire coding region of agp35 gene, or, in particular, those portions encoding amino acid numbers 1-17, 1-56, 1-78, 1-93, 8-17, 57-93, 57-64, 66-
  • a nucleic acid which is hybridizable to agp35 nucleic acid, or to a nucleic acid encoding a gp35 derivative, under conditions of low stringency is provided.
  • procedures using such conditions of low stringency are as follows (see also Shilo and Weinberg, 1981, Proc. N ⁇ tl. Ac ⁇ d. Sci. USA 78:6789-6792): Filters containing DNA are pretreated for 6 h at
  • Filters are incubated in 0 hybridization mixture for 18-20 h at 40°C, and then washed for 1.5 h at 55°C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60°C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68 °C and reexposed to film. Other conditions of low stringency which may be 5 used are well known in the art (e.g., as employed for cross-species hybridizations).
  • nucleic acid which is hybridizable to a gp35 nucleic acid under conditions of high stringency is provided (see infra).
  • the D ⁇ A may be obtained by standard procedures known in the art from, for example, by chemical synthesis or by the cloning the D ⁇ A, or fragments thereof, purified 0 from a desired cell or phage.
  • the gene should be molecularly cloned into a suitable vector for propagation ofthe gene.
  • DNA fragments are generated, some of which will encode the desired gene.
  • the DNA may be cleaved at specific sites using various restriction enzymes.
  • the linear DNA fragments can then be separated according to size by standard techniques, including, but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography. See, for example, Innis et al., 1990, PCR protocols: A Guide to Methods and Applications, Academic Press, San Diego, California; Dieffenbach et al., 1995, PCR primer, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  • identification ofthe specific DNA fragment containing the desired gene may be accomplished in a number of ways.
  • a gp35 gene ofthe present invention or its specific RNA, or a fragment thereof, such as a probe or primer may be isolated and labeled and then used in hybridization assays to detect a generated gp35 sequence (Benton, W. and Davis, R., 1977, Science 196:180; Grunstein, M., and Hogness, D., 1975, Proc. Natl. Acad. Sci. USA 72:3961).
  • Those DNA fragments sharing substantial sequence homology to the probe will hybridize, e.g., under high stringency conditions.
  • high stringency conditions refers to those hybridizing conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1 % SDS at 50 °C; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0J% Ficoll/0J% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42 °C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC and 0.1% S
  • formamide for example, 50% (vol/vol
  • DNA clones which hybrid-select the proper mRNAs can be selected which produce a protein that has similar or identical electrophoretic migration, isolectric focusing behavior, proteolytic digestion maps, binding activity or antigenic properties as known for gp35.
  • the gp35 protein may be identified by binding of labeled antibody to the putatively gp35 expressing clones, e.g., in an ELISA (enzyme-linked immunosorbent assay)-type procedure.
  • gp35 sequence can also be identified by mRNA selection by nucleic acid hybridization followed by in vitro translation. In this procedure, fragments are used to isolate complementary mRNAS by hybridization. Such DNA fragments may represent available, purified gp35 DNA of a naturally occurring or modified gp35 gene.
  • Immunoprecipitation analysis or functional assays ofthe in vitro translation products ofthe isolated products ofthe isolated mRNAS identifies the mRNA and, therefore, the complementary DNA fragments that contain the desired sequences.
  • Radiolabelled RNA or DNA may be used as a probe to identify the gp35 DNA fragments from among other DNA fragments.
  • isolating gp35 DNA examples include, but are not limited to, chemically synthesizing the gene sequence itself from a known sequence. Other methods are known to those of skill in the art and are within the scope ofthe invention.
  • the identified and isolated DNA can then be inserted into an appropriate cloning vector.
  • vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda or T4 derivatives, or plasmids such as PBR322 or pUC plasmid derivatives or the Bluescript vector (Stratagene).
  • the insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini.
  • the ends of the DNA molecules may be enzymatically modified.
  • any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized ohgonucleotides encoding restriction endonuclease recognition sequences.
  • the cleaved vector and gp35 sequence may be modified by homopolymeric tailing. Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, or other methods known to those of skill in the art, so that many copies ofthe gp35 sequence are generated.
  • the desired gp35 sequence may be identified and isolated after insertion into a suitable cloning vector in a "shot gun" approach. Enrichment for the desired DNA, for example, by size fractionization, can be done before insertion into the cloning vector.
  • host cells are transformed with recombinant DNA molecules that incorporate the isolated gp35 sequence, or synthesized DNA sequence and enables generation of multiple copies ofthe sequence.
  • the gp35 sequence may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gp35 sequence from the isolated recombinant DNA.
  • Ohgonucleotides containing a portion ofthe gp35 coding or non-coding sequences, or which encode a portion ofthe gp35 protein can be synthesized by standard methods commonly known in the art.
  • Such ohgonucleotides preferably have a size in the range of 8 to 25 nucleotides. In a specific embodiment herein, such ohgonucleotides have a size in the range of 15 to 25 nucleotides or 15 to 35 nucleotides.
  • the gp35 sequences provided by the instant invention include those nucleotide sequences encoding substantially the same amino acid sequences as found in native gp35 proteins, and those encoded amino acid sequences with functionally equivalent amino acids, as well as those encoding other gp35 derivatives or analogs, as described infra for gp35 derivatives and analogs.
  • nucleotide sequences coding for a gp35 protein, derivative (e.g. fragment) or analog thereof can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation ofthe inserted protein-coding sequence, for the generation of recombinant DNA molecules that direct the expression of a gp35 protein.
  • an appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation ofthe inserted protein-coding sequence, for the generation of recombinant DNA molecules that direct the expression of a gp35 protein.
  • Such gp35 polynucleotide sequences, as well as other polynucleotides or their complements may also be used in nucleic acid hybridization assays, Southern and Northern blot analysis, etc.
  • a bacteriophage T4 gp35 gene or a sequence encoding a functionally active portion of a bacteriophage T4 gp 35 gene is expressed.
  • a derivative (e.g., fragment) of a bacteriophage T4 gp35 gene is expressed. Due to the inherent degeneracy ofthe genetic code, other DNA sequences which encode substantially the same or a functionally equivalent gp35 amino acid sequence, is within the scope ofthe invention. Such DNA sequences include those which are capable of hybridizing to the gp35 sequence of SEQ ID NOJ under stringent conditions.
  • Altered DNA sequences which may be used in accordance with the invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent gene product.
  • the gene product itself may contain deletions, additions or substitutions of amino acid residues within an gp 35 sequence, which result in a silent change, thus, producing a functionally equivalent gp35 protein.
  • Such amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature ofthe residues involved.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine.
  • the DNA sequences ofthe invention may be engineered in order to alter agp35 coding sequence for a variety of ends including, but not limited to, alterations which modify processing and expression ofthe gene product.
  • mutations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, etc.
  • the coding sequence of gp35 is synthesized in whole or in part, using chemical methods well known in the art. See, for example, Caruthers et al., 1980, Nuc. Acids Res. Symp. Ser. 7:215-233; Crea and Horn, 1980, Nuc. Acids Res. 9(10):2331; Matteucci and Caruthers, 1980, Tetrahedron Letters 21:719; and Chow and Kempe, 1981, Nuc. Acids Res. 9(12):2807-2817.
