WO2006055040A2 - Identification de proteines dans un genome - Google Patents

Identification de proteines dans un genome Download PDF

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WO2006055040A2
WO2006055040A2 PCT/US2005/019477 US2005019477W WO2006055040A2 WO 2006055040 A2 WO2006055040 A2 WO 2006055040A2 US 2005019477 W US2005019477 W US 2005019477W WO 2006055040 A2 WO2006055040 A2 WO 2006055040A2
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site
protein
vector
proteins
soluble
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PCT/US2005/019477
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WO2006055040A3 (fr
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James L. Hartley
Dominic Esposito
Kelly Jeanne Stanard
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Government Of The United States Of America, Department Of Health And Human Services
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/90—Vectors containing a transposable element

Definitions

  • This invention pertains to methods for identifying proteins in a sample.
  • Drug discovery a process by which bioactive compounds are identified and preliminarily characterized, is a critical step in the development of treatments for human diseases.
  • Successful drug discovery depends upon the elucidation of the molecule or molecules in a human cell targeted by a particular candidate drug.
  • targets are cellular proteins
  • the primary bottleneck in protein structure identification is the difficulty in obtaining sufficient quantities of soluble proteins from mammalian cells. Indeed, when expressed recombinantly in heterologous hosts, many human proteins are expressed at low levels, are insoluble, or are soluble but difficult to purify. Obtaining sufficient quantities of soluble proteins for structural analysis, however, is difficult. Another difficulty is the lack of suitable methods for screening hundreds, or even thousands, of proteins for those that are expressed, soluble, and can be purified.
  • a method for producing soluble deletion derivatives of a protein comprises (a) preparing a vector comprising a nucleic acid sequence encoding the protein, wherein the nucleic acid sequence is flanked by a first and a second site-specific recombination site, and wherein the first and second site-specific recombination sites do not recombine with each other, (b) incubating the vector of (a) in the presence of one or more transposons and a transposase protein under conditions sufficient to cause insertion of the one or more transposons into the vector, wherein each of the one or more transposons comprises a third and a fourth site-specific recombination site, (c) transferring the nucleic acid sequence into a second vector, wherein the second vector comprises a fifth and a sixth site-specific recombination site, and propagating the second vector in a bacterium, (d) isolating one or more second vectors of (c
  • a method for identifying each of two or more soluble proteins comprises (a) preparing a mixture of two or more vectors each comprising a nucleic acid sequence encoding a soluble protein operatively linked to a promoter, (b) transferring each of the two or more vectors into one or more cells, (c) expressing the nucleic acid sequence in the one or more cells, wherein the two or more soluble proteins are produced, (d) purifying the two or more soluble proteins from the one or more cells, (e) separating the two or more soluble proteins, (f) isolating each of the two or more soluble proteins, and (g) subjecting the two or more soluble proteins to mass spectrometry, whereupon (i) each of the two or more soluble proteins is identified, and (ii) the amount of each of the two or more soluble proteins produced in the one or more cells is determined.
  • the above-described method of producing soluble deletion derivatives of a protein and the method of identifying two or more soluble proteins can be used alone or in combination.
  • Figure 1 is a diagram illustrating a method of producing an amino terminal deletion derivative of a protein in accordance with the inventive method.
  • Figure 2 is a diagram illustrating a method of producing a carboxy terminal deletion derivative of a protein in accordance with the inventive method.
  • Figure 3 is a diagram illustrating a method of producing an amino terminal deletion derivative of a protein using a transposon comprising one site-specific recombination site in accordance with the inventive method.
  • Figure 4 is a diagram illustrating a method of producing a carboxy terminal deletion derivative of a protein using a transposon comprising one site-specific recombination site in accordance with the inventive method.
  • Figure 5 is a diagram illustrating a method of producing a double deletion derivative of a protein in accordance with the inventive method.
  • Figure 6 is an image of a 2D gel on which 688 affinity-purified Caenorhabditis elegans proteins were separated.
  • Figure 7 is an image of an SDS-PAGE gel demonstrating expression of each of twelve C. elegans ORFs identified by the inventive method. Each lane of the gel represents the total protein in each cell culture.
  • Figure 8 is an image of an SDS-PAGE gel demonstrating the soluble (S) and insoluble (I) proteins present in cells transformed with vectors containing each of twelve C. elegans ORFs identified by the inventive method.
  • Figure 9 is an image of an SDS-PAGE gel demonstrating expression of C. elegans ORF #8 induced with 1 mM IPTG (I), and in the uninduced (U) state.
  • Figure 10 is an image of an SDS-PAGE gel demonstrating the soluble (S) and insoluble (I) proteins produced by C. elegans ORF #8 as compared to a negative control
  • Figure 1 Ia is an image of an SDS-PAGE gel demonstrating protein expression in whole cells transduced with C. elegans ORFs identified in accordance with the inventive method.
  • Figure 1 Ib is an image of an SDS-PAGE gel demonstrating protein expression in the soluble fraction of proteins expressed in cells transduced with C. elegans ORFs identified in accordance with the inventive method.
  • Figure 1 Ic is an image of an SDS-PAGE gel demonstrating protein expression in the insoluble fraction of proteins expressed in cells transduced with C. elegans ORFs identified in accordance with the inventive method.
  • Figure 1 Id is an image of an SDS-PAGE gel demonstrating IMAC-purified soluble proteins expressed in cells transduced with C. elegans ORFs identified in accordance with the inventive method.
  • the invention provides a method for producing soluble deletion derivatives of a protein.
  • the method comprises (a) preparing a vector comprising a nucleic acid sequence encoding the protein, wherein the nucleic acid sequence is flanked by a first and a second site-specific recombination site, and wherein the first and second site-specific recombination sites do not recombine with each other, (b) incubating the vector of (a) in the presence of one or more transposons and a transposase protein under conditions sufficient to cause insertion of the one or more transposons into the vector, wherein each of the one or more transposons comprises a third and a fourth site-specific recombination site, (c) transferring the nucleic acid sequence into a second vector, wherein the second vector comprises a fifth and a sixth site-specific recombination site, and propagating the second vector in a bacterium, (d) isolating one or more second vectors of (c) comprising the one or
  • the inventive method desirably is a high-throughput method in that it enables large-scale production of deletion derivatives (e.g., 100 or more, 500 or more, 1000 or more, or even 1,000 or more deletion derivatives). Typically and preferably, all or part of the method is automated.
  • deletion derivative refers to a variant form of a full- length protein that contains a deletion of one or more amino acids from the amino acid sequence of the full-length protein.
  • the one or more amino acids are deleted from the amino acid sequence of the full-length protein by deletion of one or more nucleotides from the nucleic acid sequence encoding the full-length protein. Any number of amino acids can be deleted from the full-length protein to produce the deletion derivative.
  • soluble is meant that the deletion derivative is capable of dissolving in a fluid, most preferably an aqueous buffer. The solubility of the deletion derivative can be determined using any suitable method known in the art, such as, for example, centrifugation- or filtration-based protein purification methods.
  • the inventive method comprises preparing vectors comprising a nucleic acid sequence encoding a protein.
  • the term "vector” refers to a nucleic acid molecule that can transfer a heterologous nucleic acid insert contained therein from one organism to another.
  • the vector desirably is a nucleic acid molecule (e.g., DNA or RNA).
  • the vector is comprised of DNA.
  • the vector can be any suitable vector, such as a plasmid, a virus, a phage, an autonomously replicating sequence (ARS), or any other sequence that is capable of replicating in vitro or in a host cell.
  • ARS autonomously replicating sequence
  • the vector is a plasmid that contains one or more restriction endonuclease sites which facilitate incorporation of the heterologous nucleic acid insert and heterologous regulatory sequences.
  • Vectors can be prepared using standard recombinant DNA and molecular biology techniques, such as those described in Sambrook et al., Molecular Cloning, a Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N. Y. (2001), Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N. Y. (1994), Hartley et al., Genome Res., 10, 1788-95 (2000) and U.S.
  • the inventive method comprises preparing a first vector comprising a nucleic acid sequence encoding the protein, wherein the nucleic acid sequence is flanked by a first and a second site-specific recombination site, and wherein the first and second site-specific recombination sites do not recombine with each other.
  • the nucleic acid sequence incorporated into the vector can encode any suitable protein from any suitable organism.
  • the protein is an animal protein.
  • the protein can be derived from any suitable animal.
