EP1066404A1 - Methods for producing libraries of expressible gene sequences - Google Patents

Methods for producing libraries of expressible gene sequences

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Publication number
EP1066404A1
EP1066404A1 EP99917335A EP99917335A EP1066404A1 EP 1066404 A1 EP1066404 A1 EP 1066404A1 EP 99917335 A EP99917335 A EP 99917335A EP 99917335 A EP99917335 A EP 99917335A EP 1066404 A1 EP1066404 A1 EP 1066404A1
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European Patent Office
Prior art keywords
protein
mrna
human
hypothetical
hypothetical protein
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Legal status (The legal status 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 status listed.)
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EP99917335A
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German (de)
French (fr)
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EP1066404A4 (en
Inventor
Joseph Manuel Fernandez
John Alastair Heyman
James Paul Hoeffler
Heather Lynn Marks-Hull
Michelle Lynn Sindici
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Life Technologies Corp
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Invitrogen Corp
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Publication of EP1066404A1 publication Critical patent/EP1066404A1/en
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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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1086—Preparation or screening of expression libraries, e.g. reporter assays
    • 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

Definitions

  • the invention disclosed herein relates to the fields of genomics and molecular biology. More specifically the invention relates to new high through-put methods of making libraries of expressed gene sequences and the libraries made using said methods.
  • the present invention comprises a method for producing libraries of expressible gene sequences.
  • the method of the invention allows for the simultaneous manipulation of multiple gene sequences and thus allows libraries to be created in an efficient and high through-put manner.
  • the expression vectors containing verified gene sequences can be used to transfect cells for the production of recombinant proteins.
  • the invention method utilizes known techniques in such a way as to create an efficient high through-put means of producing libraries of expressible gene sequences.
  • the invention further comprises libraries of expressible gene sequences produced using the method of the invention and expression vectors used in the construction of such libraries.
  • Figure 1 shows a schematic representation of the vaccinia topoisomerase type I cloning method used in the practice of the invention.
  • the present invention comprises a method for producing libraries of expressible gene sequences.
  • the invention method comprises the following steps: amplifying a plurality of gene sequences, purifying the amplified gene sequences, inserting each of the purified gene sequences into an expression vector, and verifying the size and orientation of the inserted gene sequence.
  • the gene sequences that are to be expressed are amplified.
  • amplification it is meant that the copy number of the gene sequence(s) is increased.
  • One commonly used method of amplification is the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • starter DNA is heat-denatured into single strands.
  • Two synthetic oligonucleotides, one complementary to sequence at the 3' end of the sense strand of DNA segment of interest and the other complementary to the sequence at the 3' end of the anti-sense strand of a DNA segment of interest, are added in great excess to the DNA sequence to be amplified and the temperature is lowered to 50 - 60° C.
  • the specific oligonucleotides hybridize with the complementary sequences in the DNA and then serve as primers of DNA chain synthesis, which requires the addition of a supply of deoxynucleotides and a temperature-resistant DNA polymerase, such as Taq polymerase, which can extend the primers at temperatures up to 72° C.
  • a temperature-resistant DNA polymerase such as Taq polymerase
  • the whole mixture is heated further (up to 95° C) to melt the newly formed DNA duplexes.
  • the temperature is lowered again, another round of synthesis takes place, since an excess of primer is still present. Repeated cycles of synthesis and melting quickly amplify the sequence of interest.
  • a more detailed description of PCR can be found in Erlich, Ed, PCR Technology: Principles and Applications for DNA Amplification, W.H. Freeman and Co., 1992 and Erlich, et al, Eds., Polymerase Chain Reaction, Cold Spring Harbor Laboratory, 1989, both of which are incorporated by reference herein.
  • Starter DNA can come from a variety of sources. It can be total genomic
  • DNA from an organism for example, or can be cDNA that has been synthesized from cellular mRNA using reverse transcriptase.
  • Sources of suitable RNA include normal and diseased tissues, cellular extracts, and the like.
  • the desired gene sequences can come from any source.
  • the examples presented below show the amplification of all open reading frames (ORFs) from a single organism, Saccharomyces cerevisiae, for example.
  • ORFs open reading frames
  • open reading frame it is meant a segment of DNA that exists between a start codon and a stop codon and is likely to represent a gene.
  • the examples presented below further show the amplification of a group of human genes thought to be important in the development of cancer.
  • yeast Sacharomyces cerevisiae
  • prokaryotes Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, and the like
  • fish Fegu rubripes
  • mammals human, mouse
  • plants rice, cotton
  • Well known databases include GenBank, Unigene, EMBL, IMAGE and TIGR, for example.
  • Public databases such as these can be used a source of gene sequences for use in the method of the invention.
  • the primers employed in the amplification step are specific for each desired gene sequence and include a variety of unique features.
  • the 5' "sense" primer starts with the sequence 5' -C ACC ATG... (the start codon is underlined).
  • the CACC sequence is added as a Kozak consensus that aids in translational efficiency.
  • the 3' "antisense" codon is preferably designed to make the amplification product end at the 3rd position of the last codon of the gene being amplified, plus a single adenine residue.
  • the gene sequence need not encode a full-length sequence, however, as the invention methods are equally suitable for any gene sequence, including Expressed Sequence Tags (ESTs).
  • ESTs Expressed Sequence Tags
  • the primers can be synthesized and dried in multiwell formats, such as 96-well microtiter plates to facilitate identification and further processing.
  • the amplified gene products are next isolated from the other components of the amplification reaction mixture.
  • This purification can be accomplished using a variety of methodologies such as column chromatography, gel electrophoresis, and the like.
  • a preferred method of purification utilizes low-melt agarose gel electrophoresis.
  • the reaction mixture is separated and visualized by suitable means, e.g. by ethidium bromide staining.
  • DNA bands that represent correctly sized amplification products are cut away from the rest of the gel and placed into appropriate corresponding wells of a 96-well microtiter plate. These plugs are subsequently melted and the DNA contained therein utilized as cloning inserts.
  • the use of gel electrophoresis has the advantage that the practitioner can purify the desired amplified gene sequence while additionally verifying that the sequence is of the correct size, i.e., represents the entire desired gene sequence.
  • the purified, amplified gene sequences are next inserted into an expression vector.
  • a variety of expression vectors are suitable for use in the method of the invention, both for prokaryotic expression and eukaryotic expression.
  • the expression vector will have one or more of the following features: a promoter- enhancer sequence, a selection marker sequence, an origin of replication, an affinity purification tag sequence, an inducible element sequence, an epitope-tag sequence, and the like.
  • Promoter-enhancer sequences are DNA sequences to which RNA polymerase binds and initiates transcription. The promoter determines the polarity of the transcript by specifying which strand will be transcribed.
  • Bacterial promoters consist of consensus sequences, -35 and -10 nucleotides relative to the transcriptional start, which are bound by a specific sigma factor and RNA polymerase. Eukaryotic promoters are more complex. Most promoters utilized in expression vectors are transcribed by RNA polymerase II.
  • General transcription factors (GTFs) first bind specific sequences near the start and then recruit the binding of RNA polymerase II.
  • AP-1 DNA-binding/trans-activating proteins
  • Viral promoters serve the same function as bacterial or eukaryotic promoters and either provide a specific RNA polymerase in trans (bacteriophage T7) or recruit cellular factors and RNA polymerase (SV40, RSV, CMV). Viral promoters are preferred as they are generally particularly strong promoters.