  • the protein itself could be produced using chemical methods to synthesize a gp35 amino acid sequence in whole or in part.
  • peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., see Creighton, 1983, Proteins Structures And Molecular Principles, W.H. Freeman and Co., N.Y. pp. 50-60).
  • the composition ofthe synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, 1983, Proteins, Structures and Molecular Principles, W.H. Freeman and Co., N.Y., pp. 34-49).
  • a polynucleotide sequence encoding a g ⁇ 35 protein, or derivative or analog thereof is inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation ofthe inserted coding sequence.
  • an appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation ofthe inserted coding sequence.
  • gp35 protein for use as an immunogen for generating antibodies (i.e., monoclonal or polyclonal) that immunospecifically bind a gp35 protein and providing gp35 protein building blocks for nanostructures containing bacteriophage tail fiber proteins or protein derivatives.
  • antibodies i.e., monoclonal or polyclonal
  • expression vectors containing a gp35 coding sequence of interest can be used to construct expression vectors containing a gp35 coding sequence of interest (native, modified, or recombined) and appropriate transcriptional/translational control signals.
  • These expression vectors typically contain selectable marker genes (usually conferring antibiotic resistance to transformed bacteria), sequences that allow replication ofthe plasmid to high copy number in E. coli, and a multiple cloning site immediately downstream of an inducible promoter and ribosome binding site.
  • Methods of constructing expression vectors containing a gp35 coding sequence include in vitro recombinant DNA techniques and synthetic techniques.
  • a variety of host-expression vector systems may be utilized to express a gp35 coding sequence. These systems are preferably bacteria transformed with recombinant bacteriophage DNA or plasmid DNA expression vectors containing a gp35 coding sequence, but also include, but are not limited to, yeast transformed with recombinant yeast expression vectors containing an gp35 coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculo virus) containing an gp35 coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMN; tobacco mosaic virus, TMN) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing a gp35 coding sequence; or animal cell systems.
  • yeast transformed with recombinant yeast expression vectors containing an gp35 coding sequence e.g., insect cell systems infected with
  • any of a number of suitable transcription and translation elements may be used in the expression vector.
  • inducible promoters such as PI of bacteriophage ⁇ , ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used.
  • a preferred promoter is plac (with a laci q on the vector to reduce background expression).
  • a second preferred promoter is pT7 ⁇ lO, which is specific to T7 R A polymerase and is not recognized by E. coli R ⁇ A polymerase.
  • Examples of other host systems include, but are not limited to; cloning in insect cell systems using promoters such as the baculovirus polyhedrin promoter; cloning in plant cell systems using promoters derived from the genome of plant cells (e.g., heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g., the 35S R ⁇ A promoter of CaMN; the coat protein promoter of TMN); cloning in mammalian cell systems using promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5 K promoter); and generating cell lines that contain multiple copies of a gp35 D ⁇ A, SV40-, BPN- and EBN- based vectors may be used with an appropriate selectable
  • a number of expression vectors may be advantageously selected depending upon the use intended for the gp35 protein derivative or analog expressed.
  • vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBOJ. 2:1791), in which the gp35 coding sequence may be ligated into the vector in frame with the lacZ coding region so that a hybrid AS-lacZ protein is produced; PIN vectors (Inouye & Inouye, 1985, Nucleic acids Res.
  • PGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST) (Smith and Johnson, 1988, Gene 7:31-40). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • GST glutathione S-transferase
  • the PGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety.
  • yeast a number of vectors containing constitutive or inducible promoters may be used.
  • Current Protocols in Molecular Biology Vol. 2, 1988, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant et al., 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Ed. Wu & Grossman, 1987, Acad. Press, N.Y. 153:516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in
  • the expression of a gp35 coding sequence may be driven by any of a number of promoters.
  • viral promoters such as the 35S RNA and 19S RNA promoters of CaMN (Brisson et al., 1984, Nature 310:511-514), or the coat protein promoter of TMN (Takamatsu et al., 1987, EMBO J. 3:1311) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J.
  • An alternative expression system which could be used to express a gp35 gene is an insect system.
  • Autographa californica nuclear polyhedrosis virus (AcNPN) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells.
  • a gp35 coding sequence may be cloned into non-essential regions (for example the polyhedrin gene) ofthe virus and placed under control of an Ac ⁇ PN promoter (for example, the polyhedrin promoter).
  • gp35 coding sequence Successful insertion of a gp35 coding sequence will result in inactivation ofthe polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed, (e.g., see Smith et al., 1983, J. Nirol. 46:584; Smith, U.S. Patent No. 4,215,051).
  • a number of viral based expression systems may be utilized.
  • a gp35 coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing gp35 in infected hosts, (e.g., see Logan & Shenk, 1984, Proc.
  • the vaccinia 7.5 K promoter may be used. (See, e.g., Mackett et al., 1982, Proc. Natl. Acad. Sci. USA 79:7415-7419; Mackett et al., 1984, J. Virol. 49:857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. USA 79:4927- 4931).
  • kits for use in a bacteria host include, but are not limited to, the PET system (Novagen, Inc., Madison, WI) and Superlinker vectors PSE280 and PSE380 (Invitrogen, San Diego, CA).
  • Specific initiation signals may also be required for efficient translation of an inserted gp35 coding sequence. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire gp 35 gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of a gp35 coding sequence is inserted, lacking the 5' end, exogenous translational control signals, including the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of a gp35 coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:516-544).
  • a host cell strain may be chosen which modulates the expression ofthe inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications and processing (e.g., cleavage) of protein products may be important for the function ofthe protein.
  • Different host cells have characteristic and specific mechanisms for post-transcriptional and post-translational processing and modification. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing ofthe foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing ofthe primary transcript, and phosphorylation ofthe gene product may be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, etc.
  • Preferred hosts for producing the proteins ofthe present invention are E. coli strains BL21 (DE3) and BL21 (DE/plys5) (NoVagen, Madison, Wisconsin).
  • gp35 protein, derivative or analog may be engineered.
  • host cells can be transformed with gp35 DNA controlled by appropriate expression control elements (e.g., bacterial promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), a selectable marker, and flanked by sequences that promote homologous recombination.
  • expression control elements e.g., bacterial promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • a selectable marker e.g., bacterial promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows for the stable integration ofthe plasmid into host chromosomes. This method may advantageously be used to engineer bacterial strains which express a gp35 protein.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt- cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol.
  • trpB which allows cells to utilize indole in place of tryptophan
  • hisD which allows cells to utilize histinol in place of histidine
  • ODC ornithine decarboxylase
  • 2- (difluoromethyl)-DL-ornithine 2- (difluoromethyl)-DL-ornithine