  • Suitable animals include, for example, protozoa, echinoderms (e.g., sea urchin), annelids (e.g., earthworms), nematodes (e.g., C. elegans), mollusks, arthropods (e.g., crustaceans), insects, birds, amphibians, reptiles, and mammals (e.g., primates and rodents).
  • the organism is a mammal, and most preferably the organism is a human.
  • the nucleic acid sequence encodes a protein that is a target for a therapeutic agent (i.e., a drug).
  • the nucleic acid sequence can encode a protein that is a target for a known therapeutic agent, such as those described in, for example, Physician 's Desk Reference, Medical Economics Co., Inc., Montvale, New Jersey (2004).
  • the nucleic acid sequence can a protein that is not a target for a known drug, but which serves as a target against which a new drug is developed.
  • the nucleic acid sequence in the vector is flanked by a first and a second site-specific recombination site.
  • site- specific recombination site refers to a specific sequence on a nucleic acid molecule that is recognized and bound by a specific recombination protein (also known as a recombinase), which facilitates the exchange (i.e., recombination) of nucleic acid sequences between two nucleic acid molecules.
  • Numerous site-specific recombination systems from various organisms are known in the art, and include, for example, the integrase/ ⁇ tf system derived from bacteriophage ⁇ (see, e.g., Landy, Curr. Opin. Genet. DeveL, 3, 699-707 (1993)), the Cxe/loxP system derived from bacteriophage Pl (see, e.g., Hoess et al., In: Nucleic Acids and Molecular Biology, vol. 4, Eckstein and Lilley, eds., Springer- Verlag, Berlin, pp.
  • the first and second site- specific recombination sites can be any suitable site-specific recombination site, such as, for example a wild type loxP site and a mutant loxP site (see, e.g., Sauer, Curr. Opin. Biotech, 5, 521-527 (1994)). Whatever site-specific recombinase is chosen, the first and second site- specific recombination sites do not recombine with each other.
  • the first and second site-specific recombination sites are derived from bacteriophage lambda, which uses site-specific recombination for integration into the Escherichia coli chromosome (see, e.g., Landy et al., Ann. Rev. Biochem., 58, 913-949 (1989).
  • Site-specific recombination occurs between site-specific attachment (cttt) sites.
  • site-specific recombination occurs between an attB site on the E. coli chromosome and an attP site on the lambda chromosome.
  • the first and second site-specific recombination sites flanking the nucleic acid sequence are each an attL site.
  • the inventive method further comprises incubating a first vector in the presence of one or more transposons and a transposase protein under conditions sufficient to cause insertion of the transposon into the vector.
  • transposon refers to a mobile genetic element.
  • Transposons are structurally variable, and can encode a transposition catalyzing enzyme, called a transposase, flanked by DNA sequences organized in inverted orientations (see, e.g., Sherratt, ed., Mobile Genetic Elements, Oxford University Press (1995), and Berg and Howe, eds., Mobile DNA, American Society for Microbiology, Washington, DC (1989)).
  • Transposons are used in the art to insert DNA into target DNA sequences.
  • Transposons typically contain additional genes not related to transposition, such as antibiotic resistance genes, and are classified as “simple” (class 1) and "complex” (class 2).
  • class 1 the insertion of transposons into target DNA is a random event, but transposon insertion can also occur with some sequence specificity.
  • transposon Tn7 can integrate itself into a specific site in the E. coli genome as one part of its life cycle (see, e.g., Stellwagen et al., Trends in Biochemical Sciences, 23, 486-490 (1998)).
  • the Tn5 transposon is known to preferentially insert at GC rich sequences (see, e.g., Herron et al., Nucl.
  • transposons include, for example, Tn7, TnS, TnI, Tn3, Tn4, Tn7, Tn501, Tn551, Tn9, Tn903, Tnl681, TnIO, and Tn5.
  • incubation of the first vector with the one or more transposons can occur in vitro or in vivo (e.g., in a bacterium).
  • the first vector and the one or more transposons are incubated in vitro.
  • Each of the one or more transposons preferably is a Tn5 transposon.
  • the use of multiple transposons that vary in reading frame can be employed in the inventive method.
  • any number of transposons can be employed in the inventive method.
  • the first vector is incubated with at least one transposon (e.g., 1, 3, 5, or more transposons).
  • the first vector is incubated with three transposons, each of which comprises a different reading frame (e.g., two additional transposons having one or two extra nucleotides at the 3' ends).
  • the use of three transposons can yield a viable in-frame deletion in accordance with the inventive method. Whereas a single transposon may insert frequently at a location out of frame, three transposons inserting at the same site will improve the likelihood of at least one in-frame deletion.
  • the transposon comprises a third and a fourth site-specific recombination site, each of which can be any suitable site-specific recombination site such as those described herein.
  • the third and the fourth site- specific recombination sites are derived from phage lambda. More preferably, the third and fourth site-specific recombination sites are each an attB site.
  • the third and fourth site- specific recombination sites can be different attB sites (e.g., an ottB3 site and an attB4 site) (see, e.g., Cheo et al., Genome Res., 14, 2111-20 (2004)), but preferably they are identical and inverted.
  • the transposon can comprise other elements that facilitate identification of cells that contain the transposon.
  • Such elements are typically referred to in the art as "selectable marker genes," and include, for example, antibiotic resistance genes, genes encoding protein products which are otherwise lacking in a recipient cell (e.g., herpes simples virus thymidine kinase (HSV-TK)), and genes encoding protein products that can be visually identified (e.g., ⁇ - galactosidase, luciferase, and green fluorescent protein (GFP)).
  • the transposon can comprise any suitable number of selectable marker genes.
  • the transposon comprises one selectable marker gene.
  • the transposon comprises an antibiotic resistance gene.
  • the transposon can comprise any suitable antibiotic resistance gene. Suitable antibiotic resistance genes are known in the art and include, for example, ampicillin resistance, kanamycin resistance, chloramphenicol resistance, tetracycline resistance, spectinomycin resistance, streptomycin resistance, and sulfonamide resistance genes.
  • the transposon can further comprise a nucleic acid sequence that encodes a protein product that is toxic to cells harboring the transposon when grown under specific conditions. In this manner, cells containing the transposon are killed (i.e., "selected against") when grown under such conditions.
  • tetracycline resistance gene also confers sensitivity to nickel (see, e.g., Podolsky et al., Plasmid, 36, 112-115 (1996)).
  • nickel e.g. nickel sulphate
  • cells containing a tetracycline resistance gene do not survive, and are selected against.
  • mutant phenylalanine tRNA synthetase (PheS) protein causes the incorporation of chlorophenylalanine (Cl-Phe) into proteins, which is toxic to cells.
  • the transposon comprises a kanamycin resistance gene (kan R ) or a tetracycline resistance gene (tet R ) (see, e.g., Podolsky et al., supra).
  • the first vector can be incubated with the transposon and the transposase using any suitable molecular biology technique known in the art, so long as the conditions under which incubation occurs are sufficient to cause insertion of the transposon into the vector. Suitable methods are disclosed in, for example, Miller et al., eds., Mobile Genetic Elements: Protocols and Genomic Applications (Methods in Molecular Biology), Humana Press, Totowa, New Jersey (2004). As a result of incubation of the first vector with the transposon and transposase, the transposon integrates into the vector. In accordance with the inventive method, two types of integration events are generated. First, the transposon can integrate within the nucleic acid sequence in the vector that encodes the protein of interest. Alternatively, the transposon can integrate within the vector but not within the nucleic acid sequence encoding the protein of interest.
  • the invention further comprises transferring the nucleic acid sequence into a second vector, and propagating the second vector in a bacterium, wherein the second vector comprises a fifth and a sixth site-specific recombination site.
  • the second vector carries a selectable marker gene, and preferably carries a different selectable marker gene (e.g., a different antibiotic resistance gene) than the selectable marker gene(s) carried by the transposon and by the first vector.
  • Bacterial cells are then transfected with the second vector, and grown under conditions such that only those cells containing both the second vector, and the transposon integrated therein, are able to grow.
  • bacterial cells are grown in the presence of an antibiotic against which the transposon is resistant, and an antibiotic against which the second vector is resistant.
  • Any suitable bacterium known in the art may be used to propagate the second vector.
  • the bacterium used to propagate the second vector is E. coli.
  • the second vector can be introduced into the bacterium and propagated therein using routine molecular biology and recombinant DNA techniques, such as, for example, chemical transformation or electroporation (see, e.g., Sambrook et al., supra).
  • the nucleic acid sequence, and transposon integrated therein, can be transferred to the second vector using routine recombinant DNA and molecular biology techniques, such as those known in the art and described herein.