  • Promoters may be, furthermore, either constitutive or, more preferably, regulatable (i.e., inducible or derepressible).
  • Inducible elements are DNA sequence elements which act in conjunction with promoters and bind either repressors (e.g. lacO/LAC Iq repressor system in E. coli) or inducers (e.g. gall/GAL4 inducer system in yeast). In either case, transcription is virtually “shut off until the promoter is derepressed or induced, at which point transcription is "turned-on".
  • constitutive promoters include the int promoter of bacteriophage ⁇ , the bla promoter of the ⁇ -lactamase gene sequence of pBR322, the CAT promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, and the like.
  • inducible prokaryotic promoters include the major right and left promoters of bacteriophage (P L and P R ), the trp, reca, lacZ, Lad, AraC and gal promoters of E. coli, the ⁇ -amylase (Ulmanen et al, J. Bacteriol. 162:176-182. 1985) and the sigma-28-specific promoters of B.
  • subtilis (Gilman et al, Gene sequence 32:11-20(1984)), the promoters of the bacteriophages of Bacillus (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), Streptomyces promoters (Ward et al, Mol. Gen. Genet. 20J.:468-478, 1986), and the like.
  • prokaryotic promoters are reviewed by Glick (J. Ind. Microbiol. 1:277- 282, 1987); Cenatiempo (Biochimie 68_:505-516, 1986); and Gottesman (Ann. Rev. Genet. 18:415-442, 1984).
  • Preferred eukaryotic promoters include, for example, the promoter of the mouse metallothionein I gene sequence (Hamer et al, J. Mol. Appl. Gen. 1:273-288, 1982); the TK promoter of Herpes virus (McKnight, Cell 11:355-365, 1982); the SV40 early promoter (Benoist et al, Nature (London) 290:304-310, 1981); the yeast gall gene sequence promoter (Johnston et al, Proc. Natl Acad. Sci. (USA) 72:6971-6975, 1982); Silver et al, Proc. Natl. Acad. Sci. (USA) 81:5951-5955, 1984), the CMV promoter, the EF-1 promoter, Ecdysone-responsive promoter(s), and the like.
  • Selection marker sequences are valuable elements in expression vectors as they provide a means to select for growth only those cells which contain a vector.
  • markers are of two types: drug resistance and auxotrophic.
  • a drug resistance marker enables cells to detoxify an exogenously added drug that would otherwise kill the cell.
  • Auxotrophic markers allow cells to synthesize an essential component (usually an amino acid) while grown in media which lacks that essential component.
  • Common selectable marker gene sequences include those for resistance to antibiotics such as ampicillin, tetracycline, kanamycin, streptomycin, bleomycin, hygromycin, neomycin, ZeocinTM, and the like.
  • Selectable auxotrophic gene sequences include, for example, hisD, which allows growth in histidine free media in the presence of histidinol.
  • a preferred selectable marker sequence for use in yeast expression systems is URA3.
  • Laboratory yeast strains carrying mutations in the gene which encodes orotidine-5' -phosphate decarboxylase, an enzyme essential for uracil biosynthesis, are unable to grow in the absence of exogenous uracil.
  • a copy of the wild-type gene (ura4+ in S. pombe and URA3 in S. cerevisiae) will complement this defect in trans.
  • a further element useful in an expression vector is an origin of replication sequence.
  • Replication origins are unique DNA segments that contain multiple short repeated sequences that are recognized by multimeric origin-binding proteins and which play a key role in assembling DNA replication enzymes at the origin site.
  • Suitable origins of replication for use in expression vectors employed herein include E. coli oriC, 2 ⁇ and ARS (both useful in yeast systems), sfl, SV40 (useful in mammalian systems), and the like.
  • Affinity purification tags are generally peptide sequences that can interact with a binding partner immobilized on a solid support. Synthetic DNA sequences encoding multiple consecutive single amino acids, such as histidine, when fused to the expressed protein, may be used for one-step purification of the recombinant protein by high affinity binding to a resin column, such as nickel sepharose. An endopeptidase recognition sequence is often engineered between the polyamino acid tag and the protein of interest to allow subsequent removal of the leader peptide by digestion with a specific protease.
  • Sequences encoding peptides such as the chitin binding domain (which binds to chitin), glutathione-S-transferase (which binds to glutathione), biotin (which binds to avidin or strepavidin), and the like can also be used for facilitating purification of the protein of interest.
  • the affinity purification tag can be separated 8
  • inteins protein self-splicing elements
  • Epitope tags are short peptide sequences that are recognized by epitope specific antibodies.
  • a fusion protein comprising a recombinant protein and an epitope tag can be simply and easily purified using an antibody bound to a chromatography resin.
  • the presence of the epitope tag furthermore allows the recombinant protein to be detected in subsequent assays, such as Western blots, without having to produce an antibody specific for the recombinant protein itself.
  • Examples of commonly used epitope tags include V5, glutathione-S-transferase (GST), hemaglutinin (HA), the peptide Phe-His-His-Thr-Thr, chitin binding domain, and the like.
  • a further useful element in an expression vector is a multiple cloning site or polylinker.
  • Synthetic DNA encoding a series of restriction endonuclease recognition sites is inserted into a plasmid vector downstream of the promoter element. These sites are engineered for convenient cloning of DNA into the vector at a specific position.
  • Suitable prokaryotic vectors include plasmids such as those capable of replication in E. coli (for example, pBR322, ColEl, pSClOl, PACYC 184, itVX, pRSET, pBAD (Invitrogen, Carlsbad, CA) and the like). Such plasmids are disclosed by Sambrook (cf. "Molecular Cloning: A Laboratory
  • Bacillus plasmids include pC194, pC221, ⁇ T127, and the like, and are disclosed by Gryczan (In: The Molecular Biology of the Bacilli, Academic Press, NY (1982), pp. 307-329).
  • Suitable Streptomyces plasmids include plJlOl (Kendall et al, J. Bacteriol.
  • Pseudomonas plasmids are reviewed by John et al. (Rev. Infect. Dis. &693-704, 1986), and Izaki (Jpn. J. Bacte ⁇ ol. 33:729-742, 1978).
  • Suitable eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 2- micron circle, pcDNA3.1, pCDNA3.1/GS, pYES2/GS, pMT, p LND, pIND(Spl), pVgRXR (Invitrogen), and the like, or their derivatives.
  • Such plasmids are well known in the art (Botstein et al, Miami Wntr. Symp. 19:265-274, 1982); Broach, In: "The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p.
  • DNA ligase has limitations, however, in that it is relatively slow acting and temperature sensitive.
  • any site-specific enzyme of this type is suitable, for example, a type I topoisomerase or a site-specific recombinase.
  • suitable site- specific recombinases include lambda integrase, FLP recombinase, Pl-Cre protein, Kw recombinase, and the like (Pan, et al, J. Biol. Chem.
  • a particularly suitable enzyme for use in the invention method is a type I topoisomerase, particularly vaccinia DNA topoisomerase.
  • topoisomerase binds to duplex DNA and cleaves the phosphodiester backbone of one strand.
  • the enzyme exhibits a high level of sequence specificity, akin to that of a restriction endonuclease. Cleavage occurs at a consensus pentapyrimidine element 5'-(C/T)CCTT in the scissile strand. In the cleavage reaction, bond energy is conserved via the formation of a covalent adduct between the 3' phosphate of the incised strand and a tyrosyl residue of the protein.
  • Vaccinia topoisomerase can religate the covalently held strand across the same bond originally cleaved (as occurs during DNA relaxation) or it can religate to a heterologous acceptor DNA and thereby create a recombinant molecule.