  • DFMO McConlogue, L., 1987, In: Current 5 Communications in Molecular Biology, Cold Spring Harbor Laboratory, Ed.
  • the present invention provides a method for producing a recombinant gp35 protein, derivative or analog comprising culturing a host cell transformed with a recombinant expression vector encoding a gp35 protein, derivative or analog, such that the gp35 protein, derivative or analog is expressed by the cell and recovering the expressed gp35 protein, 0 derivative or analog.
  • the host cells which contain the coding sequence and which express the gp35 g product or functionally active derivatives or analogs thereof may be identified by at least four general approaches; (a) DNA-DNA or DNA-RNA hybridization; (b) the presence or absence of "marker" gene functions; (c) assessing the level of transcription as measured by the expression of gp35 mRNA transcripts in the host cell; and (d) detection ofthe gene product as measured by immunoassay or by its biological activity.
  • the presence ofthe gp35 coding sequence inserted in the expression vector can be detected by DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide sequences that are homologous to the gp35 coding sequence, respectively, or derivatives (e.g., fragments) or analogs thereof.
  • the recombinant expression vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., resistance to antibiotics). For example, if the gp35 coding sequence is inserted within a marker gene sequence ofthe vector, recombinant cells containing the gp35 coding sequence can be identified by the absence ofthe marker gene function.
  • certain "marker" gene functions e.g., resistance to antibiotics.
  • a marker gene can be placed in tandem with a gp35 coding sequence under the control ofthe same or different promoter used to control the expression ofthe gp35 coding sequence. Expression ofthe marker in response to induction or selection indicates expression ofthe gp35 coding sequence.
  • transcriptional activity o ⁇ gp35 can be assessed by hybridization assays.
  • RNA can be isolated and analyzed by Northern blot using a probe having sequence homology to a gp35 coding sequence or transcribed noncoding sequence or particular portions thereof.
  • total nucleic acid ofthe host cell may be extracted and quantitatively assayed for hybridization to such probes.
  • the levels of a gp35 protein, derivative or analog product can be assessed immunologically, for example by Western blots, immunoassays such as radioimmuno-precipitation, enzyme-linked immunoassays and the like.
  • the gene product can be analyzed. This is achieved by assays based on the physical or functional properties ofthe product, including radioactive labelling ofthe product followed by analysis by gel electrophoresis, immunoassay, or other detection methods known to those of skill in the art.
  • the gp35 protein may be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • the functional properties may be evaluated using any suitable assay.
  • the amino acid sequence ofthe protein can be deduced from the nucleotide sequence ofthe chimeric gene contained in the recombinant.
  • the protein can be synthesized by standard chemical methods known in the art (e.g., see Hunkapiller et al., 1984, Nature 310:105-111).
  • the invention relates to a purified gp35 protein that is not contained in a gel suitable for electrophoresis.
  • the purified gp35 protein is not denatured.
  • the invention relates to a composition containing at least 1, 10, 50, 100 or 500 nanogram(s), 1, 10, 50, 100 or 500 microgram(s), or 1, 10, 50, 100 or 500 milligram(s), of purified non-denatured gp35 protein.
  • this composition is not a gel suitable for electrophoresis.
  • such gp35 proteins whether produced by recombinant DNA techniques or by chemical synthetic methods include, but are not limited to, those containing, as a primary amino acid sequence, all or part ofthe amino acid sequence substantially as depicted in Figure 2 (SEQ ID NO:2), as well as fragments and other derivatives, and analogs thereof.
  • gp35 protein may be used as an immunogen to generate antibodies which recognize such an immunogen.
  • antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library.
  • gp35 specific antisera is prepared according to procedures as described by Edgar (1965, Genetics 52: 1187) and Ward (1970, J. Mol. Biol. 54:15). Briefly, whole T4 bacteriophage are used as an immunogen; the resulting antiserum is then adsorbed with tail-less phage particles, thus removing all antibodies except those directed against the tail fiber proteins. In a subsequent step, different aliquots ofthe antiserum are adsorbed individually with extracts that each lack a particular tail fiber protein.
  • an extract containing only tail fiber components gp34, gp36 and gp37 (derived from a cell infected with a mutant T4 that does not produce gp35) is used for adsorption, the resulting antiserum will recognize only mature gp35 and dimerized gp35- P36 or gp35-P34.
  • antibody is raised against purified tail fiber halves, e.g., gp35-gp36-gp37.
  • anti gp35-gp36-gp37 is then adsorbed with gp36-gp37 to produce anti-gp35.
  • anti-gp35 is produced directly using purified gp35 proteins, derivatives or analogs thereof, as an immunogen.
  • monoclonal antibodies are generated against a gp35 protein sequence or analog thereof using techniques known in the art.
  • various host animals can be immunized by injection with the native gp35 protein, or a synthetic version, derivative (e.g., fragment) or analog thereof, including, but not limited to, rabbits, mice, rats, etc.
  • Various adjuvants may be used to increase the immunological response, depending on the host species, and including, but not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, BCG (bacille Calmette-Guerin) and corynebacterium parvum.
  • any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used.
  • the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBN-hybridoma technique to produce human monoclonal antibodies Colde et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • monoclonal antibodies can be produced in germ-free animals utilizing recent technology (PCT/US90/02545).
  • a molecule comprising a fragment ofthe gp35 protein is used as an immunogen.
  • the fragment used as the immunogen has a sequence that is all or a portion of amino acid residues 1 to 93, and lacks amino acid residues 94 to 373 in Figure 2. Since hydrophilic regions are believed most likely to contain antigenic determinants, a peptide corresponding to or containing a hydrophilic portion of a gp35 protein is preferably used as immunogen.
  • Antibody fragments which contain the idiotype ofthe molecule can be generated by known techniques.
  • such fragments include, but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion ofthe antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges ofthe F(ab') 2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • screening for the desired antibody can be accomplished by techniques known in the art, e.g. , ELISA (enzyme-linked immunosorbent assay).
  • ELISA enzyme-linked immunosorbent assay
  • the foregoing antibodies can be used in methods known in the art relating to the localization and activity ofthe protein sequences ofthe invention, e.g., for imaging these proteins, measuring levels thereof, in diagnostic methods, etc.
  • a non- limiting method by which anti-gp35 may also be used to detect gp35 tail fiber proteins, derivatives or analogs involves screening for bacterial colonies expressing proteins, derivatives or analogs by directly transferring the colonies, or, alternatively, samples of lysed or unlysed cultures, to nitrocellulose filters, lysing the bacterial cells on the filter if necessary, and incubating with specific antibodies. Formation of immune complexes may then be detected by methods widely used in the art (e.g., secondary antibody conjugated to a chromogenic enzyme or radiolabelled Staphylococcal Protein A.). This method is particularly useful to screen large numbers of colonies.
  • bacterial cells expressing the protein, derivative, or analog of interest are first metabolically labelled with 35 S-methionine, followed by preparation of extracts and incubation with antiserum. The immune complexes may then be recovered by incubation with immobilized Protein A followed by centrifugation and resolution by SDS-PAGE. 5.5. STRUCTURE OF THE ⁇ p35 GENE AND PROTEIN
  • the structure ofthe gp35 gene and protein can be analyzed by any of various methods known in the art. Representative methods are set forth below.