  • the nucleic acid sequence is transferred to the second vector utilizing site-specific recombination provided by the Gateway ® Technology cloning system (Invitrogen Life Technologies, Carlsbad, CA).
  • the first vector when the first and second site-specific recombination sites flanking the nucleic acid sequence are each an attL site, the first vector is combined with a second vector containing two attR site-specific recombination sites, as well as an ampicillin resistance gene, in the presence of a suitable recombinase protein.
  • a suitable recombinase protein In this manner, recombination between the attL and attR sites results in transfer of the nucleic acid sequence to the second vector, and the creation of attB site-specific recombination sites flanking the nucleic acid sequence.
  • the inventive method further comprises isolating the second vector comprising the transposon inserted into the nucleic acid sequence.
  • the second vector can be isolated using routine molecular biology techniques, such as those described in Ausubel et al., supra, Sambrook et al., supra.
  • the inventive method further comprises combining the second vector with a third vector to produce a mixture, m a preferred embodiment of the invention, the third vector comprises a seventh and an eighth site-specific recombination site.
  • the seventh and eighth site-specific recombination sites each can be any suitable site-specific recombination sites described herein. As discussed above, recombination between the attL sites on the first vector and the attR sites on the second vector creates attB sites on the second vector.
  • the seventh and eighth site-specific recombination sites on the third vector are preferably each an attP site- specific recombination site.
  • the third vector preferably carries a selectable marker gene (e.g., an antibiotic resistance gene).
  • the third vector can comprise any suitable selectable marker gene, so long as the selectable marker gene of the third vector is different than the selectable marker genes carried by the transposon and the second vector.
  • the third vector comprises a kanamycin resistance gene as a selectable marker gene.
  • the seventh and eighth site-specific recombination sites of the third vector preferably are attP site-specific recombination sites (e.g., attPl and attP2)
  • the fifth and sixth site-specific recombination sites of the second vector, which flank the nucleic acid sequence preferably are attB site-specific recombination sites (e.g., attBl and attBl)
  • the third and fourth site-specific recombination sites on the transposon preferably are attB site-specific recombination sites (e.g., two inverted attBl sites).
  • the mixture of the second and third vectors are incubated in the presence of at least one recombinase protein under conditions sufficient to cause recombination of the attP sites on the third vector with one attB site on the second vector, and one attB site on the transposon.
  • deletion of the amino terminus of a protein of interest can be performed as illustrated in Figure 1.
  • the third, fourth, and fifth site- specific recombination sites are each an attBl site
  • the sixth site-specific recombination site is an attB2 site.
  • the seventh site-specific recombination site on the third vector is an attPl site, while the eighth site-specific recombination site on the third vector is an attP2 site.
  • an attBl site will recombine with an attPl site of appropriate orientation
  • an attB2 site will recombine with an attP2 site of appropriate orientation.
  • the seventh and eighth site- specific recombination sites of the third vector can recombine with (a) the fourth site- specific recombination site of the transposon and the sixth site-specific recombination site of the second vector, respectively, or (b) the fifth and sixth site-specific recombination sites of the second vector, respectively.
  • Recombination of the seventh and eighth site-specific recombination sites of the third vector with the fourth site-specific recombination site of the transposon and the sixth site-specific recombination site of the second vector, respectively, is preferred when generating an amino terminal deletion derivative of a protein of interest.
  • Such a recombination event transfers the portion of the nucleic acid sequence encoding the carboxy portion of the protein into a separate vector, eliminating the transposon but retaining one transposase recognition sequence.
  • the method set forth in Figure 1, however, is merely an exemplary embodiment of the present invention, and should not be construed as limiting the scope of the inventive method.
  • the third, fourth, and sixth site-specific recombination sites can each be an attB2 site, while the fifth site-specific recombination site can be an attBl site.
  • the seventh and eighth site-specific recombination sites of the third vector can be an attPl site and an attP2 site, respectively.
  • the seventh and eighth site-specific recombination sites of the third vector can recombine with (a) the fifth site-specific recombination site of the second vector and the third site-specific recombination site of the transposon, respectively, or (b) the fifth and sixth site-specific recombination sites of the second vector, respectively.
  • Recombination of the seventh and eighth site-specific recombination sites of the third vector with the fifth site-specific recombination site of the second vector and the third site-specific recombination site of the transposon, respectively, is preferred when generating a carboxy terminal deletion derivative of a protein of interest.
  • Such a recombination event transfers the portion of the nucleic acid sequence encoding the amino portion (i.e., fragment) of the protein into a separate vector, eliminating the transposon.
  • the method set forth in Figure 2, however, is merely an exemplary embodiment of the present invention, and should not be construed as limiting the scope of the inventive method.
  • the transposon preferably comprises two site-specific recombination sites (i.e., a third and fourth site-specific recombination site), a transposon comprising only one site-specific recombination site (i.e., a third site-specific recombination site) also is within the scope of the inventive method.
  • the second vector comprises a fourth and a fifth site-specific recombination site
  • the third vector comprises a sixth and a seventh site-specific recombination site.
  • the sixth and seventh site-specific recombination sites of the third vector preferably are attP site-specific recombination sites (e.g., attPl and attPI), the fourth and fifth site-specific recombination sites of the second vector, which flank the nucleic acid sequence, preferably are attB site-specific recombination sites (e.g., attBl and attBl), and the third site-specific recombination site on the transposon preferably is an attB site-specific recombination site (e.g., attBl or attBI).
  • attP site-specific recombination sites e.g., attPl and attPI
  • the fourth and fifth site-specific recombination sites of the second vector, which flank the nucleic acid sequence preferably are attB site-specific recombination sites (e.g., attBl and attBl)
  • the third vector and the transposon each possesses one or more selectable marker gene(s) that allow selection of bacterial cells containing a third vector that harbors a desired deletion derivative.
  • the transposon carries a tetracycline resistance gene, while the third vector carries a kanamycin resistance gene.
  • the desired recombination event is selected for by introducing the recombination products into suitable bacterial cells, and growing such cells in the presence of nickel and kanamycin.
  • cells that are resistant to kanamycin and nickel i.e., cells which have lost the transposon comprising the tetracycline resistance gene and part of the nucleic acid sequence as a result of recombination, are selected for.
  • recombination of the seventh and eighth site- specific recombination sites of the third vector with the fourth site-specific recombination site of the transposon and the sixth site-specific recombination site of the second vector, respectively results in the transfer of a portion of the nucleic acid sequence into the third vector, which is equivalent to deletion of one or more nucleotides of the nucleic acid sequence.
  • a suitable host e.g., a bacteria
  • the nucleic acid sequence is expressed, and a soluble deletion derivative of the protein of interest is produced.
  • the inventive method can result in deletion of amino acids at the amino terminus (N-terminus) or at the carboxy terminus (C-terminus) (see Figures 1 and 2).
  • the inventive method of producing two or more soluble deletion derivatives of a protein can be used to generate deletions at both the N-terminus and the C-terminus ("double deletions").
  • a nucleic acid sequence encoding a protein of interest is subjected to two successive cycles of the inventive method of producing one or more soluble deletion derivatives.
  • an N-terminal deletion derivative of a protein of interest generated in accordance with the inventive method can be subjected to a second cycle of the inventive method to generate a C-terminal deletion of the protein, and vice versa (see Figure 5).
  • the deletion derivative(s) can be sequenced, screened for solubility, and/or screened for proper reading frame.
  • the nucleic acid sequence encoding the deletion derivative can be sequenced using DNA sequencing methods known in the art. By sequencing the deletion derivative, the exact deletion endpoint can be determined by comparing the nucleic acid sequence of the deletion derivative to the nucleic acid sequence encoding the protein from which the deletion derivative is derived.
  • the solubility of the deletion derivative(s) also can be determined using methods known in the art, such as gel electrophoresis or mass spectrometry.
  • the solubility of the deletion derivative(s) can be determined by screening specially-designed expression vectors encoding carboxy-terminal fusion proteins of the deletion derivative(s) of interest and a fluorescent protein or an antibiotic resistance gene.
  • nucleic acid sequences encoding deletion derivatives generated by the inventive method must be in the correct reading frame, so as to produce a soluble deletion derivative protein.
  • the nucleic acid sequences encoding the deletion derivatives generated in accordance with the inventive method preferably are screened for the proper reading frame. Suitable screening methods are known in the art, and include, for example, the use of software packages that translate a given nucleic acid sequence into all possible open reading frames (e.g., Transeq, European Bioinformatics Institute) and compare open reading frame sequences to sequences of known proteins (e.g., BLAST).