  • cleavage is accompanied by the spontaneous dissociation of the downstream portion of the cleaved strand.
  • the resulting topoisomerase-DNA complex containing a 5' single-stranded tail, can religate to an acceptor DNA if the acceptor molecule has a 5' OH tail complementary to that of the activated donor complex.
  • this reaction has been optimized for joining PCR-amplified DNA fragments into plasmid vectors (See Figure 1).
  • PCR fragments are naturally good surrogate substrates for the topoisomerase I religation step because they generally have 5' hydroxyl residues from the primers used for the amplification reaction. The 5' hydroxyl is the substrate for the religation reactions.
  • the use of vaccinia topoisomerase type I for cloning is described in detail in copending US patent application serial number 08/358,344, filed 12/19/94, incorporated by reference herein in its entirety.
  • the gene sequence being inserted into the expression vector can insert in either the sense or antisense direction. Therefore, the invention method provides for verification of both the size and orientation of the insert to insure that the gene sequence will express the desired protein.
  • the insert plus vector is utilized in a standard bacterial transformation reaction and the contents of the transformation 11
  • Bacterial transformation and growth selection procedures are well known in the art and described in detail in, for example, Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed. 1995.
  • Performing the PCR reaction directly from the cultured cell lysates, rather than first preparing DNA from the bacteria, is a particular advantage of the invention method as it significantly reduces both the time needed to generate the required data and the cost of doing so.
  • Plasmid DNA is prepared for use in the transformation of host cells for expression.
  • Methods of preparing plasmid DNA and transformation of cells are well known to those skilled in the art. Such methods are described, for example, in Ausubel, et al, supra.
  • Prokaryotic hosts are, generally, very efficient and convenient for the production of recombinant proteins and are, therefore, one type of preferred expression system. Prokaryotes most frequently are represented by various strains of E. coli. However, other organisms may also be used, including other bacterial strains.
  • prokaryotic hosts include bacteria such as E. coli and those from genera such as Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. However, under such conditions, the polypeptide will not be glycosylated.
  • the prokaryotic host selected for use herein must be compatible with the replicon and control sequences in the expression plasmid. 12
  • Suitable hosts may often include eukaryotic cells.
  • Preferred eukaryotic hosts include, for example, yeast, fungi, insect cells, and mammalian cells either in vivo, or in tissue culture.
  • Mammalian cells which may be useful as hosts include HeLa cells, cells of fibroblast origin such as VERO, 3T3 or CHOK1, HEK 293 cells or cells of lymphoid origin (such as 32D cells) and their derivatives.
  • Preferred mammalian host cells include nonadherent cells such as CHO, 32D, and the like.
  • Preferred yeast host cells include S. pombe, Pichi pastoris, S. cerevisiae (such as INVScl), and the like.
  • plant cells are also available as hosts, and control sequences compatible with plant cells are available, such as the cauliflower mosaic virus 35S and 19S, nopaline synthase promoter and polyadenylation signal sequences, and the like.
  • Another preferred host is an insect cell, for example the Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase or MT promoter can be used. Rubin, Science 240:1453-1459, 1988).
  • baculovirus vectors can be engineered to express large amounts of peptide encoded by a desire gene sequence in insects cells (Jasny, Science 23 !:1653, 1987); Miller et al., In: Genetic Engineering (1986), Setlow, J.K., et al., eds., Plenum, Vol. 8, pp. 277-297).
  • libraries of expressible gene sequences produced by the methods of the invention comprise gene sequences from a variety of sources such as yeast, mammals (including humans), and the like.
  • the present invention also features the purified, isolated or enriched versions of the expressed gene products produced by the methods described above.
  • Kits comprising one or more containers or vials containing components for using the libraries of the present invention are also within the scope of the invention.
  • Kits can comprise any one or more of the following elements: one or more expressible gene sequences, cells which are or can be transfected with said gene sequences, and antibodies recognizing the expressed gene product or an epitope tag associated therewith.
  • Cells suitable for inclusion in such a kit include bacterial cells, yeast cells (such as INVScl), insect cells or mammalian cells (such as CHO). 13
  • such a kit can comprises a detergent solution, preferably the Trax® lysing reagent (6% NP-40 and 9% Triton X-100 in IX PBS). Also included in the kit can be one or more binding partners, e.g., an antibody or antibodies, preferably a pair of antibodies to the same expressed gene product, which preferably do not compete for the same binding site on the expressed gene product.
  • a detergent solution preferably the Trax® lysing reagent (6% NP-40 and 9% Triton X-100 in IX PBS).
  • binding partners e.g., an antibody or antibodies, preferably a pair of antibodies to the same expressed gene product, which preferably do not compete for the same binding site on the expressed gene product.
  • a kit can comprise more than one pair of such antibodies or other binding partners, each pair directed against a different target molecule, thus allowing the detection or measurement of a plurality of such target molecules in a sample.
  • one binding partner of the kit may be pre-adsorbed to a solid phase matrix, or alternatively, the binding partner and matrix are supplied separately and the attachment is performed as part of the assay procedure.
  • the kit preferably contains the other necessary washing reagents well- known in the art.
  • the kit contains the chromogenic substrate as well as a reagent for stopping the enzymatic reaction when color development has occurred.
  • the substrate included in the kit is one appropriate for the enzyme conjugated to one of the antibody preparations. These are well-known in the art, and some are exemplified below.
  • the kit can optionally also comprise a target molecule standard; i.e., an amount of purified target molecule that is the target molecule being detected or measured.
  • a kit of the invention comprises in one or more containers: (1) a solid phase carrier, such as a microtiter plate coated with a first binding partner; (2) a detectably labeled second binding partner which binds to the same expressed gene product as the first binding partner; (3) a standard sample of the expressed gene product recognized by the first and second binding partners; (4) concentrated detergent solution; and (5) optionally, diluent.
  • a solid phase carrier such as a microtiter plate coated with a first binding partner
  • a detectably labeled second binding partner which binds to the same expressed gene product as the first binding partner
  • a standard sample of the expressed gene product recognized by the first and second binding partners (4) concentrated detergent solution
  • diluent optionally, diluent.
  • the following example illustrates the creation of a library of expressible yeast gene sequences.
  • Amplification - 6,032 yeast ORFs and a corresponding gene-specific primer of the 3' end of each were obtained from Research Genetics (Huntsville, AL) in a 96-well microtiter plate format at a concentration of 0.3 ng/ ⁇ l.
  • Each gene specific primer was designed to exclude the gene's stop codon. Since the templates each contain a common sequence immediately 5' of the start ATG (5'- GCAGTCCTGGAATTCCAGCTGACCACC) (SEQ ID NO:l), it was possible to amplify each template with a common 5' primer.
  • ORF template 5 ⁇ l was added to a fresh 96-well microtiter plate (polycarbonate Thermowell Thinwall, Model M. Cat # 6511) using a 12 channel pipetter. 6 ⁇ l of specific 3' primer solution (2 ⁇ M) was added and the total volume per well brought to 30 ⁇ l with PCR cocktail, immediately after which the plate was placed on ice.
  • Hybaid Micromat lid was washed by soaking in 0.1 M HC1, the rinsed for 2 minutes with distilled water and dried completely before applying to the 96-well plate.
  • the PCR reaction was performed using a Hybaid, Ltd. (Middlesex, UK) thermo-cycler according to the manufacturer's instructions.