  • the cloned DNA co ⁇ esponding to gp35 can be analyzed by methods including, but not limited to, Southern hybridization (Southern, E.M., 1975, J. Mol. Biol. 98:503-517), Northern hybridization (see, e.g., Freeman et al., 1983, Proc. Natl. Acad. Sci. USA 80:4094- 4098), restriction endonuclease mapping (Maniatis, T., 1982, Molecular Cloning, A Laboratory, Cold Spring Harbor, New York), and DNA sequence analysis. Polymerase chain reaction (PCR; U.S. Patent Nos. 4,683,202, 4,683,195, and 4,889,818; Gyllenstein et al., 1988, Proc.
  • PCR Polymerase chain reaction
  • Restriction endonuclease mapping can be used to roughly determine the genetic structure of gp35. Restriction maps derived by restriction endonuclease cleavage can be confirmed by DNA sequence analysis.
  • DNA sequence analysis can be performed by any techniques known in the art, including, but not limited to, the method of Maxam and Gilbert (1980, Meth. Enzymol. 65:499-560), the Sanger dideoxy method (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74:5463), the use of T7 DNA polymerase (Tabor and Richardson, U.S. Patent No. 4,795,699), or use of an automated DNA sequenator (e.g., Applied Biosystems, Foster City, CA).
  • the nucleotide sequence of a representative gp35 gene comprises the sequence substantially as depicted in Figure 2 (SEQ ID NOJ), and described in Section 6, infra.
  • the amino acid sequence of a gp35 protein, derivative, fragment or analog can be derived by deduction from the DNA sequence, or alternatively, by direct sequencing ofthe protein, e.g., with an automated amino acid sequencer.
  • the amino acid sequence of a representative gp35 protein comprises the sequence substantially as depicted in Figure 2 (SEQ ID NO:2), and detailed in Section 6, infra, with the representative protein that is shown by amino acid numbers 1-372.
  • the gp35 protein sequence can be further characterized by a hydrophilicity analysis
  • a hydrophilicity profile can be used to identify the hydrophobic and hydrophilic regions ofthe gp35 protein and the corresponding regions ofthe DNA sequence which encode such regions. Hydrophilic regions are predicted to be antigenic/immunogenic. Secondary structural analysis (Chou, P., and Fasman, G., 1974, Biochemistry
  • JJ:222 JJ:222
  • Manipulation, translation, and secondary structure prediction, as well as open reading frame prediction and plotting, can also be accomplished using computer software programs available in the art.
  • the invention further relates to gp35 proteins, derivatives (including, but not limited to, fragments) and analogs of gp35 proteins.
  • Nucleic acids encoding gp35 proteins, derivatives and analogs are also provided.
  • Molecules comprising gp35 proteins, derivatives or analogs are also provided.
  • the gp35 proteins, derivatives or analogs are encoded by the gp35 nucleic acids described in Section 5J supra.
  • the derivative or analog is functionally active, i.e., capable of exhibiting one or more functional activities associated with a full-length, wild- type gp35 protein.
  • such derivatives or analogs which have the desired immunogenicity or antigenicity can be used, for example, in immunoassays, for inhibition of gp35 activity, etc.
  • such derivatives or analogs which are able to bind bacteriophage T4 tail fiber proteins P36 and/or P34 are provided.
  • Derivatives or analogs that retain a desired gp35 property of interest can be used as inhibitors of such property and its physiological correlates.
  • a specific embodiment relates to a gp35 fragment that can be bound by an anti-gp35 antibody.
  • Derivatives or analogs of gp35 can be tested for the desired activity by procedures known in the art, including, but not limited to, the assays described infra.
  • g ⁇ 35 derivatives can be made by altering gp35 sequences by substitutions, additions or deletions that provide for functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as a gp35 gene may be used in the practice of the present invention. These include, but are not limited to, nucleotide sequences comprising all or portions oigp35 which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
  • the gp35 derivatives ofthe invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part ofthe amino acid sequence of a gp35 protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.
  • Conservative substitutes for an amino acid within the sequence may be selected from other members ofthe class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • the invention relates to non-native bacteriophage T4 gp35 proteins, derivatives or analogs in which only conservative substitutions relative to the sequence in Figure 2 are made.
  • the invention also relates to non-native molecules encoded by a nucleic acid that is capable of hybridizing to gp35 coding sequence (SEQ ID NOJ), under stringent, moderately stringent, or nonstringent conditions.
  • the invention relates to proteins, derivatives or analogs comprising the amino acid sequence depicted in Figure 2 (SEQ ID NO:2) from amino acid residues 1-17, 1-56, 1-78, 1-93, 8-17, 57-93, 57-93, 57-64, 66-79, or 81-93.
  • these proteins contain only conservative substitutions relative to the sequence in Figure 2.
  • the invention additionally relates to proteins, derivatives or analogs, comprising an amino acid sequence that has at least 60%, 65%, 70%, 75%, 80%, 85%, or 90% amino acid sequence homology, to bacteriophage T4 gp35 amino acids number 1 to 100 in Figure 2 over a 100 amino acid sequence.
  • the invention further relates to proteins, derivatives, fragments or analogs comprising an amino acid sequence sharing at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% homology to amino acids numbers 57 to 93 in Figure 2 over a 36 amino acid sequence.
  • the invention further provides derivatives, fragments or analogs of a gp35 protein consisting of at least 8, 15, or 20 contiguous amino acids ofthe gp35 protein sequence depicted in Figure 2 (SEQ ID NO:2) from amino acids numbers 1 to 24.
  • the derivative, fragment or analog is not native and contains only conservative substitutions relative to the sequence in Figure 2.
  • the derivative or analog additionally displays one or more functional activities of a gp35 protein.
  • the derivative, fragment or analog specifically binds P34 and or P36.
  • the derivative or analog is able to be bound by an antibody directed against a gp35 protein in which only conservative substitutions relative to the sequence in Figure 2 are made.
  • the invention also provides derivatives or analogs of a gp35 protein consisting of at least 40, 45, 50, 60, or 70 contiguous amino acid residues ofthe gp35 protein sequence depicted in Figure 2 (SEQ ID NO:2) from amino acids numbers 1 to 100. In a specific embodiment, this derivative lacks amino acid residues 93 to 372.
  • Tail fiber assembly takes place in a predetermined, ordered interaction of specific bacteriophage protein subunits.
  • the angled joint ofthe tail fiber is formed by the two step process in which first, the N- terminus of P36 attaches to the carboxy terminal region of a gp35 monomer and second the N-terminal region of gp35-P36 oligomer then attaches to the C-terminus of P34.
  • a gp35 mutant/derivative or analog is provided in which the interaction ofthe gp35 derivative or analog with P34 is independent
  • gp35 derivatives or analogs form average angles with other tail fiber proteins that are different from the native angle of 137° or 158°.
  • the angle joint forms average angles of less than about 90°, 100°, 110°, 120°, or 125°, or more than about 145°, 155°, 165°, under conditions wherein the
  • g ⁇ 35 protein forms an angle of 137° when combined with P36-P37 and P34 dimers or trimers.
  • the angle joint of gp35 proteins, derivatives or analogs exhibit more or less flexibility than the native polypeptide.
  • gp35 sequence variants can be screened for the ability to form such an angle.
  • Thermolabile structures have many uses in nanostructure construction, such as, for example
  • gp35 derivatives and analogs exhibit thermolabile interactions with cognate partners.
  • the interaction of a gp35 derivative with a P36 protein oligomer of bacteriophage T4 is unstable at a temperature of about 40°C, 45°C, 50°C,
  • the interaction of a gp35 derivative with a P34 protein oligomer of bacteriophage T4 is unstable at a temperature of about 40°C, 45 °C, 50°C, 55 °C or 60°C (see Section 7).
  • the thermolabile interaction between gp35 and cognate partners is reversible, thereby permitting reattachment ofthe appropriate termini when the lower temperature is restored, in another