  • a nucleic acid sequence encoding a deletion derivative can be engineered to comprise a selectable marker gene as part of a fusion protein (e.g., an antibiotic resistance gene), such that selection of the phenotype of the selectable marker gene will co-select for deletion derivatives in the proper reading frame.
  • a selectable marker gene as part of a fusion protein (e.g., an antibiotic resistance gene)
  • the invention further provides a method for identifying one or more protein portions (i.e., fragments) in a sample.
  • the method comprises (a) preparing a mixture of two or more (e.g., 2, 10, 50, 100, 500, or 1000 or more) soluble deletion derivatives of one or more proteins in accordance with the inventive method, (b) separating the two or more soluble deletion derivatives, (c) isolating each of the two or more separated soluble deletion derivatives, and (d) subjecting each of the two or more soluble deletion derivatives to mass spectrometry, whereupon the one or more protein portions is identified.
  • Separation of the two or more soluble deletion derivatives can be performed using any suitable method known in the art.
  • the two or more soluble deletion derivatives preferably are produced in and isolated from host cells (e.g., bacterial cells) prior to separation.
  • cells preferably are harvested and lysed using routine molecular biology techniques.
  • Insoluble proteins are then removed from the cell lysate using any suitable method, such as, for example, centrifugation, and can be further processed in accordance with other embodiments of the invention.
  • the soluble deletion derivatives are then purified from the cell lysate, by, for example, affinity purification.
  • affinity purification Methods for isolating and purifying recombinantly produced proteins are described in, for example, Sambrook et al., supra, and Ausubel et al., supra.
  • suitable protein separation methods include, but are not limited to, centrifugation, ion exchange chromatography, isoelectric focusing, reversed-phase liquid chromatography, and gel electrophoresis.
  • the two or more soluble deletion derivatives are separated using gel electrophoresis (e.g., one-dimensional or two-dimensional gel electrophoresis).
  • gel electrophoresis e.g., one-dimensional or two-dimensional gel electrophoresis
  • the two or more soluble deletion derivatives are separated using two- dimensional gel electrophoresis (2DGE).
  • 2DGE typically involves separation of proteins in a first dimension by charge using isoelectric focusing (IEF).
  • the charge-focused proteins are then separated in a second dimension according to size by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (see, e.g., Lin et al., Biochimica et Biophysica Acta, 1646, 1- 10 (2003) and Ong et al., Biomol Eng., 18, 195-205 (2001)).
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • the separated proteins can be digested with a protease (e.g., trypsin) to aid in protein sequencing.
  • protease e.g., trypsin
  • protein digestion is performed in the gel once the two or more soluble deletion derivatives have been separated.
  • each of the two or more deletion derivatives is isolated from the separation medium.
  • the deletion derivatives can be isolated using any suitable technique, such as by extracting the protein "spots" from the gel. Extraction of protein spots from a gel typically involves the physical cutting of the spot from the gel.
  • the isolated deletion derivatives preferably are subjected to mass spectrometry.
  • mass spectrometry a substance is ionized, and the positive fragments which are produced (cations and radical cations) are accelerated in a vacuum through a magnetic field and are sorted on the basis of mass-to-charge ratio (m/z). Since the bulk of the ions produced in the mass spectrometer carry a unit positive charge, the value m/z is equivalent to the molecular weight of the fragment. Any suitable mass spectrometry method can be used in connection with the inventive method.
  • suitable mass spectrometry methods include matrix-assisted laser desorption/ionization mass spectrometry (MALDI), matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, plasma desorption/ionization mass spectrometry (PDI), electrospray ionization mass spectrometry (ESI), surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry, and liquid chromatography mass spectrometry (LC-MS or LC-MS-MS).
  • MALDI matrix-assisted laser desorption/ionization mass spectrometry
  • MALDI-TOF matrix-assisted laser desorption/ionization-time of flight
  • PDI plasma desorption/ionization mass spectrometry
  • ESI electrospray ionization mass spectrometry
  • SELDI-TOF surface enhanced laser desorption/ionization-time of flight
  • time-of-flight (TOF) methods of mass spectrometry charged (ionized) molecules are produced in a vacuum and accelerated by an electric field produced by an ion-optic assembly into a free-flight tube or drift time.
  • the velocity to which the molecules may be accelerated is proportional to the square root of the accelerating potential, the square root of the charge of the molecule, and inversely proportional to the square root of the mass of the molecule.
  • the charged molecules travel down the TOF tube to a detector.
  • LC-MS-MS liquid chromatography is used to separate the two or more deletion derivatives, which are then subject to mass spectrometry.
  • Computer software selects the fragments from the mass spectrometer, channels them for ionization via collision-induced dissociation (CID), and a second mass spectrometer detects the fragments.
  • CID collision-induced dissociation
  • the fragmentation caused by these collisions gives fragments with a "ladder" of masses, each "rung” of which corresponds to the next amino acid in the chain.
  • the difference in masses between the fragments provides the identity of each amino acid, and thus the sequence of the deletion derivative.
  • Mass spectrometry methods are further described in, for example, International Patent Application Publication No. WO 93/24834, U.S. Patent 5,792,664, U.S. Patent Application Publication No.
  • the one or more nucleic acid sequences can be derived from any suitable organism, and need not be derived from the same organism.
  • the inventive method for identifying one or more protein portions in a sample can be used to identify one or more proteins from multiple different organisms.
  • the inventive method for identifying one or more protein portions in a sample can be used to identify one or more proteins derived from the same organism.
  • the organism is an animal.
  • the two or more proteins can be derived from any suitable animal. Suitable animals include, for example, protozoa, echinoderms (e.g., sea urchin), annelids (e.g., earthworms), nematodes (e.g., C.
  • the invention further provides a method for identifying two or more soluble proteins, or peptide fragments thereof.
  • the method comprises (a) preparing a mixture of two or more vectors each comprising a nucleic acid sequence encoding a soluble protein operatively linked to a promoter, (b) transferring each of the two or more vectors into one or more cells, (c) expressing the nucleic acid sequence in each of the one or more cells, wherein the two or more soluble proteins are produced, (d) purifying the two or more soluble proteins from the one or more cells, (e) separating the two or more soluble proteins, (f) isolating each of the two or more soluble proteins, and (g) subjecting the two or more soluble proteins to mass spectrometry, whereupon the each of the two or more soluble proteins is identified, and the amount of each of the two or more soluble proteins produced in the one or more cells is determined.
  • Descriptions of the vector, soluble protein, and mass spectrometric analysis set forth above in connection with other embodiments of the invention also are applicable to those same aspects of the aforesaid inventive method for
  • the inventive method for each of two or more soluble proteins can be used to identify any number of proteins (e.g., 2, 10, 20, 50, 100, or 1000 or more) simultaneously.
  • the method comprises preparing two or more vectors (e.g., 2, 10, 20, 50, 100, or 1000 or more), comprising a nucleic acid sequence encoding a soluble protein operatively linked to a promoter.
  • each vector comprises a nucleic acid sequence encoding one soluble protein.
  • the nucleic acid is operatively linked to (i.e., under the transcriptional control of) one or more promoter and/or enhancer elements. Techniques for operatively linking sequences together are well known in the art.
  • a "promoter” is a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis.
  • a promoter can be native or non-native to the nucleic acid sequence to which it is operably linked.
  • Any promoter i.e., whether isolated from nature or produced by recombinant DNA or synthetic techniques
  • the promoter preferably is capable of directing transcription in a cell (e.g., a prokaryotic or eukaryotic cell).
  • the functioning of the promoter can be altered by the presence of one or more enhancers and/or silencers present on the vector.
  • Enhanccers are cis-acting elements of DNA that stimulate or inhibit transcription of adjacent genes.
  • Enhancers differ from DNA-binding sites for sequence-specific DNA binding proteins found only in the promoter (which also are termed “promoter elements”) in that enhancers can function in either orientation, and over distances of up to several kilobase pairs (kb), even from a position downstream of a transcribed region.
  • promoter elements DNA-binding sites for sequence-specific DNA binding proteins found only in the promoter (which also are termed “promoter elements") in that enhancers can function in either orientation, and over distances of up to several kilobase pairs (kb), even from a position downstream of a transcribed region.
  • Any suitable promoter or enhancer sequence can be used in the context of the invention.
  • the nucleic acid sequence can be operatively linked to a constitutive promoter.
  • Any suitable constitutive promoter can be used in connection with the inventive method.