  • the conditions used were as follows: pre-melt step: 94° C x 4 min; melt step: 94° C x 30 sec, anneal step: 58° C x 45 sec, extend step: 72° C x 3 min - repeated for 25 cycles; final extension: 72° C x 4 min; final block temperature set to room temp (approx. 22° C).
  • the plates were stored at 4° C. 15
  • each lane containing the amplified gene sequence was cut from the gel and transferred to a well in a 96-well microtiter plate, melted on a heat block (75° C), and a portion of the melt multi-channel pipetted into a 96-well microtiter plate (7 ⁇ l/well) containing one of two expression vectors: TOPO-adapted pcDNA3.1/GS or pYES2/GS (Invitrogen, Carlsbad, CA) previously digested with Hindlll. The plate was covered with parafilm and incubated at 37° C for 7 minutes.
  • the contents of each well were plated onto a LB(10g tryptone, 5g yeast extract, lOg NaCl per liter)/1.5% agar petrie plate containing the appropriate selection marker (ampicillin (50 ⁇ g/ml) for pYES2/GS and ZeocinTM (25 ⁇ g/ml) for pcDNA3.1/GS).
  • the petrie plates were grown overnight at 37° C.
  • Contamination is a potentially serious problem in this step. Care should be taken to guard against contaminating the process through airborne contamination, unsterile reagents or equipment, or well-to-well contamination.
  • Each well contained 100 ⁇ l of 2X LB plus 100 ⁇ g/ml ampicillin or 50 ⁇ g/ml ZeocinTM as appropriate for the expression vector used. The plates were incubated overnight at 37° C.
  • the plates were spun briefly at 1000 rpm.
  • the cells were stirred by pipetting up and down in a pipetter, then 2 ⁇ l from each well was transferred to a corresponding well in a PCR reaction plate containing 28 ⁇ l/well PCR cocktail (PCR cocktail for 840 reactions - 5040 ⁇ l 5X Buffer J, 336 ⁇ l dNTPs (50mM stock), 84 ⁇ l common 5' primer (1 ⁇ g/ ⁇ l stock, Dalton Chemical Lab. Inc, Ont. CAN), 84 ⁇ l 3' H ⁇ stopprevu primer (1 ⁇ g/ ⁇ l, Dalton Chemical Lab. Inc, Ont.
  • H ⁇ stopprevu primer has the sequence 5 ' AAA CTC AAT GGT GAT GGT GAT GAT GACC - 3') (SEQ ID NO:2).
  • the PCR reaction was run essentially as described above with the following cycle: pre-melt step: 94° C x 10 min; melt step: 94° C x 1 min, anneal step: 67° C x 1 min, extend step: 72° C x 3 min - 35 cycles; final extension: 72° C x 4 min; final block temp set to room temp (approximately 22° C).
  • pre-melt step 94° C x 10 min
  • melt step 94° C x 1 min
  • anneal step 67° C x 1 min
  • extend step 72° C x 3 min - 35 cycles
  • final extension 72° C x 4 min
  • final block temp set to room temp (approximately 22° C).
  • the plates were spun briefly at 100 rpm and 6 ⁇ l of 6X gel loading dye added to each well. Samples were run on a 1% agarose gel which was subsequently stained with ethidium bromide. Only plasmids with correctly oriented inserts give
  • the location of the positive clones was entered into a database and a spreadsheet of positive clones generated.
  • the spreadsheet was downloaded onto a Qiagen BioRobot 9600TM to direct the re-racking of the positive cultures into deep- 17
  • CHO cells were transfected with the prepared plasmid DNA using the Pfx-6 PerFect Lipid system (Invitrogen, Cat #T930-16).
  • Yeast cells (INVScl) were transfected using the S.C. EasyComp Transformation kit (Invitrogen, Cat #K5050- 01). Expression was verified by Western blot using anti-V5 antibody to detect the epitope tag. A total of 558 clones expressing a correct protein were obtained after a single pass.
  • Fetal human heart tissue was obtained from the International Institute for the Advancement of Medicine (HAM).
  • Poly A+ mRNA was isolated using the FastTrackTM 2.0 Kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.
  • the mRNA was converted to first-strand cDNA using a cDNA Cycle® Kit (Invitrogen) using the oligo dT primer provided and the protocols suggested.
  • a single cDNA synthesis reaction was split into 12 separate wells of a 96-well PCR amplification plate, and PCR amplifications were performed using specific primer sets, essentially as described above, with the exception that the ratio of Taq to Pfu was 50:1 in the initial amplification (final cone. 2 U Taq:0.04 U Pfu/well).
  • Primers were synthesized using a Primerstation 960 (Intelligent Automation Systems, Inc.) used according to the manufacturer's instructions and were designed from sequences downloaded from Unigene and sent directly to the synthesizer. Approximately 15 nMoles of each primer, having an average length of 25 basepairs, was synthesized in a 96-well format. After synthesis, the primers were cleaved from the supports, deprotected and dried in the same 96-well format (see manufacturer's instructions). 18
  • the amplified gene sequences were purified and inserted into the pcDNA3.1/GS expression vector essentially as described above.
  • the expression vectors containing sequences verified to be in the correct orientation were transfected into CHO cells in 96-well deep-well blocks using the Pfx-6 PerFect Lipid system (Invitrogen, Cat #T930-16). Cell lysates were made 48 hours after transfection, and the lysates were separated by SDS-PAGE and analyzed by Western blot according to standard protocols using an anti-V5 epitope tag Mab/horseradish peroxidase conjugate. Table 1 lists the human proteins successfully expressed using this methodology. A total of 66 clones expressing a correct protein, out of 118, were obtained after a single pass.
  • H3 H-AB006969 Homo sapiens hGAAl mRNA, 68.42 70 complete eds
  • G3 H-AD001528 Homo sapiens spermidine 40.37 40 aminopropyltransferase mRNA, complete eds
  • H4 H-AF008936 Homo sapiens syntaxin-16B 35.75 47 mRNA, complete eds
  • H5 H-AF009243 Homo sapiens proline-rich Gla 22.33 36 protein 2 (PRGP2) mRNA, complete eds
  • H6 H-AF024714 Homo sapiens interferon- 37.84 48 inducible protein (AIM2) mRNA, complete eds
  • H5 H-AF026071 Homo sapiens soluble death 30.58 50 receptor 3 beta (DR3) mRNA, complete eds
  • VTI1 Homo sapiens vesicle soluble 25.63 36.0kDa NSF attachment protein receptor (VTI1) mRNA, complete eds
  • F3 H-AF037335 Homo sapiens carbonic anhydrase 39.05 39 precursor (CA 12) mRNA, complete eds
  • GI H-AF039019 Homo sapiens zinc finger DNA 87.45 87 binding protein 89 kDa (ZBP-89) mRNA, complete eds
  • E2 H-AJ001340 Homo sapiens mRNA for U3 52.36 60 snoRNP associated 55 kDa protein
  • M236 B2 H-D45248 proteasome activator hPA28, 26.4 38 subunit bet may be cell adhesion protein
  • H2 H-D86322 Homo sapiens mRNA for 67.21 64 calmegin, complete eds
  • E2 H-D88308 Homo sapiens mRNA for very- 68.31 64 long-chain acyl-CoA synthetase, complete eds
  • E3 H-J02854 Human 20-kDa myosin light 19.03 31 chain (MLC-2) mRNA, complete eds
  • M311 F2 H-L11245 complement component 4-binding 27.83 30 protein, beta
  • JNK2 human protein kinase
  • E3 H-L40802 Homo sapiens 17-beta- 42.68 60 hydroxysteroid dehydrogenase (17-HSD) gene
  • G6 H-M33680 Human 26-kDa cell surface 26.07 24 protein TAPA-1 mRNA, complete eds
  • HAP1 apurinic/apyrimidinic endonuclease
  • proS prolyl-tRNA synthetase
  • VSPIM adenine specific DNA 60.06 methyltransferase
  • H-PHIMC cytosine specific DNA 36.3 methyltransferase
  • Rho Rho

Abstract

The present invention comprises a method for producing libraries of expressible gene sequences. The method of the invention allows for the simultaneous manipulation of multiple gene sequences and thus allows libraries to be created in an efficient and high throughput manner. The expression vectors containing verified gene sequences can be used to transfect cells for the production of recombinant proteins. The invention further comprises libraries of expressible gene sequences produced using the method of the invention and expression vectors used in the construction of said libraries.