  • the gp35 derivative or analog interacts with only mutant cognate partners (e.g., see Section 7).
  • gp35 derivatives or analogs contain a mutant amino acid sequence, or are conjugated to a fixed group, that confers specific binding properties on the
  • sequences derived from avidin that recognize biotin sequences derived from immunoglobulin heavy chain that recognize Staphylococcal A protein, sequences derived from the Fab portion ofthe heavy chain of monoclonal antibodies to which their respective Fab light chain counterparts could attach and form an antigen-binding site, immunoactive sequences that recognize specific antibodies, or sequences that bind specific metal ions (e.g., divalent metal ions).
  • ligands may be immobilized to facilitate purification and/or assembly.
  • the fragment consists of at least 15 or 20 amino acids ofthe gp35 protein depicted in Figure 2 from amino acids number 1-24.
  • the invention also provides fragments of a gp35 protein consisting of at least 40, 45,
  • such fragments are not larger than 75, 100 or 150 amino acids. In other specific embodiments, such fragments lack amino acid number 93 to 372 in Figure 2.
  • Derivatives or analogs of gp35 include, but are not limited to, those molecules comprising regions that are substantially homologous to gp35 or fragments thereof (e.g., in various embodiments, at least 60% or 70% or 80% or 90% or 95% identity over an amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art) or whose encoding nucleic acid is capable of hybridizing to agp35 coding sequence, under stringent, moderately stringent, or nonstringent conditions.
  • the gp35 derivatives and analogs ofthe invention can be produced by various methods known in the art.
  • the manipulations which result in their production can occur at the gene or protein level.
  • the cloned gp35 sequence can be modified by any of numerous strategies known in the art (Maniatis, T., 1990, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).
  • the sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro.
  • the gp35-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
  • mutagenesis Any technique for mutagenesis known in the art can be used, including, but not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson, C, et al., 1978, J. Biol. Chem 253:6551), PCR amplification using primers with altered sequences, etc.
  • Manipulations ofthe gp35 sequence may also be made at the protein level. Included within the scope ofthe invention are gp35 protein fragments or other derivatives or analogs which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.
  • gp35 can be chemically synthesized.
  • a peptide corresponding to a specific portion of a gp35 protein see Section 5.6J, or which mediates the desired activity in vitro, can be synthesized by use of a peptide synthesizer.
  • nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the gp35 sequence.
  • Non- classical amino acids include, but are not limited to, the D-isomers ofthe common amino acids, ⁇ -amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, ⁇ -Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t- butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, fluoro-amino acids, designer amino acids such as ⁇ -methyl amino acids, C ⁇ -methyl amino acids, N ⁇ - methyl amino acids, N
  • the gp35 derivative is a molecule comprising a region of homology with a gp35 protein.
  • a first protein region can be considered "homologous" to a second protein region when the amino acid sequence ofthe first region is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95% identical, when compared to any sequence in the second region of an equal number of amino acids as the number contained in the first region or when compared to an aligned sequence ofthe second region that has been aligned by a computer homology program known in the art.
  • a molecule can comprise one or more regions homologous to a gp35 region (see Section 5.6J) or a full-length gp35 protein.
  • the gp35 proteins, derivatives, fragments or analogs ofthe invention are combined with other tail fiber proteins, derivatives, fragments and/or analogs, to form polygons.
  • a polygon is formed using the gp35 protein, derivative, or analog ofthe invention in combination with a P36-34 chimer rod unit as described in PCT Publication WO 96/11947, dated April 25, 1996.
  • gp35 proteins, derivatives and analogs can be assayed by various methods.
  • various immunoassays known in the art can be used, including, but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, gel
  • antibody binding is detected by
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labelled. Many means are known in the art for detecting binding in an immunoassay and are within the scope ofthe present invention.
  • gp35-binding protein e.g., P34 and
  • the binding can be assayed, by means well-known in the art.
  • a nonlimiting method by which antibodies specific to g ⁇ 35 proteins may be used to assay for the ability of gp35 proteins, derivatives or analogs to associate with other tail fiber proteins involves screening for bacterial colonies expressing mature tail fiber proteins by directly transferring the colonies, or, alternatively, samples of lysed or unlysed cultures, to nitrocellulose filters, lysing the bacterial cells on the filter if necessary, and incubating with antibodies specific for gp35 and its binding partner and detecting the formation of immune complexes by methods widely used in the art (e.g., secondary antibody conjugated to a chromogenic enzyme or radiolabelled Staphylococcal Protein A).
  • Another nonlimiting method involves metabolically labelling bacterial cells expressing gp35 with 35 S- methionine, preparing and incubating extracts of these cells with gp35 antiserum, recovering immune complexes by incubation with immobilized Protein A followed by centrifugation, and resolving the proteins by SDS-polyacrylamide gel electrophoresis.
  • a nonlimiting competitive assay for testing whether gp35 derivatives or analogs such as internally deleted tail fiber proteins that do not permit phage infection nonetheless retain the ability to associate with their appropriate partners utilizes an in vitro, complementation system which involves mixing a bacterial extract containing the modified gp35 tail fiber protein with a second extract prepared from cells infected with a phage that is agp35 null mutant and therefore does not produce gp35. After several hours of incubation, a third extract is added that contains wild-type gp35, and incubation is continued for several additional hours. Finally, the extract is titered for infectious phage particles by infecting E. coli and quantifying the phage plaques that result.
  • a modified gp35 protein, derivative or analog that correctly associates with its tail fiber partners is incorporated into tail fibers in a non- functional manner in the first mixture, thereby preventing the incorporation ofthe wild-type version ofthe protein after addition ofthe third extract; the result is a reduction in the titer ofthe resulting phage sample.
  • the modified gp35 protein, derivative or analog is unable to associate with its binding partner, it will not be incorporated into phage particles in the first mixture and, thus, will not compete with assembly of intact phage particles when the third extract is added; the phage titer should thus be equivalent to that observed when no modified gp35 is added in to the first mixture (a negative control).
  • Assays for testing whether gp35 proteins, derivatives, such as internally deleted proteins, or analogs that do not permit phage infection nonetheless retain the ability to associate with appropriate tail fiber partners can also be performed in vivo. These assays detect the ability of gp35 proteins, derivatives, or analogs to compete with normal phage parts for assembly, thus reducing the burst size of a wild-type phage infecting the same host cell in which g ⁇ 35 proteins, derivatives, or analogs are recombinantly expressed. Thus, expression from an expression vector encoding the gp35 proteins, derivative, or analogs is induced inside a cell, which cell is then infected by a wild-type phage. Inhibition of wild- type phage production demonstrates the ability ofthe recombinant gp35 protein, derivative, or analog to associate with the appropriate tail fiber proteins ofthe phage.
  • the gp35 proteins, derivatives, and analogs ofthe invention have use in the construction of nanostructures.
  • the uses of such nanostructures are manifold and include applications that require highly regular, well-defined arrays of fibers, cages, or solids, which may include specific attachment sites that allow them to associate with other materials.