  • Suitable constitutive promoters include, for example, cytomegalovirus (CMV) promoters, such as the CMV immediate-early promoter (described in, for example, U.S. Patents 5,168,062 and 5,385,839), promoters derived from human immunodeficiency virus (HIV), Rous sarcoma virus (RSV) promoters, such as the RSV long terminal repeat, mouse mammary tumor virus (MMTV) promoters, HSV promoters, such as the Lap2 promoter or the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. ScI, 78, 144-145 (1981)), promoters derived from SV40 or Epstein Barr virus, the YYl promoter, the ubiquitin promoter, and the like.
  • CMV cytomegalovirus
  • CMV CMV immediate-early promoter
  • RSV Rous sarcoma virus
  • the promoter is a regulatable promoter, i.e., a promoter that is up- and/or down-regulated in response to appropriate signals.
  • Suitable regulatable promoter systems include, but are not limited to, the tetracycline expression system, the IL-8 promoter, the metallothionine inducible promoter system, the bacterial lacZYA expression system, and the T7 polymerase system.
  • promoters that are selectively activated at different developmental stages e.g., globin genes are differentially transcribed from globin- associated promoters in embryos and adults
  • the promoter sequence can contain at least one regulatory sequence responsive to regulation by an exogenous agent.
  • the regulatory sequences are preferably responsive to exogenous agents such as, but not limited to, drugs, hormones, radiation, sugars, salts, and the like.
  • the promoter can be a cell-specific or tissue-specific promoter, i.e., a promoter that is preferentially activated in a given cell or tissue and results in expression of a gene product in the tissue where activated.
  • a cell-specific or tissue-specific promoter suitable for use in the invention can be chosen by the ordinarily skilled artisan based upon the target tissue or cell-type.
  • the two or more vectors are transferred into one or more cells.
  • each cell is transduced with only one vector.
  • the two or more vectors can be transferred into one or more cells using any suitable method known in the art. Suitable methods include, for example, chemical transformation, electroporation, high velocity bombardment with DNA-coated microprojectiles, incubation with calcium phosphate-DNA precipitate, direct microinjection into single cells, calcium phosphate or DEAE-dextran-mediated transfection, polybrene transfection, protoplast fusion, liposome-mediated transfection, and the like (see, e.g., Sambrook et al., supra).
  • each of the two or more vectors need not be transferred to two or more cells in a one-to-one ratio.
  • one or more vectors can be transferred into a single cell.
  • the two or more vectors can be transferred into any cells capable of transduction which permit expression of the nucleic acid sequence encoding the soluble protein and can be readily propagated (i.e., cultured).
  • suitable cells include bacterial cells, such as E. co ⁇ i, yeast cells (e.g. Pichia pastoris and Saccharomyces cerevisiae), and animal cells, such as insect cells and C. elegans cells, and mammalian cloned cells, such as HeLa cells, CHO cells, and VERO cells.
  • the one or more cells are each bacterial cells.
  • the two or more vectors can be introduced into the one or more cells as described herein, and the one or more cells can be combined to form a single sample. In a preferred embodiment, the two or more vectors are combined into a single sample, and the mixture of vectors is transduced into host cells.
  • the inventive method further comprises expressing the nucleic acid sequence in each of the one or more cells, wherein the two or more soluble proteins are produced.
  • expression of a nucleic acid sequence can be regulated (e.g., induced) when the nucleic acid sequence is operatively linked to a regulatable promoter.
  • the nucleic acid sequence is operatively linked to an inducible promoter.
  • the nucleic acid sequence can be operatively linked to any suitable inducible promoter, such as those described herein. In the presence of an appropriate signal, the inducible promoter is activated, and the nucleic acid sequence encoding the soluble protein is expressed.
  • each of the two or more nucleic acids results in production of the two or more soluble proteins, or peptide fragments thereof.
  • each of the two or more soluble proteins is purified from the one or more cells.
  • the two or more soluble proteins can be purified using any suitable method known in the art, such as those described herein. Specifically, cells preferably are harvested and lysed using routine molecular biology techniques. Insoluble proteins are then removed from the cell lysate using any suitable method, such as, for example, centrifugation, and can be further processed in accordance with other embodiments of the invention. The soluble proteins are then purified from the cell lysate, by, for example, affinity purification. Methods for isolating and purifying recombinantly produced proteins are described in, for example, Sambrook et al., supra, and Ausubel et al., supra.
  • the inventive method further comprises separating the two or more soluble proteins, isolating each of the two or more soluble proteins, and subjecting the two or more soluble proteins to mass spectrometry.
  • the two or more soluble proteins can be separated using any suitable technique known in the art.
  • the two or more soluble proteins are separated by electrophoresis, more preferably two-dimensional gel electrophoresis (2DGE).
  • the separated proteins can be digested with a protease (e.g., trypsin) to aid in protein sequencing.
  • protein digestion is performed in the gel once the two or more soluble deletion derivatives have been separated.
  • each of the two or more soluble proteins is isolated from the separation medium.
  • the soluble proteins can be isolated using any suitable technique, such as by extracting the protein "spots" from the gel. Extraction of protein spots from a gel typically involves the physical cutting of the spot from the gel.
  • each of the two or more soluble proteins, or peptide fragments thereof is identified by subjecting the two or more soluble proteins to mass spectrometry. Any suitable mass spectrometry method can be used in connection with the inventive method.
  • Suitable mass spectrometry methods include, for example, matrix-assisted laser desorption/ionization mass spectrometry (MALDI), matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF or MALDI-TOF- TOF) mass spectrometry, plasma desorption/ionization mass spectrometry (PDI), electrospray ionization mass spectrometry (ESI), surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry, and liquid chromatography mass spectrometry (LC-MS or LC-MS-MS).
  • MALDI matrix-assisted laser desorption/ionization mass spectrometry
  • MALDI-TOF or MALDI-TOF- TOF MALDI-TOF-TOF
  • PDI plasma desorption/ionization mass spectrometry
  • ESI electrospray ionization mass spectrometry
  • SELDI-TOF surface enhanced laser de
  • the inventive method also encompasses the identification of the sequence of each of the two or more soluble proteins, or peptide fragments thereof.
  • the proteins can be sequenced using any suitable method known in the art. Suitable protein sequencing methods include, for example, Edman sequencing (see, e.g., Gevaert et al., Electrophoresis, 21, 1145-1154 (2000)), and sequencing by mass spectrometry (see, e.g., Kinter and Sherman, Protein Sequencing and Identification Using Tandem Mass Spectrometry, Wiley- Interscience (2000), and U.S. Patents 6,632,339 and 6,706,529).
  • Mass spectrometry-based protein identification methods include, for example, MALDI-MS peptide mass fingerprinting (MALDI-MS-PMF), MALDI-MS post-source decay analysis (MALDI-MS- PSD), and liquid chromatography mass spectrometry (LC-MS or LC-MS-MS).
  • MALDI- MS-PMF a protein of interest, which typically is purified by 2-D gel electrophoresis, is either enzymatically or chemically cleaved and an aliquot of the obtained peptide mixture is analyzed by mass spectrometric techniques, thereby generating a mass "fingerprint" of the protein.
  • PSD fragments are generated in the field-free drift region after MALDI. It has been postulated that PSD primarily is the result of amide bond cleavages. Due to the high complexity of PSD spectra, however, MALDI- MS-PSD is not frequently used in the art to identify proteins (see, e.g., Gevaert et al., supra).
  • LC-MS-MS involves mass spectroscopic analysis of proteins separated by liquid chromatography.
  • Protein fragments selected using computer software are channeled for ionization via collision-induced dissociation (CID), and a second mass spectrometer detects the fragments.
  • CID collision-induced dissociation
  • the fragmentation caused by these collisions gives fragments with a "ladder" of masses, each "rung” of which corresponds to the next amino acid in the chain.
  • the difference in masses between the fragments provides the identity of each amino acid, and thus the sequence of the protein.
  • ELISA enzyme-linked immunosorbent assay
  • Western blot analysis Western blot analysis
  • immunoprecipitation immunoprecipitation
  • isoelectric focusing followed by 2-D gel electrophoresis.
  • the amount of each of the two or more soluble proteins, or peptide fragments thereof, produced in the one or more cells can be determined prior to or following mass spectrometry of the two or more soluble proteins, or peptide fragments thereof.
  • the intensity of the "spots" on the 2D gel corresponding to each of the two or more soluble proteins analyzed by mass spectrometry can be measured.
  • the intensity of a particular protein spot on the 2D gel is approximately proportional to the amount of that protein present in a cell.