Description

METHODS FOR PRODUCING LIBRARIES OF EXPRESSIBLE GENE
SEQUENCES
Field of the Invention
The invention disclosed herein relates to the fields of genomics and molecular biology. More specifically the invention relates to new high through-put methods of making libraries of expressed gene sequences and the libraries made using said methods.
Background of the Invention
Recent breakthroughs in nucleic acid sequencing technology have made possible the sequencing of entire genomes from a variety of organisms, including humans. The potential benefits of a complete genome sequence are many, ranging from applications in medicine to a greater understanding of evolutionary processes. These benefits cannot be fully realized, however, without an understanding of how and where these newly sequenced genes function.
Traditionally, functional understanding started with recognizing an activity, isolating a protein associated with that activity, then identifying and isolating the gene, or genes, encoding that protein. Each gene of interest was identified, isolated and expressed separately, a relatively time consuming process.
Recently, breakthroughs in high through-put DNA sequencing technology have allowed massive amounts of gene sequence information to become available to the public. Yet methods of expressing these sequences to produce the proteins encoded by them for study have still required that each sequence be manipulated one at a time. Accordingly, a need exists for the development of methods for the rapid, simultaneous expression of large numbers of gene sequences. The invention described herein addresses this and related needs as will become apparent upon inspection of the specification and the appended claims. Brief Description of the Invention
The present invention comprises a method for producing libraries of expressible gene sequences. The method of the invention allows for the simultaneous manipulation of multiple gene sequences and thus allows libraries to be created in an efficient and high through-put manner. The expression vectors containing verified gene sequences can be used to transfect cells for the production of recombinant proteins. The invention method utilizes known techniques in such a way as to create an efficient high through-put means of producing libraries of expressible gene sequences.
The invention further comprises libraries of expressible gene sequences produced using the method of the invention and expression vectors used in the construction of such libraries.
Brief Description of the Figure
Figure 1 shows a schematic representation of the vaccinia topoisomerase type I cloning method used in the practice of the invention.
Detailed Description of the Invention
The present invention comprises a method for producing libraries of expressible gene sequences. The invention method comprises the following steps: amplifying a plurality of gene sequences, purifying the amplified gene sequences, inserting each of the purified gene sequences into an expression vector, and verifying the size and orientation of the inserted gene sequence.
In the first step, the gene sequences that are to be expressed are amplified. By "amplification" it is meant that the copy number of the gene sequence(s) is increased. One commonly used method of amplification is the polymerase chain reaction (PCR). In brief, starter DNA is heat-denatured into single strands. Two synthetic oligonucleotides, one complementary to sequence at the 3' end of the sense strand of DNA segment of interest and the other complementary to the sequence at the 3' end of the anti-sense strand of a DNA segment of interest, are added in great excess to the DNA sequence to be amplified and the temperature is lowered to 50 - 60° C. The specific oligonucleotides hybridize with the complementary sequences in the DNA and then serve as primers of DNA chain synthesis, which requires the addition of a supply of deoxynucleotides and a temperature-resistant DNA polymerase, such as Taq polymerase, which can extend the primers at temperatures up to 72° C. When synthesis is complete, the whole mixture is heated further (up to 95° C) to melt the newly formed DNA duplexes. When the temperature is lowered again, another round of synthesis takes place, since an excess of primer is still present. Repeated cycles of synthesis and melting quickly amplify the sequence of interest. A more detailed description of PCR can be found in Erlich, Ed, PCR Technology: Principles and Applications for DNA Amplification, W.H. Freeman and Co., 1992 and Erlich, et al, Eds., Polymerase Chain Reaction, Cold Spring Harbor Laboratory, 1989, both of which are incorporated by reference herein.
Starter DNA can come from a variety of sources. It can be total genomic
DNA from an organism, for example, or can be cDNA that has been synthesized from cellular mRNA using reverse transcriptase. Sources of suitable RNA include normal and diseased tissues, cellular extracts, and the like.
In practicing the method of the invention, the desired gene sequences can come from any source. The examples presented below show the amplification of all open reading frames (ORFs) from a single organism, Saccharomyces cerevisiae, for example. By "open reading frame" it is meant a segment of DNA that exists between a start codon and a stop codon and is likely to represent a gene. The examples presented below further show the amplification of a group of human genes thought to be important in the development of cancer.
Public databases exist that contain the entire or partial genome of a particular organism, for example yeast (Saccharomyces cerevisiae), prokaryotes (Bacillus subtilis, E. coli, Borrelia burgdorferi, Helicobacter pylori, Mycoplasma genitalium, and the like), fish (Fugu rubripes), mammals (human, mouse), plants (rice, cotton) and the like. Well known databases include GenBank, Unigene, EMBL, IMAGE and TIGR, for example. Public databases such as these can be used a source of gene sequences for use in the method of the invention.
The primers employed in the amplification step are specific for each desired gene sequence and include a variety of unique features. For example, the 5' "sense" primer starts with the sequence 5' -C ACC ATG... (the start codon is underlined). The CACC sequence is added as a Kozak consensus that aids in translational efficiency. When the gene sequence being amplified represents a full-length gene, the 3' "antisense" codon is preferably designed to make the amplification product end at the 3rd position of the last codon of the gene being amplified, plus a single adenine residue. This facilitates the fusion of the coding region in-frame with a heterologous peptide sequence such as an epitope tag, an affinity purification tag, and the like (see below). The gene sequence need not encode a full-length sequence, however, as the invention methods are equally suitable for any gene sequence, including Expressed Sequence Tags (ESTs). The primers can be synthesized and dried in multiwell formats, such as 96-well microtiter plates to facilitate identification and further processing.
The amplified gene products are next isolated from the other components of the amplification reaction mixture. This purification can be accomplished using a variety of methodologies such as column chromatography, gel electrophoresis, and the like. A preferred method of purification utilizes low-melt agarose gel electrophoresis. The reaction mixture is separated and visualized by suitable means, e.g. by ethidium bromide staining. DNA bands that represent correctly sized amplification products are cut away from the rest of the gel and placed into appropriate corresponding wells of a 96-well microtiter plate. These plugs are subsequently melted and the DNA contained therein utilized as cloning inserts. The use of gel electrophoresis has the advantage that the practitioner can purify the desired amplified gene sequence while additionally verifying that the sequence is of the correct size, i.e., represents the entire desired gene sequence. The purified, amplified gene sequences are next inserted into an expression vector. A variety of expression vectors are suitable for use in the method of the invention, both for prokaryotic expression and eukaryotic expression. In general, the expression vector will have one or more of the following features: a promoter- enhancer sequence, a selection marker sequence, an origin of replication, an affinity purification tag sequence, an inducible element sequence, an epitope-tag sequence, and the like.