  • a three-dimensional hexagonal array of tubes is used as a molecular sieve or filter, providing regular vertical pores of precise diameter for selective separation of particles by size.
  • filters can be used for sterilization of solutions (i.e., to remove microorganisms or viruses), or as a series of molecular- weight cut-off filters.
  • the protein components ofthe pores may be modified so as to provide specific surface properties (i.e., hydrophilicity or hydrophobicity, ability to bind specific ligands, etc.).
  • specific surface properties i.e., hydrophilicity or hydrophobicity, ability to bind specific ligands, etc.
  • long one-dimensional fibers are incorporated, for example, into paper or cement or plastic during manufacture to provide added wet and dry tensile strength.
  • different nanostructure arrays are impregnated into paper and fabric as anti-counterfeiting markers. In this case, a simple color-linked antibody reaction (such as those commercially available in kits) is used to verify the origin ofthe material.
  • such nanostructure arrays could bind dyes or other substances, either before or after incorporation to color the paper or fabrics or modify their appearance or properties in other ways.
  • the nanostructures comprising recombinant gp35 and its derivatives, fragments and analogs include, but are not limited to, other polygonal structures such as octagons, as well as open solids such as tetrahedrons and icosahedrons formed from triangles and boxes formed from squares and rectangles.
  • the range of structures is limited only by the types of angle units and the substituents that can be engineered on the different axes ofthe rod units.
  • other naturally occurring angles are found in the fibers of bacteriophage T7, which has a 90° angle (Steven et al., J. Mol. Biol. 200: 352-365, 1988).
  • the use of bacteriophage tail fiber components in the construction of nanostructures is further described in PCT Publication WO 96/11947, dated April 25, 1996, which is incorporated by reference herein in its entirety.
  • gp35 proteins, derivatives, fragments and analogs ofthe invention have use in the study and research ofthe bacteriophage T4 life cycle.
  • T4 tail fiber proteins gp34, gp35, gp36, and gp37 are produced naturally following infection of E. coli cells by intact T4 phage particles.
  • P34, P36, and P37 homooligomers are stiff and rod-shaped proteins in which two identical ⁇ sheets, oriented in the same direction, are fused face-to-face by hydrophobic interactions between the sheets juxtaposed with a 180° rotational axis of symmetry through the long axis ofthe rod.
  • gp35 is a monomeric polypeptide that attaches specifically to the N-terminus of a P36 homooligomer and then to the C-terminus of a P34 homooligomer and forms an angle joint between two rods at an average angle of 137° ( ⁇ 7°) or 156° ( ⁇ 12°).
  • coli, gp37 (the monomeric 109 Kda translation product of gene 37) forms the homooligomer P37, with the aid of 2 accessory (chaperon) proteins, gp57 and gp38; this process is believed to initiate near the C-terminus of gp37.
  • the N-terminus of P37 initiates the oligomerization of two g ⁇ 36 molecules of 23 Kda each, in a butt-end joint to form the P36 homooligomer rod.
  • the N-terminus of P36 then attaches to the carboxy terminal region of a gp35 monomer; this interaction stabilizes P36 and forms the flexible angle joint ofthe tail fiber.
  • the nanostructures ofthe invention are composed of tail fiber chimers, such as for example, P36-34, which is an oligomer ofthe fusion protein gp36-34; gp36-34 consists of a portion of gp36 containing the amino terminus fused to a portion of gp34 containing the carboxy terminus.
  • Expression vectors encoding such chimers may be constructed using recombinant technology known in the art.
  • Such chimers have novel functional properties, including but not limited to rod domains and/or N- and C-termini combinations that are different from native tail fiber proteins. Chimers having novel N- and C- termini combinations allow for new patterns for joining different rod segments.
  • polygon nanostructures may be generated using P36-34 chimeric fusion proteins and gp35.
  • the creation of constructs encoding tail fiber fusion chimers, such as P36-34, and their use in generating nanostructures, is further described in PCT Publication WO 96/11947, dated April 25, 1996, which is incorporated by reference herein in its entirety.
  • Recombinant expression ofthe proteins ofthe present invention in E. coli as described above results in the synthesis of large quantities of protein, and allows the simultaneous expression and assembly of different components in the same cells.
  • the methods for scale-up of recombinant protein production are straightforward and widely known in the art, and many standard protocols can be used to recover native and modified tail fiber proteins from a bacterial culture.
  • recombinant gp35 is isolated for use by growing host cells transformed or adsorbed with nucleotide sequence encoding a gp35 protein having the amino acid sequence depicted in Figure 2, operably linked to a heterologous promoter, under conditions in which the gp35 encoding nucleic acid is expressed, and isolating gp35 from the resulting culture by standard methods.
  • P34, P36-P37, P37 and chimers derived therefrom are purified from phage-infected (or recombinant) E. coli cultures as mature oligomers.
  • gp35 protein, derivatives or analogs thereof are purified as monomers.
  • Standard methods may be utilized to isolate and purify the nanostructure components, these methods include but are not limited to: chromatography on molecular sieve, ion-exchange, and/or hydrophobic matrices; preparative ultracentrifugation; and affinity chromatography, using as the immobilized ligand specific antibodies or other specific binding.
  • the proteins have been engineered to include heterologous domains that act as ligands or binding sites
  • the cognate partner may be immobilized on a solid matrix and used in affinity purification.
  • a heterologous domain can be avidin, which binds to a biotin-coated solid phase.
  • phage tail fiber components and where necessary, chaperon proteins such as gp57 and gp37 required for homooligomerization, are co-expressed in the same bacterial cells, and sub-assemblies of larger nanostructures are purified subsequent to limited in vivo assembly, using the methods enumerated above.
  • the purified nanostructure components and/or subassemblies are combined in vitro under conditions where assembly ofthe desired nanostructure occurs at temperatures between about 4°C and about 37°C, and at pH's between about 5 and about 9.
  • optimal conditions for assembly i.e., type and concentration of salts and metal ions
  • one or more crude bacterial extracts are prepared, mixed, and assembly reactions are allowed to proceed prior to purification.
  • one or more purified components assemble spontaneously into the desired structure, without the necessity for initiators. In other cases, an initiator is required to nucleate the polymerization ofthe nanostructure.
  • polygons are assembled using gp35 and P36-34 chimer.
  • gp57 is used to chaperon the homodimerization of gp36-34 to P36-34.
  • P36-34 chimer is added to a solution containing a gp35 initiator that optionally is reversibly immobilized using methods known in the art, so as to allow binding of P36-34 chimer.
  • gp35 and P36-34 are administered as a mixture or sequentially to form the desired polygon structure.
  • the type of polygon that is formed using this protocol depends upon the length of rod units and the angle formed by the angle joint. For example, alternating rod units of different sizes can be used.
  • variant gp35 polypeptides that form angles different than the natural angle can be used, allowing the formation of different regular polygons.
  • the sides in either half can be of any size provided the two halves are symmetric.
  • thermolabile a mutant (thermolabile) gp34 that can be made to detach upon exposure to a higher temperature (e.g., 40°C).
  • Such a mutant gp34 termed T4 tsB45, having a mutation at its C-terminal end such that gp34 attaches to the distal tail fiber half at 30°C, but can be separated from it in vitro by incubation at 40 °C in the presence of 1% SDS (unlike wild- type T4 which are stable under these conditions), has been reported (Seed, 1980, Studies of the Bacteriophage T4 Proximal Half Tail Fiber, Ph.D. Thesis, California Institute of Technology), and can be used.