  • the protein "spots" on the 2D gel can be analyzed using various commercially-available computer software packages, such as, for example, PDQuest 7.3.0 Software (Bio-Rad Laboratories, Hercules, CA), Phoretix 2D Gel Image Analysis Software (United Bioinformatica Inc., Calgary, AB, Canada), and ProFINDER 2D Image Analysis Software (PerkinElmer Life & Analytical Science, Inc., Boston, MA).
  • PDQuest 7.3.0 Software Bio-Rad Laboratories, Hercules, CA
  • Phoretix 2D Gel Image Analysis Software United Bioinformatica Inc., Calgary, AB, Canada
  • ProFINDER 2D Image Analysis Software PerkinElmer Life & Analytical Science, Inc., Boston, MA.
  • Insoluble proteins that are not identified by the method of identifying two or more soluble proteins can be treated with solubilizing agents and used to generate soluble deletion derivatives as described herein. Such deletion derivatives can then be further analyzed by the method of identifying two or more soluble proteins, to determine if fragments of the insoluble proteins are soluble.
  • the inventive method provides an improved method for identifying soluble proteins. The inventive method thus will aid in the discovery of cellular targets against which new disease therapeutics can be developed. In addition, the invention will enable researches to elucidate the molecular pathways underlying specific diseases. The inventive method also will aid in methods for enhancing the solubility of insoluble proteins.
  • the deletion derivatives described herein can be used as solubility enhancement tags by creating fusion proteins comprising a soluble deletion derivative and an insoluble protein.
  • the Gateway ® entry clones typically contain an attLl site and an attL2 site flanking the ORF of interest, as well as an antibiotic resistance gene, such as a spectinomycin resistance (Sp R ) gene.
  • the DNA concentrations of Gateway ® entry clones in eight 96-well plates i.e., plates 11084, 11085, 11086, 11087, 11094, 11095, 11096, and 11099) were determined by PicoGreen ® fluorescence (Molecular Probes, Inc., Eugene, OR), and used to calculate the molar concentration of each plasmid, which ranged from 0 to 8.73 nM.
  • Each 96-well plate contained 94 Gateway ® entry clones encoding a C. elegans ORF and two control plasmids.
  • plasmid DNA was generated in one of two ways: for liquid pooling, a complete transformation sample was diluted into 100 mL of LB medium supplemented with necessary antibiotics, and grown overnight at 37 °C. For plate-based pooling, the transformation mixture was centrifuged and dissolved in 100 ⁇ l LB and plated on 100 mm LB-Agar plates supplemented with necessary antibiotics, and grown overnight at 37 °C. Colonies from the plates were pooled by scraping the plate into 1 mL of LB, which was used immediately to prepare plasmid, or by dilution into a larger volume for overnight growth prior to preparation of plasmid. Plasmid DNA was prepared using the FastPlasmid Mini kit (Eppendorf) per manufacturer's instructions.
  • Eppendorf FastPlasmid Mini kit
  • the tetracycline resistance gene from the TET-I transposon was amplified using oligonucleotide primers containing Tn5 mosaic end (ME) sites and Gateway ® attBl sites.
  • This amplification product was purified using the QiaQuick PCR purification system (Qiagen, Inc., Valencia, CA) and transposed into pUC19 DNA using the Epicentre EZ::TN system according to the manufacturer's instructions. Positive colonies were selected using 100 ⁇ g/mL ampicillin and 12.5 ⁇ g/mL tetracycline, and plasmid DNA was prepared by standard methods.
  • pUC DNA containing the transposon was sequence verified throughout to confirm the DNA sequence of the entire transposon.
  • the pUC-transposon DNA was used as a template in a PCR amplification using ME primers. Amplified DNA was digested with Dpnl (New England Biolabs, Beverly, MA) for one hour at 37 °C to remove residual plasmid template DNA, heat inactivated by incubation at 80 °C for 20 minutes, and purified using the QiaQuick PCR purification system.
  • transposon containing attBl sites is used to generate an amino-terminal deletion, while a transposon containing attBl sites is used to generate a carboxy-terminal deletion.
  • Electroporation of DNA was carried out by mixing 1 ⁇ l of a DNA mixture with 20 ⁇ l of Electromax DH5 ⁇ -E competent cells (Invitrogen), incubated on ice for 10 minutes, and electroporated in a 0.1 cm gap cuvette using the BioRad (Herecules, CA) MicroPulser according to manufacturer's settings for Escherichia coli. After electroporation, samples were diluted in 1 ml of LB or SOC and grown at 200 rpm at 37 0 C for 1 hour. Transposition Reactions
  • transposition reactions 0.2 ⁇ g target DNA and 2-fold molar equivalent of transposon was incubated with 1 unit (U) transposase (Epicentre Technologies, Madison, WI) in 10 ⁇ l total volume in Ix transposition buffer (Epicentre Technologies, Madison, WI) for 5-6 hours at 37 0 C. The reaction was stopped by addition of 1 ⁇ l of 10x stop solution (Epicentre Technologies, Madison, WI) followed by incubation at 70 0 C for 10 minutes.
  • U unit
  • 10x stop solution Epicentre Technologies, Madison, WI
  • the reaction was precipitated with one tenth volume of 3 M sodium acetate and 2.5 volumes of 100% ethanol, centrifuged for 20 minutes, dried, and dissolved in 10 ⁇ l TE (10 mM Tris-Cl, pH 7.5, 0.1 mM EDTA). 1 ⁇ l of the dissolved DNA was electroporated into Electromax DH5 ⁇ -E cells (Invitrogen, Inc.). After electroporation, samples were diluted in 1 rnL of LB, grown at 200 rpm at 37 0 C for 1 hour, and plated or diluted for liquid growth using LB supplemented with 100 ⁇ g/ml spectinomycin and 12.5 ⁇ g/ml tetracycline. Plasmid DNA was prepared as described above.
  • a Gateway ® LR reaction was performed using pooled transposed clones and the plasmid pDest-6 (Invitrogen Corp.), which is a Gateway ® "Destination Vector" containing an ampicillin resistance (Ap R ) gene, in a 10 ⁇ L reaction per the manufacturer's specifications, but with an increased reaction time of 16-18 hours. After stopping the reaction with proteinase K, the reaction was precipitated with ethanol, and dissolved in 10 ⁇ l TE. 1 ⁇ L of the reaction mixture was electroporated into Electromax DH5 ⁇ -E cells.
  • pDest-6 Invitrogen Corp.
  • Plasmid DNA was prepared as described above.
  • pooled transposed expression clones were transferred to a pDonr201 vector via Gateway ® BP recombination in a 10 ⁇ L reaction per the manufacturer's specifications, but with an increased reaction time of 16-18 hours.
  • the pDonr201 vector contains the toxic ccdB gene flanked by attP sites (i.e., attPl and attP2), as well as a kanamycin resistance gene or a spectinomycin resistance gene. 1 ⁇ L of the reaction mixture was electroporated into Electromax DH5 ⁇ -E cells.
  • the remaining 99% of the expression mixture was added to 50 mL CircleGrow ® media (QBiogene, Inc., Irvine, CA) containing ampicillin, and grown overnight at 37 0 C. Plasmid DNA was then purified, and about 100 ng of pooled expression plasmids were electroporated into E. coli Rosetta (DE3) strain (Novagen, Madison, WI), resulting in about 1.4 million transformants. The 1 mL of SOC expression mixture was diluted into 50 mL of CircleGrow® containing 100 ⁇ g/mL ampicillin and 15 ⁇ g/mL chloramphenicol, and grown overnight at 37 0 C.
  • the overnight culture was diluted 1:100 into IL of CircleGrow ® containing 100 ⁇ g/mL ampicillin and 15 ⁇ g chloramphenicol, grown at 37 0 C to an A600 concentration of 0.5, and cooled to 16 0 C. Protein expression was induced by adding IPTG to 1 mM. After 16 hours at 16 0 C, cells were harvested and frozen at -80 0 C.
  • Deletion derivative clones were transferred to final expression plasmids using the Gateway ® LR reaction according to the manufacturer's instructions. 1 ⁇ L of the reaction mixture was electroporated into Electromax DH5 ⁇ -E cells. After electroporation, samples were diluted in 1 mL of LB, grown at 200 rpm at 37 0 C for 1 hour, and plated or diluted for liquid growth using LB supplemented with 100 ⁇ g/mL ampicillin. Plasmid DNA was prepared as described above. For expression experiments, plasmid DNA was transformed into E. coli Rosetta (DE3) cells (Novagen, Madison, WI) and grown in 100 ⁇ g/mL ampicillin and 15 ⁇ g/mL chloramphenicol.