Promoter-enhancer sequences are DNA sequences to which RNA polymerase binds and initiates transcription. The promoter determines the polarity of the transcript by specifying which strand will be transcribed. Bacterial promoters consist of consensus sequences, -35 and -10 nucleotides relative to the transcriptional start, which are bound by a specific sigma factor and RNA polymerase. Eukaryotic promoters are more complex. Most promoters utilized in expression vectors are transcribed by RNA polymerase II. General transcription factors (GTFs) first bind specific sequences near the start and then recruit the binding of RNA polymerase II. In addition to these minimal promoter elements, small sequence elements are recognized specifically by modular DNA-binding/trans-activating proteins (e.g. AP-1, SP-1) which regulate the activity of a given promoter. Viral promoters serve the same function as bacterial or eukaryotic promoters and either provide a specific RNA polymerase in trans (bacteriophage T7) or recruit cellular factors and RNA polymerase (SV40, RSV, CMV). Viral promoters are preferred as they are generally particularly strong promoters.
Promoters may be, furthermore, either constitutive or, more preferably, regulatable (i.e., inducible or derepressible). Inducible elements are DNA sequence elements which act in conjunction with promoters and bind either repressors (e.g. lacO/LAC Iq repressor system in E. coli) or inducers (e.g. gall/GAL4 inducer system in yeast). In either case, transcription is virtually "shut off until the promoter is derepressed or induced, at which point transcription is "turned-on". Examples of constitutive promoters include the int promoter of bacteriophage λ, the bla promoter of the β-lactamase gene sequence of pBR322, the CAT promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, and the like. Examples of inducible prokaryotic promoters include the major right and left promoters of bacteriophage (PL and PR), the trp, reca, lacZ, Lad, AraC and gal promoters of E. coli, the α-amylase (Ulmanen et al, J. Bacteriol. 162:176-182. 1985) and the sigma-28-specific promoters of B. subtilis (Gilman et al, Gene sequence 32:11-20(1984)), the promoters of the bacteriophages of Bacillus (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), Streptomyces promoters (Ward et al, Mol. Gen. Genet. 20J.:468-478, 1986), and the like.
Exemplary prokaryotic promoters are reviewed by Glick (J. Ind. Microbiol. 1:277- 282, 1987); Cenatiempo (Biochimie 68_:505-516, 1986); and Gottesman (Ann. Rev. Genet. 18:415-442, 1984).
Preferred eukaryotic promoters include, for example, the promoter of the mouse metallothionein I gene sequence (Hamer et al, J. Mol. Appl. Gen. 1:273-288, 1982); the TK promoter of Herpes virus (McKnight, Cell 11:355-365, 1982); the SV40 early promoter (Benoist et al, Nature (London) 290:304-310, 1981); the yeast gall gene sequence promoter (Johnston et al, Proc. Natl Acad. Sci. (USA) 72:6971-6975, 1982); Silver et al, Proc. Natl. Acad. Sci. (USA) 81:5951-5955, 1984), the CMV promoter, the EF-1 promoter, Ecdysone-responsive promoter(s), and the like.
Selection marker sequences are valuable elements in expression vectors as they provide a means to select for growth only those cells which contain a vector. Such markers are of two types: drug resistance and auxotrophic. A drug resistance marker enables cells to detoxify an exogenously added drug that would otherwise kill the cell. Auxotrophic markers allow cells to synthesize an essential component (usually an amino acid) while grown in media which lacks that essential component.
Common selectable marker gene sequences include those for resistance to antibiotics such as ampicillin, tetracycline, kanamycin, streptomycin, bleomycin, hygromycin, neomycin, Zeocin™, and the like. Selectable auxotrophic gene sequences include, for example, hisD, which allows growth in histidine free media in the presence of histidinol.
A preferred selectable marker sequence for use in yeast expression systems is URA3. Laboratory yeast strains carrying mutations in the gene which encodes orotidine-5' -phosphate decarboxylase, an enzyme essential for uracil biosynthesis, are unable to grow in the absence of exogenous uracil. A copy of the wild-type gene (ura4+ in S. pombe and URA3 in S. cerevisiae) will complement this defect in trans.
A further element useful in an expression vector is an origin of replication sequence. Replication origins are unique DNA segments that contain multiple short repeated sequences that are recognized by multimeric origin-binding proteins and which play a key role in assembling DNA replication enzymes at the origin site. Suitable origins of replication for use in expression vectors employed herein include E. coli oriC, 2μ and ARS (both useful in yeast systems), sfl, SV40 (useful in mammalian systems), and the like.
Additional elements that can be included in expression vectors employed in the invention method are sequences encoding affinity purification tags or epitope tags. Affinity purification tags are generally peptide sequences that can interact with a binding partner immobilized on a solid support. Synthetic DNA sequences encoding multiple consecutive single amino acids, such as histidine, when fused to the expressed protein, may be used for one-step purification of the recombinant protein by high affinity binding to a resin column, such as nickel sepharose. An endopeptidase recognition sequence is often engineered between the polyamino acid tag and the protein of interest to allow subsequent removal of the leader peptide by digestion with a specific protease. Sequences encoding peptides such as the chitin binding domain (which binds to chitin), glutathione-S-transferase (which binds to glutathione), biotin (which binds to avidin or strepavidin), and the like can also be used for facilitating purification of the protein of interest. The affinity purification tag can be separated 8
from the protein of interest by methods well known in the art, including the use of inteins (protein self-splicing elements, Chong, et al, Gene 192:271-281. 1997).
Epitope tags are short peptide sequences that are recognized by epitope specific antibodies. A fusion protein comprising a recombinant protein and an epitope tag can be simply and easily purified using an antibody bound to a chromatography resin. The presence of the epitope tag furthermore allows the recombinant protein to be detected in subsequent assays, such as Western blots, without having to produce an antibody specific for the recombinant protein itself. Examples of commonly used epitope tags include V5, glutathione-S-transferase (GST), hemaglutinin (HA), the peptide Phe-His-His-Thr-Thr, chitin binding domain, and the like.
A further useful element in an expression vector is a multiple cloning site or polylinker. Synthetic DNA encoding a series of restriction endonuclease recognition sites is inserted into a plasmid vector downstream of the promoter element. These sites are engineered for convenient cloning of DNA into the vector at a specific position.
The foregoing elements can be combined to produce expression vectors useful in the practice of the present invention. Suitable prokaryotic vectors include plasmids such as those capable of replication in E. coli (for example, pBR322, ColEl, pSClOl, PACYC 184, itVX, pRSET, pBAD (Invitrogen, Carlsbad, CA) and the like). Such plasmids are disclosed by Sambrook (cf. "Molecular Cloning: A Laboratory
Manual", second edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, (1989)). Bacillus plasmids include pC194, pC221, ρT127, and the like, and are disclosed by Gryczan (In: The Molecular Biology of the Bacilli, Academic Press, NY (1982), pp. 307-329). Suitable Streptomyces plasmids include plJlOl (Kendall et al, J. Bacteriol. 169:4177-4183,1987), and streptomyces bacteriophages such as φC31 (Chater et al, In: Sixth International Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54). 9
Pseudomonas plasmids are reviewed by John et al. (Rev. Infect. Dis. &693-704, 1986), and Izaki (Jpn. J. Bacteήol. 33:729-742, 1978).