  • the polymer can be easily separated from the matrix-bound initiator, thereby permitting: easy preparation of stock solutions of uniform parts or subassemblies, and re-use ofthe matrix-bound initiator for multiple cycles of polymer initiation, growth, and release.
  • T4 bacteriophage gene 35 a gene encoding a tail fiber protein which functions to join the rodlike proximal and distal halves ofthe bacteriophage tail fibers.
  • Phage T4 gp35 is located between gene 34 and gene 36.
  • a sequence for gp35 is available on the NCBI database (NCBI.NIH.GOV) within the sequence T4g34-t (nucleotides 4188-5075; see Figure 3). The NCBI sequence predicts that the gp35 open reading frame, ORF35 encodes a putative protein having a molecular weight of 32,334 Daltons.
  • ORF34J encodes the N-terminus of gp35.
  • gp35 contains a single ORF of 1, 119 nucleotide pairs having 373 codons, of which 372 encode a protein having a putative molecular weight of 40,096 Daltons.
  • the terminal codon ofthe gp35 open reading frame is the ochre stop codon, TAA.
  • This 1,119 nucleotide sequence was compared with the 1 ,121 nucleotide sequence from the NCBI database using the FASTA program. Six differences were detected between the sequence and that ofthe NCBI sequence.
  • deletion ofthe adenine at nucleotide 22 ofthe NCBI sequence insertion of a thymine between the adenine at nucleotide 49 and the thymine at nucleotide 50 ofthe NCBI sequence; deletion ofthe cytosine at nucleotide 170 ofthe NCBI sequence; change of nucleotide 238 from a thymine to a cytosine ofthe NCBI sequence; deletion ofthe thymine at nucleotide 280 ofthe NCBI sequence; and change of nucleotide 557 ofthe NCBI sequence from an adenine to a guanine.
  • the sequence ofthe N-terminal 10 residues ofthe induced protein generated from the expression vector construct were determined to be identical to the first ten residues the inventor predicted for the new gp35 ORF.
  • the determination of residues 8, 9 and 10 in the induced protein to be phenylalanine, glycine and glutamine, instead ofthe isoleucine, tryptophan and threonine residues respectively predicted for ORF 34J ofthe NCBI database sequence proves that the new gp35 ORF sequence is correct and that the adenine located at nucleotide 22 in the NCBI sequence, is the result of a sequencing error and is not actually present in bacteriophage T4 gene 35.
  • the inventor has therefore shown that the correct gp35 sequence is not that previously reported, but actually is a larger protein with a different N-terminus, that is 24% heavier than that predicted from the published sequence.
  • the correct gp35 sequence encodes 77 more N-terminal amino acid residues than the NCBI sequence. Additionally, 15 ofthe first 16 N-terminal residues encoded by the NCBI sequence are incorrect.
  • a variant (temperature-sensitive) gp35 that permits heat induced separation ofthe Q gp35-P36 junction may be formed by mutagenizing the 3' region of gp35 DNA (encoding the carboxy terminal region of gp35) with randomly doped ohgonucleotides. Randomly doped ohgonucleotides are prepared during chemical synthesis of ohgonucleotides, by adding a trace amount (up to a few percent) ofthe other three nucleotides at a given position, so that the resulting ohgonucleotide mix has a small percentage of incorrect 5 nucleotides at that position (Hutchison et al., 1991, Methods Enzymol. 202:356).
  • the mutagenized DNA fragment is then recombined into T4 phage by infection ofthe cell containing the mutagenized DNA by a T4 phage containing two amber mutations flanking the mutagenized region.
  • non-amber phage are selected at low temperature on E. coli Su° at 30°C.
  • the progeny of these plaques are Q resuspended in a buffered solution and challenged by heating at 60°C. At this temperature, wild-type tail fibers remain intact and functional, whereas the thermolabile versions release the P36 units and thus render those phage non-infectious.
  • wild type phage are removed either by adsorbing the wild type phage to sensitive bacteria and sedimenting (or filtering out) the bacteria with the adsorbed wild 5 tyP e phage or by reacting the lysate with anti-gp35-P36 specific antibody, followed by immobilized Protein A and removal of adsorbed wild type phage. Either of these methods leaves the noninfectious mutant phage particles in the supernatant fluid or filtrate, from which they can be recovered.
  • non-infectious phage lacking terminal gp35-P36 moieties are then urea treated with 6M urea, and mixed with bacterial spheroplasts to permit Q infection at low multiplicity whereupon they replicate at low temperature and release progeny.
  • infectious phage are reconstituted by in vitro incubation ofthe mutant phage with wild type P36 at 30 °C; this is followed by infection of intact bacterial cells using a standard protocol. The latter method of infection specifically selects mutant phage in which the thermolability ofthe gp35-P36 junction is reversible.
  • the phage populations are subjected to multiple rounds of selection, after which individual phage particles are isolated by plaque purification at 30°C.
  • the putative mutants are evaluated individually for: loss of infectivity after incubation at high temperatures (40-60 °C), as measured by a decrease in titer; loss of P36 after incubation at high temperature, as measured by decrease in binding of gp35-P36-specific antibody to phage particles; and morphological changes in the tail fibers after incubation at high temperatures, as assessed by electron microscopy.
  • the mutants are isolated and their phenotypes confirmed, the gp35 gene is preferably sequenced. If the mutations localize to particular regions or residues, those sequences are preferably targeted for site-directed mutagenesis to optimize the desired characteristics.
  • mutant gene 35 is cloned into an expression plasmid and expressed individually in E. coli.
  • the mutant gp35 protein is then purified from bacterial extracts and used in vitro assembly reactions.
  • gp35 variants can be isolated that exhibit a thermolabile interaction with P34.
  • the screen for gp35 mutants exhibiting a thermolabile interaction with P34 involves random doped ohgonucleotide mutagenesis ofthe entire gp35 gene. Mutants generated according to the experimental protocol described above are incubated at a high temperature, resulting in the loss ofthe entire distal half of the tail fiber (i.e., gp35-P36-P37) in the thermolabile mutants.
  • Wild-type phage (and distal half-fibers from thermolabile mutants) are then separated from thermolabile mutant phage that have been inactivated at high temperature (but still have proximal half tail fibers attached) by precipitating both the distal half- fibers and the phage particles containing intact tail fibers with any ofthe anti-distal half tail-fiber antibodies and protein-A beads. Mutant phage remaining in the supernatant are then reactivated by incubation at low temperature with bacterial extracts containing wild type intact distal half fibers. The thermolabile gene 35 mutants grown at 30 °C can be tested for reversible thermolability by inactivation at 60°C and reincubation at 30°C.
  • Inactivation is performed on a concentrated suspension of phage, and reincubation at 30 °C is performed either before or after dilution. If phage are successfully reactivated before, but not after dilution, this indicates that their gp35 is reversibly thermolabile.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne des séquences de nucléotides de gènes gp35, des séquences d'acides aminés de leurs protéines codées, ainsi que leurs dérivés et analogues, et leurs anticorps. Elle concerne également l'utilisation de séquences de nucléotides du gène 35 du bactériophage T4 et de séquences d'acides aminés de sa protéine codée, et leurs dérivés, variantes et analogues dans la construction de nanostructures.
PCT/US1999/013024 1999-06-11 1999-06-11 Gene et sequences de proteines du gene 35 de phage t4 WO2000077196A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
AU46781/99A AU4678199A (en) 1999-06-11 1999-06-11 Gene and protein sequences of phage t4 gene 35
PCT/US1999/013024 WO2000077196A1 (fr) 1999-06-11 1999-06-11 Gene et sequences de proteines du gene 35 de phage t4
CA002375998A CA2375998A1 (fr) 1999-06-11 1999-06-11 Gene et sequences de proteines du gene 35 de phage t4
JP2001503640A JP2003507007A (ja) 1999-06-11 1999-06-11 ファージｔ４の遺伝子３５の遺伝子配列およびタンパク質配列
EP19990930192 EP1185638A4 (fr) 1999-06-11 1999-06-11 Gene et sequences de proteines du gene 35 de phage t4
HK02104995.5A HK1043155A1 (zh) 1999-06-11 2002-07-03 噬菌體t4基因35的基因及蛋白質排列