  • E. coli Rosetta DE3 cells (Novagen, Madison, WI)
  • Cells were grown overnight at 37 °C, diluted 1:100 into CircleGrow ® media and grown at 37 °C to an OD600 of 0.5. Expression was induced by addition of 0.5 mM IPTG and growth was continued at 37 °C for 2.5 hours or 16 °C overnight. Cells were harvested after induction and 0.05 OD600 units were spun down and dissolved in Ix SDS-loading buffer prior to SDS-PAGE analysis.
  • E. coli cell pastes were resuspended with two volumes of extraction buffer per gram of wet weight to achieve a final concentration of 20 mM sodium phosphate buffer, pH 7.5, 100 mM NaCl, 5 mM MgCl 2 , 5% glycerol, 45 mM imidazole and Complete protease inhibitor with EDTA (Roche Diagnostics Corp., Indianapolis, IN) (1 tablet per 50 mL of extract). Extracts were treated with lysozyme (0.5 mg/niL) for 30 minutes and with Benzonase (Novagen, Madison, WI) (10 U/mL) for an additional 20 minutes.
  • lysozyme 0.5 mg/niL
  • Benzonase Novagen, Madison, WI
  • Samples were sonicated to lyse the cells (verified by microscopic examination) and adjusted to 500 mM NaCl with solid NaCl. Following centrifugation (111,000 x g for 30 minutes), samples were filtered (0.45 ⁇ m, PES membrane), applied to 1 mL HisTrap columns (GE Healthcare, Piscataway, NJ) (0.6 mL/min), and equilibrated with extraction buffer in 500 mM NaCl and 45 mM imidazole (binding buffer). After washing the columns with binding buffer, proteins were eluted with binding buffer and 500 mM imidazole, collected in 1 mL fractions, and analyzed by SDS- PAGE.
  • the protein pools were precipitated as follows: (1) trichloroacetic acid was added to 6% (v/v) final concentration and vortexed, (2) five minute incubation on ice, (3) centrifugation at 16,100 x g for 10 minutes, (4) removal of supernatant, (5) five minute incubation of pellets with ice cold acetone, (6) centrifugation at 16,100 x g for five minutes, (7) removal of supernatant, and (8) drying of pellet (two minutes, 70 °C).
  • Precipitated proteins were dissolved in room-temperature solubilization buffer (8 M urea, 4% CHAPS, 50 mM Tris, pH 8.5) to a concentration of 20 mg/mL and stored in 50 ⁇ l aliquots at -80 °C.
  • Purified proteins were separated in the first dimension according to their net charge by isoelectric focusing, and were separated in the second dimension according to size (see Figure 6).
  • the nominal isoelectric point (pi) range of the gel was 4 to 7, however the effective pi range for the proteins was about 4 to 6. All the proteins with pis greater than about 6 appeared along the right edge of the gel.
  • the nominal size range of the gel was 10 to 10O kDa.
  • the rehydration buffer-protein mixture was placed in a 24 cm ceramic strip holder and a 24 cm IPG strip, pH 4-7, was glided, gel side down, into the strip holder. Mineral oil was placed on top of the gel to minimize evaporation and covered with the strip holder plastic cover.
  • the ceramic strip holder was placed in the IPGphor unit for isoelectric focusing under the following conditions: 30 volts for 12 hours, 500 volts for 1 hour, 1000 volts for 1 hour and 8000 volts for 8 hours. [0085] Prior to the separation in the second dimension, the IPG strip was equilibrated with an SDS buffer system.
  • the equilibration solution contained 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and a trace of bromophenol blue.
  • 100 mg of DTT was added in 10 mL equilibration buffer.
  • the IPG strips were placed in individual tubes containing the buffer. The tubes were then placed on a rocker and equilibrated for 12 minutes. A second equilibration was performed with 250 mg iodoacetamide solution (instead of DTT) and incubated for another 12 minutes.
  • the equilibrated IPG strip was then inserted into a cassette containing a pre-cast Ettan DALT II 12.5% polyacrylamide gel, and contact was made with the 2-D gel. Melted agarose was added to cover the IPG strip.
  • the 2DGE chamber was filled with anode buffer (0.5 M diethanolamine, 0.5 M acetic acid). Cathode buffer (0.1% SDS, 0.192 M glycine, 0.025 M Tris) was added to the top chamber.
  • the running conditions were set in the power supply (phase I, 5 Watts (W) per gel/15min; phase 2, 150W/gel), and electrophoresis continued until the bromophenol blue dye front reached the bottom of the gel (approximately 4-5 hours).
  • Coommassie blue stained protein gel spots were digested as described by WiIm et al., Nature, 379, 466-469 (1996). Samples were desalted with Cl 8 Zip Tips (Millipore,
  • RPLC Microcapillary reverse-phase liquid chromatography
  • the columns were connected via a stainless steel union to an Agilent 1100 nanoflow LC system (Agilent Technologies, Paolo Alto, CA), which was used to deliver solvents A (0.1% HCOOH in water) and B (0.1% HCOOH in CH 3 CN).
  • Agilent 1100 nanoflow LC system Agilent Technologies, Paolo Alto, CA
  • solvents A 0.1% HCOOH in water
  • B 0.1% HCOOH in CH 3 CN
  • the column was washed for 15 minutes with 98% mobile phase B and re-equilibrated with 98% mobile phase A prior to subsequent sample loading.
  • the ⁇ RPLC column was coupled online to an LIT-MS using the manufacturer's nanoelectrospray source with an applied electrospray potential of 1.5 kV and capillary temperature of 160 0 C.
  • the LIT-MS was operated in a data-dependent mode where each full MS scan was followed by five MS/MS scans, in which the five most abundant peptide molecular ions detected from the MS scan were dynamically selected for five subsequent MS/MS scans using a collisional-induced dissociation (CID) energy of 35%.
  • CID collisional-induced dissociation
  • an additional affinity tag can be used.
  • a Strep2 affinity tag IBA GmbH, G ⁇ ttingen, Germany
  • His6 affinity tag a Strep2 affinity tag
  • the four most intense spots on the gel were E. coli proteins and contained three proteins: slyD (proline cis-trans isomerase), dnaK, and groEL. These proteins are stress response proteins presumably induced by overexpression of C. elegans proteins in the E. coli cells.
  • Some C. elegans proteins were found in a single spot, while others were found in up to 13 spots. Proteins found in intense spots tended to be found in multiple spots, most of which clustered together. Some spots were pure protein, others contained mixtures of proteins from host E. coli cells or C. elegans.
  • C. elegans proteins found by searching predicted C. elegans proteins (including those not cloned in the version 1.1 ORFeome), 34 were found in the plates that were combined to make the pooled proteins, and another 5 were found in these plates as "related proteins.” Ten proteins were not in the original pool of 688 C. elegans ORFs, and of these seven were identified on the basis of a single peptide in a single spot. [0098] Twelve C. elegans proteins were chosen on the basis of both spot intensities and potential biological interest for individual expression (see Table 1). All twelve were found in the subset of the 752 originally screened C. elegans ORFs.
  • Each of the twelve ORFs were subcloned into pDest527 and expressed in E. coli Rosetta (DE3) cells (700 ⁇ l cultures in a 24 well dish). Once the cultures reached an absorbance A600 of 0.5, they were transferred to 17 x 100 mm polypropylene tubes (Falcon 2059), cooled to 16 0 C, induced with IPTG (1 mM), and expressed overnight at 16 0 C. To examine total expressed protein, 0.1 absorbance units of cells were pelleted, lysed, and applied to a 4 to 20% SDS-PAGE gel (Criterion, Bio-Rad) (see Figure 7).
  • ORF #8 Eight of the twelve ORFs yielded significant soluble protein.
  • ORFs Twelve positives (i.e., ORFs identified from the most intense spots on the 2D gel) and twelve negatives (i.e., proteins absent from the 2D gel) were isolated from the eight 96-well plates described above. Each ORF was tested individually for expression, solubility, and purification from E. coli cells. Specifically, each of the 24 C elegans ORFs was cloned into pDest527, and each of these plasmids was transformed into E. coli cells (Rosetta (DE3) strain, Novagen, Madison, WI). Cultures were grown at 37 0 C in 0.7 mL cultures until OD 0.5 was reached. Expression was induced with 1 mM IPTG at 16 0 C overnight (20 hours).