Suitable eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 2- micron circle, pcDNA3.1, pCDNA3.1/GS, pYES2/GS, pMT, p LND, pIND(Spl), pVgRXR (Invitrogen), and the like, or their derivatives. Such plasmids are well known in the art (Botstein et al, Miami Wntr. Symp. 19:265-274, 1982); Broach, In: "The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470, 1981; Broach, Cell 28:203-204, 1982; Dilon et α/., J. Clin. Hematol Oncol. 10:39-48, 1980; Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression, Academic Press, NY, pp. 563-608, 1980).
Construction of chimaeric DNA molecules in vitro relies traditionally on two enzymatic steps catalyzed by separate protein components. PCR amplification or site- specific restriction endonucleases are used to generate linear DNAs with defined termini that can then be joined covalently at their ends via the action of DNA ligase. DNA ligase has limitations, however, in that it is relatively slow acting and temperature sensitive.
Thus, when inserting the purified, amplified gene sequence into the expression vector the use of an enzyme that can both cleave and religate DNA in a site specific manner is preferred. Any site-specific enzyme of this type is suitable, for example, a type I topoisomerase or a site-specific recombinase. Examples of suitable site- specific recombinases include lambda integrase, FLP recombinase, Pl-Cre protein, Kw recombinase, and the like (Pan, et al, J. Biol. Chem. 261:3683-3689, 1993; Nunes-Duby, et al, EMBO J.13:4421-4430, 1994; Hallet and Sherratt, FEMS Microbio. Revs 21:157-178, 1997; Ringrose, et al, Eur J. Biochem 248:903-912, 1997).
A particularly suitable enzyme for use in the invention method is a type I topoisomerase, particularly vaccinia DNA topoisomerase. Vaccinia DNA 10
topoisomerase binds to duplex DNA and cleaves the phosphodiester backbone of one strand. The enzyme exhibits a high level of sequence specificity, akin to that of a restriction endonuclease. Cleavage occurs at a consensus pentapyrimidine element 5'-(C/T)CCTT in the scissile strand. In the cleavage reaction, bond energy is conserved via the formation of a covalent adduct between the 3' phosphate of the incised strand and a tyrosyl residue of the protein. Vaccinia topoisomerase can religate the covalently held strand across the same bond originally cleaved (as occurs during DNA relaxation) or it can religate to a heterologous acceptor DNA and thereby create a recombinant molecule.
When the substrate is configured such that the scissile bond is situated near
(within 10 basepairs of) the 3' end of a DNA duplex, cleavage is accompanied by the spontaneous dissociation of the downstream portion of the cleaved strand. The resulting topoisomerase-DNA complex, containing a 5' single-stranded tail, can religate to an acceptor DNA if the acceptor molecule has a 5' OH tail complementary to that of the activated donor complex.
In accordance with the present invention, this reaction has been optimized for joining PCR-amplified DNA fragments into plasmid vectors (See Figure 1). PCR fragments are naturally good surrogate substrates for the topoisomerase I religation step because they generally have 5' hydroxyl residues from the primers used for the amplification reaction. The 5' hydroxyl is the substrate for the religation reactions. The use of vaccinia topoisomerase type I for cloning is described in detail in copending US patent application serial number 08/358,344, filed 12/19/94, incorporated by reference herein in its entirety.
The gene sequence being inserted into the expression vector can insert in either the sense or antisense direction. Therefore, the invention method provides for verification of both the size and orientation of the insert to insure that the gene sequence will express the desired protein. Preferably, the insert plus vector is utilized in a standard bacterial transformation reaction and the contents of the transformation 11
plated onto selective growth media. Bacterial transformation and growth selection procedures are well known in the art and described in detail in, for example, Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed. 1995.
Individual bacterial colonies are picked and grown in individual wells of a multiwell microtiter plate containing selective growth media. An aliquot of these cells is used directly in a diagnostic PCR reaction. Primers for this reaction are designed such that only plasmids with correctly oriented inserts give amplification product. The amplified DNA is separated and visualized by SDS-PAGE gel electrophoresis using standard protocols (see Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed. 1995).
Performing the PCR reaction directly from the cultured cell lysates, rather than first preparing DNA from the bacteria, is a particular advantage of the invention method as it significantly reduces both the time needed to generate the required data and the cost of doing so.
Once plasmids containing the gene sequence insert in the correct orientation have been identified, plasmid DNA is prepared for use in the transformation of host cells for expression. Methods of preparing plasmid DNA and transformation of cells are well known to those skilled in the art. Such methods are described, for example, in Ausubel, et al, supra.
Prokaryotic hosts are, generally, very efficient and convenient for the production of recombinant proteins and are, therefore, one type of preferred expression system. Prokaryotes most frequently are represented by various strains of E. coli. However, other organisms may also be used, including other bacterial strains.
Recognized prokaryotic hosts include bacteria such as E. coli and those from genera such as Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. However, under such conditions, the polypeptide will not be glycosylated. The prokaryotic host selected for use herein must be compatible with the replicon and control sequences in the expression plasmid. 12
Suitable hosts may often include eukaryotic cells. Preferred eukaryotic hosts include, for example, yeast, fungi, insect cells, and mammalian cells either in vivo, or in tissue culture. Mammalian cells which may be useful as hosts include HeLa cells, cells of fibroblast origin such as VERO, 3T3 or CHOK1, HEK 293 cells or cells of lymphoid origin (such as 32D cells) and their derivatives. Preferred mammalian host cells include nonadherent cells such as CHO, 32D, and the like. Preferred yeast host cells include S. pombe, Pichi pastoris, S. cerevisiae (such as INVScl), and the like.
In addition, plant cells are also available as hosts, and control sequences compatible with plant cells are available, such as the cauliflower mosaic virus 35S and 19S, nopaline synthase promoter and polyadenylation signal sequences, and the like. Another preferred host is an insect cell, for example the Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase or MT promoter can be used. Rubin, Science 240:1453-1459, 1988). Alternatively, baculovirus vectors can be engineered to express large amounts of peptide encoded by a desire gene sequence in insects cells (Jasny, Science 23 !:1653, 1987); Miller et al., In: Genetic Engineering (1986), Setlow, J.K., et al., eds., Plenum, Vol. 8, pp. 277-297).
In a further embodiment of the invention, there are provided libraries of expressible gene sequences produced by the methods of the invention. As shown in more detail in the Examples presented below, such libraries comprise gene sequences from a variety of sources such as yeast, mammals (including humans), and the like. The present invention also features the purified, isolated or enriched versions of the expressed gene products produced by the methods described above.
Kits comprising one or more containers or vials containing components for using the libraries of the present invention are also within the scope of the invention. Kits can comprise any one or more of the following elements: one or more expressible gene sequences, cells which are or can be transfected with said gene sequences, and antibodies recognizing the expressed gene product or an epitope tag associated therewith. Cells suitable for inclusion in such a kit include bacterial cells, yeast cells (such as INVScl), insect cells or mammalian cells (such as CHO). 13
In one embodiment, such a kit can comprises a detergent solution, preferably the Trax® lysing reagent (6% NP-40 and 9% Triton X-100 in IX PBS). Also included in the kit can be one or more binding partners, e.g., an antibody or antibodies, preferably a pair of antibodies to the same expressed gene product, which preferably do not compete for the same binding site on the expressed gene product.