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1999/013024 WO2000077196A1 (fr) 1999-06-11 1999-06-11 Gene et sequences de proteines du gene 35 de phage t4

Publications (1)

Publication Number Publication Date
WO2000077196A1 true WO2000077196A1 (fr) 2000-12-21

Family

ID=22272920

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/013024 WO2000077196A1 (fr) 1999-06-11 1999-06-11 Gene et sequences de proteines du gene 35 de phage t4

Country Status (6)

Country Link
EP (1) EP1185638A4 (fr)
JP (1) JP2003507007A (fr)
AU (1) AU4678199A (fr)
CA (1) CA2375998A1 (fr)
HK (1) HK1043155A1 (fr)
WO (1) WO2000077196A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1572897A2 (fr) * 2001-11-08 2005-09-14 United States of America, as represented by the Administrator of the National Aeronautics and Space Administration ( NASA) Nanostructures biologiques ordonnees produites a partir de polypeptides de chaperonine
US7795388B2 (en) 2001-11-08 2010-09-14 The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration (Nasa) Versatile platform for nanotechnology based on circular permutations of chaperonin protein
WO2016055870A1 (fr) * 2014-10-10 2016-04-14 Universidad Del Valle Bactériophage artificiel à base de nanostructures de carbone pour l'administration de médicaments

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996011947A1 (fr) * 1994-10-13 1996-04-25 Goldberg Edward B Materiaux pour la production de structures nanometriques et leur utilisation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996011947A1 (fr) * 1994-10-13 1996-04-25 Goldberg Edward B Materiaux pour la production de structures nanometriques et leur utilisation
US5864013A (en) * 1994-10-13 1999-01-26 Nanoframes, Llc Materials for the production of nanometer structures and use thereof
US5877279A (en) * 1994-10-13 1999-03-02 Nanoframes, Llc Materials for the production of nanometer structures and use thereof

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DICKSON R. C.: "Assembly of Bacteriophage T4 Tail Fibers, IV. Subunit Composition of Tail Fibers and Fiber Precursors", JOURNAL OF MOLECULAR BIOLOGY, vol. 79, no. 4, 5 October 1973 (1973-10-05), pages 633 - 647, XP002924018 *
OLIVER D. B.: "DNA Sequence of the Tail Fibre Genes 36 and 37 of Bacteriophage T4", JOURNAL OF MOLECULAR BIOLOGY, vol. 153, 1981, pages 545 - 568, XP002924020 *
REVEL H R: "ORGANIZATION OF THE BACTERIOPHAGE T4 TAIL FIBER GENE CLUSTER 34-34", BACTERIOPHAGE ASSEMBLY, XX, XX, 1 January 1981 (1981-01-01), XX, pages 353 - 364, XP002924017 *
REVEL H. R.: "Molecular Cloning of the T4 Genome: Organization and Expression of the Tail Fiber Gene Cluster 34-38", MOLECULAR AND GENERAL GENETICS, vol. 182, no. 3, 1981, pages 445 - 455, XP002924019 *
See also references of EP1185638A4 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1572897A2 (fr) * 2001-11-08 2005-09-14 United States of America, as represented by the Administrator of the National Aeronautics and Space Administration ( NASA) Nanostructures biologiques ordonnees produites a partir de polypeptides de chaperonine
EP1572897A4 (fr) * 2001-11-08 2007-06-13 Nasa Nanostructures biologiques ordonnees produites a partir de polypeptides de chaperonine
US7795388B2 (en) 2001-11-08 2010-09-14 The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration (Nasa) Versatile platform for nanotechnology based on circular permutations of chaperonin protein
US7816491B2 (en) 2001-11-08 2010-10-19 The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration Ordered biological nanostructures formed from chaperonin polypeptides
WO2016055870A1 (fr) * 2014-10-10 2016-04-14 Universidad Del Valle Bactériophage artificiel à base de nanostructures de carbone pour l'administration de médicaments

Also Published As

Publication number Publication date
HK1043155A1 (zh) 2002-09-06
EP1185638A1 (fr) 2002-03-13
CA2375998A1 (fr) 2000-12-21
JP2003507007A (ja) 2003-02-25
AU4678199A (en) 2001-01-02
EP1185638A4 (fr) 2002-10-18

Similar Documents

Publication Publication Date Title
KR100425966B1 (ko) 미소구조생산용물질과이의용도
Zhao et al. In vitro assembly of cowpea chlorotic mottle virus from coat protein expressed in Escherichia coli and in vitro-transcribed viral cDNA
MXPA97002657A (es) Materiales para la produccion de estructuras nanometricas y uso de las mismas
JPH07502640A (ja) シグナルペプチド、選択的相互作用ポリペプチド及び膜固定配列をコードする組換えｄｎａ配列
JPH05501062A (ja) Ｈｉｖ―１のウイルス性コンプレックス抗原に結合する組換え蛋白質
WO1989009277A1 (fr) Angiogenine humaine mutante (facteur d'angiogenese avec activite plus intense de l'angiogenine), gene s'y rapportant et procedes d'expression
JPH10507933A (ja) 可溶性で活性なｃ型肝炎ウイルスプロテアーゼ
KR20220101738A (ko) 생체자기 마이크로스피어 및 이의 제조 방법과 용도
US6084069A (en) Autotaxin: motility stimulating protein useful in cancer diagnosis and therapy
JPH03155795A (ja) マウス・インターロイキン―６レセプター蛋白質
JP2708046B2 (ja) 融合タンパク質および粒子
KR102128453B1 (ko) 황열 바이러스 유래 ns1 단백질, 이에 특이적으로 결합하는 단일클론항체 및 이의 용도
EP4228779A1 (fr) Agents d'affinité aav8
EP1185638A1 (fr) Gene et sequences de proteines du gene 35 de phage t4
Wang et al. Cloning of the J gene of bacteriophage lambda, expression and solubilization of the J protein: first in vitro studies on the interactions between J and LamB, its cell surface receptor
CA2139517A1 (fr) Conjugues immunologiques d'epitopes de neutralisation principaux, propres au vih
JPH10508479A (ja) 天然疎水性ペプチドアナログとしての組換えペプチドの産生
CA2183362A1 (fr) Polypeptide en mosaique et procedes de detection du virus de l'hepatite e
WO1992009633A1 (fr) Proteines de liaison d'immunoglobuline et molecules d'adn de recombinaison codant pour ces proteines
WO1997046583A1 (fr) Nouvelle proteine de cytokine feline
US7507790B2 (en) Fiber-shaping peptides capable of interacting with self-assembling peptides
JPH11124398A (ja) Ｃ型肝炎ウイルス由来の抗原ペプチドとそれを用いる抗体検査薬
WO2024102190A2 (fr) Agents d'affinité
JPH0430791A (ja) 構造蛋白質遺伝子、組換えベクター、組換えウイルス、ポリペプチドおよびポリペプチドの製造方法
JP2002510967A (ja) 多発性硬化症に罹患している患者の抗体との反応が可能なポリペプチド及び使用

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
ENP Entry into the national phase

Ref document number: 2375998

Country of ref document: CA

Ref country code: CA

Ref document number: 2375998

Kind code of ref document: A

Format of ref document f/p: F

ENP Entry into the national phase

Ref country code: JP

Ref document number: 2001 503640

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 10009874

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 1999930192

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1999930192

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWW Wipo information: withdrawn in national office

Ref document number: 1999930192

Country of ref document: EP