  • a vector is engineered to contain a nucleic acid sequence labeled with an affinity tag, and is introduced into a suitable cell as described herein.
  • subcellular fractions are prepared from the from the host cell.
  • Affinity tagged proteins are purified from the subcellular fractions and are subjected to 2D gel electrophoresis. Proteins in each subcellular fraction are analyzed by mass spectrometry and compared to other 2D gels containing other subcellular fractions, allowing estimation of the distribution of each protein among the various cellular compartments, in parallel, for the entire pool.
  • This example demonstrates using the inventive method of identifying one or more soluble proteins to identify a ligand cell surface binding site.
  • a ligand for a cell surface binding site is immobilized on a solid support.
  • a pool of ORFs is generated and expressed as part of an Ebna vector in 293E cells as described herein. The pool of cells is passed over the immobilized ligand. Cells bound to the ligand are recovered, and the identity of the ORF mediating binding to the ligand is determined using methods known in the art.
  • a pellet of insoluble proteins isolated in accordance with the above-described methods is dissolved in 8M Urea, and subjected to 2D gel electrophoresis.
  • the spots expressed at the highest levels represent proteins that are well expressed, but are insoluble.
  • These insoluble proteins can serve as likely targets for solubility enhancement by solubility tags.
  • the insoluble proteins also serve as targets for the generation and analysis of deletion derivatives in accordance with the invention.
  • fragments of the proteins may serve as useful starting points for structural or functional studies.
  • solubility enhancers e.g., maltose-binding protein (MBP), NusA, etc.
  • MBP maltose-binding protein
  • NusA NusA
  • the pool of 16 clones was recombined by Gateway ® LR recombination into a Gateway ® destination plasmid conferring ampicillin resistance and selected on medium containing ampicillin and tetracycline to eliminate transposition events outside of the folliculin gene.
  • the three in-frame individual clones from the pool were transferred to pDest-586 individually.
  • the individual clones and the pooled clones were transformed into Rosetta (DE3) E. coli cells (Epicentre Technologies, Madison, WI), and expressed.
  • Rosetta DE3 E. coli cells
  • Expression of the individual clones produced proteins of the expected molecular weight for the His6-MBP fusions to the deletion fragments.
  • Two of the three individual in- frame fragments expressed significant levels of protein, while the third produced a much lower level of protein and induced significant toxicity in the cells, as seen by a dramatic decrease in growth rate post-induction.
  • the pooled sample showed induction of at least six protein bands, including bands which corresponded to the three individual proteins, and others which were the expected size for several of the out-of-frame deletions.
  • a Gateway ® entry clone containing the folliculin gene was used as a target for transposition using the methods described above.
  • the transposon used in this experiment contained attBl sites at both ends to permit productive recovery of deletions from all transposition events.
  • Transposition and selection on plates resulted in more than 2000 Tet- resistant colonies representing clones which contained a transposon insertion somewhere in the entry clone in one orientation or the other.
  • a pool of these clones was used to transfer the transposed genes into an expression vector to select for transposition events in the gene of interest. The clones were then pooled and transferred back into entry clones by recombination, and plated on selective media to select for deletions.
  • a pool of deletion derivatives produced using the methods in Example 6 are screened by transferring the pool to specially designed expression clones containing carboxy terminal fluorescent protein fusions.
  • the fluorescence of the partner protein is linked to the solubility of the deletion fragment (see, e.g., Waldo et al., Nat. Biotechnol, 17, 691-695 (1999)). Therefore, only clones which are both in-frame and soluble produce fluorescent colonies, which can be easily screened.
  • Soluble deletion derivatives also can be screened by transferring a pool of deletion derivatives to an expression vector which contains a carboxy terminal fusion with an antibiotic resistance gene (e.g., chloramphenicol acetyl transferase (CAT)).
  • an antibiotic resistance gene e.g., chloramphenicol acetyl transferase (CAT)
  • soluble fusions produce a protein that provides resistance to chloramphenicol, and data shows that levels of resistance correlate with levels of solubility (see, e.g., Maxwell et al., Protein Science, 8, 1908-1911 (1999)). This provides a direct readout of solubility of the deletion derivatives.
  • a pool of deletion derivatives can be screened using the method described in Example 3 by transferring the pool to a suitable expression vector, followed by pooled growth and purification. Samples can then be separated on 2D gels to identify soluble fragments.
  • a pool of deletion derivatives also can be used as solubility enhancement tags. For example, large numbers of deletion derivatives of proteins of interest can be screened to identify proteins which have the best chance of enhancing the solubility of their partners. To this end, new destination vectors can be developed which produce carboxyterminal fusions to strongly insoluble proteins (e.g., ketosteroid isomerase (KSI)). The pool of deletion fragments fused to the insoluble protein can then be screened using the methods described in Examples 1-3 to identify fragments which provide the highest level of solubility enhancement. [0119] While the inventive methods preferably are practiced in vivo, the inventive methods can also be practiced in vitro with only minor modifications that are well within the skill in the art.
  • KKI ketosteroid isomerase

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Abstract

La présente invention concerne un procédé de production de protéines solubles. Dans un mode de réalisation, cette invention concerne un procédé de production de dérivés de délétion solubles d'une protéine comprenant l'intégration de transposon et le clonage de recombinaison au moyen de recombinases spécifiques de site. Dans un autre mode de réalisation, cette invention concerne un procédé d'identification de chacune des au moins deux protéines solubles, qui consiste à analyser simultanément des cassettes de lecture ouvertes groupées. Ces deux modes de réalisation comprennent la purification de protéines, la séparation de protéines et l'analyse spectroscopique de masse des protéines séparées. Cette invention concerne aussi un transposon comprenant un gène marqueur sélectionnable flanqué par des séquences de recombinaison spécifiques du site inversées identiques.
PCT/US2005/019477 2004-11-19 2005-06-03 Identification de proteines dans un genome WO2006055040A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8071364B2 (en) 2003-12-24 2011-12-06 Transgenrx, Inc. Gene therapy using transposon-based vectors
US8283518B2 (en) 2002-06-26 2012-10-09 Transgenrx, Inc. Administration of transposon-based vectors to reproductive organs

Families Citing this family (3)

* Cited by examiner, † Cited by third party
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WO2010036978A2 (fr) 2008-09-25 2010-04-01 Transgenrx, Inc. Nouveaux vecteurs pour la production d'hormone de croissance
US9150880B2 (en) 2008-09-25 2015-10-06 Proteovec Holding, L.L.C. Vectors for production of antibodies
WO2010118360A1 (fr) 2009-04-09 2010-10-14 The Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Production de protéines au moyen de vecteurs à base de transposon

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BUESSOW KONRAD ET AL: "A human cDNA library for high-throughput protein expression screening" GENOMICS, vol. 65, no. 1, 1 April 2000 (2000-04-01), pages 1-8, XP004439385 ISSN: 0888-7543 *
CHATTERJEE PRADEEP K ET AL: "Mutually exclusive recombination of wild-type and mutant loxP sites in vivo facilitates transposon-mediated deletions from both ends of genomic DNA in PACs" NUCLEIC ACIDS RESEARCH, vol. 32, no. 18, 2004, pages 5668-5676, XP002380388 ISSN: 0305-1048 *
HONORÉ BENT ET AL: "Functional genomics studied by proteomics." BIOESSAYS : NEWS AND REVIEWS IN MOLECULAR, CELLULAR AND DEVELOPMENTAL BIOLOGY. AUG 2004, vol. 26, no. 8, August 2004 (2004-08), pages 901-915, XP009053681 ISSN: 0265-9247 *
WALHOUT A J M ET AL: "GATEWAY RECOMBINATIONAL CLONING: APPLICATION TO THE CLONING OF LARGE NUMBERS OF OPEN READING FRAMES OR ORFEOMES" METHODS IN ENZYMOLOGY, ACADEMIC PRESS INC, SAN DIEGO, CA, US, vol. 328, 2000, pages 575-592, XP001056139 ISSN: 0076-6879 *
ZHANG SEN ET AL: "Resources for targeted insertional and deletional mutagenesis in Arabidopsis." PLANT MOLECULAR BIOLOGY, vol. 53, no. 1-2, September 2003 (2003-09), pages 133-150, XP002380387 ISSN: 0167-4412 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8283518B2 (en) 2002-06-26 2012-10-09 Transgenrx, Inc. Administration of transposon-based vectors to reproductive organs
US8071364B2 (en) 2003-12-24 2011-12-06 Transgenrx, Inc. Gene therapy using transposon-based vectors

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