In another embodiment, a kit can comprise more than one pair of such antibodies or other binding partners, each pair directed against a different target molecule, thus allowing the detection or measurement of a plurality of such target molecules in a sample. In a specific embodiment, one binding partner of the kit may be pre-adsorbed to a solid phase matrix, or alternatively, the binding partner and matrix are supplied separately and the attachment is performed as part of the assay procedure. The kit preferably contains the other necessary washing reagents well- known in the art. For EIA, the kit contains the chromogenic substrate as well as a reagent for stopping the enzymatic reaction when color development has occurred. The substrate included in the kit is one appropriate for the enzyme conjugated to one of the antibody preparations. These are well-known in the art, and some are exemplified below. The kit can optionally also comprise a target molecule standard; i.e., an amount of purified target molecule that is the target molecule being detected or measured.
In a specific embodiment, a kit of the invention comprises in one or more containers: (1) a solid phase carrier, such as a microtiter plate coated with a first binding partner; (2) a detectably labeled second binding partner which binds to the same expressed gene product as the first binding partner; (3) a standard sample of the expressed gene product recognized by the first and second binding partners; (4) concentrated detergent solution; and (5) optionally, diluent.
The invention will now be described in greater detail by reference to the following non-limiting examples. 14
Example 1 - High-throughput Expression of Yeast ORFs
The following example illustrates the creation of a library of expressible yeast gene sequences.
Amplification - 6,032 yeast ORFs and a corresponding gene-specific primer of the 3' end of each were obtained from Research Genetics (Huntsville, AL) in a 96-well microtiter plate format at a concentration of 0.3 ng/μl. Each gene specific primer was designed to exclude the gene's stop codon. Since the templates each contain a common sequence immediately 5' of the start ATG (5'- GCAGTCCTGGAATTCCAGCTGACCACC) (SEQ ID NO:l), it was possible to amplify each template with a common 5' primer.
5 μl of ORF template was added to a fresh 96-well microtiter plate (polycarbonate Thermowell Thinwall, Model M. Cat # 6511) using a 12 channel pipetter. 6 μl of specific 3' primer solution (2 μM) was added and the total volume per well brought to 30 μl with PCR cocktail, immediately after which the plate was placed on ice. (PCR cocktail for 120 reactions - 720 μl 5X Buffer J, 48 μl dNTPs (50mM stock), 12 μl common 5' primer (1 μg/μl stock), 48 μl Taq DNA polymerase (Boeringer-Mannheim or Promega, 5 units/μl), 1.92 μl Pfu DNA polymerase (Stratgene, cat. # 600153-81, 2.5 units/μl) and 1464 μl distilled water. 5X Buffer J: 300 mM Tris (pH 9.5), 75 mM ammonium sulfate, 10 mM MgCl2). The rubber
Hybaid Micromat lid was washed by soaking in 0.1 M HC1, the rinsed for 2 minutes with distilled water and dried completely before applying to the 96-well plate.
The PCR reaction was performed using a Hybaid, Ltd. (Middlesex, UK) thermo-cycler according to the manufacturer's instructions. The conditions used were as follows: pre-melt step: 94° C x 4 min; melt step: 94° C x 30 sec, anneal step: 58° C x 45 sec, extend step: 72° C x 3 min - repeated for 25 cycles; final extension: 72° C x 4 min; final block temperature set to room temp (approx. 22° C). The plates were stored at 4° C. 15
Purification -
The plates were spun briefly at 1000 rpm, then 10 μl of 6X gel loading dye was added to each well (6X gel loading dye: 6 mM Tris (pH 8), 6 mM EDTA, 0.03% Bromphenol Blue, 30% glycerol). The entire contents of each well were loaded onto a 1% low melt agarose (Invitrogen # 46-0150) gel (plus ethidium bromide at 20 μl of a 10 mg/ml solution added to 400 mis of agarose) in IX TAE (50X TAE = 242g Tris base, 57.1 ml glacial acetic acid, 100 ml 0.5 M EDTA, pH 8.0 per liter (water)) and run at 110 - 120 volts for 1.25 to 1.5 hours. A UV light box was used to visualize the amplification products and ensure that only correct-sized PCR products are used in the insertion step.
Insertion into expression vector(s) -
The portion of each lane containing the amplified gene sequence was cut from the gel and transferred to a well in a 96-well microtiter plate, melted on a heat block (75° C), and a portion of the melt multi-channel pipetted into a 96-well microtiter plate (7 μl/well) containing one of two expression vectors: TOPO-adapted pcDNA3.1/GS or pYES2/GS (Invitrogen, Carlsbad, CA) previously digested with Hindlll. The plate was covered with parafilm and incubated at 37° C for 7 minutes. Top 10 Chemically Competent Cells (Invitrogen) were added to each well (45 μl/well, O.D.=4.7), whereupon the plate was re-covered and incubated on ice for 5 minutes. The cells were then heat shocked on a 42° C block for 1 minute and returned to ice for 1 minute. An aliquot of SOC medium was added to each well (150 μl, 20g tryptone, 5g yeast extract, 0.5g NaCl, 250 mM KC1, 20 ml 1M glucose/liter), and the plate was incubated at 37° C for 90 to 120 minutes.
The contents of each well were plated onto a LB(10g tryptone, 5g yeast extract, lOg NaCl per liter)/1.5% agar petrie plate containing the appropriate selection marker (ampicillin (50 μg/ml) for pYES2/GS and Zeocin™ (25 μg/ml) for pcDNA3.1/GS). The petrie plates were grown overnight at 37° C.
Verification of size and orientation - 16
Contamination is a potentially serious problem in this step. Care should be taken to guard against contaminating the process through airborne contamination, unsterile reagents or equipment, or well-to-well contamination.
Eight colonies were picked from each petrie plate and placed in eight individual wells of a 96-well microtiter plate. Each well contained 100 μl of 2X LB plus 100 μg/ml ampicillin or 50 μg/ml Zeocin™ as appropriate for the expression vector used. The plates were incubated overnight at 37° C.
The plates were spun briefly at 1000 rpm. The cells were stirred by pipetting up and down in a pipetter, then 2 μl from each well was transferred to a corresponding well in a PCR reaction plate containing 28 μl/well PCR cocktail (PCR cocktail for 840 reactions - 5040 μl 5X Buffer J, 336 μl dNTPs (50mM stock), 84 μl common 5' primer (1 μg/μl stock, Dalton Chemical Lab. Inc, Ont. CAN), 84 μl 3' Hόstopprevu primer (1 μg/μl, Dalton Chemical Lab. Inc, Ont. CAN), 336 μl Taq DNA polymerase (Boeringer-Mannheim or Promega, 5 units/μl), and 17.64 mis distilled water. Hόstopprevu primer has the sequence 5 ' AAA CTC AAT GGT GAT GGT GAT GAT GACC - 3') (SEQ ID NO:2).
The PCR reaction was run essentially as described above with the following cycle: pre-melt step: 94° C x 10 min; melt step: 94° C x 1 min, anneal step: 67° C x 1 min, extend step: 72° C x 3 min - 35 cycles; final extension: 72° C x 4 min; final block temp set to room temp (approximately 22° C). The plates were spun briefly at 100 rpm and 6 μl of 6X gel loading dye added to each well. Samples were run on a 1% agarose gel which was subsequently stained with ethidium bromide. Only plasmids with correctly oriented inserts give an amplification product in this step.
The location of the positive clones was entered into a database and a spreadsheet of positive clones generated. The spreadsheet was downloaded onto a Qiagen BioRobot 9600™ to direct the re-racking of the positive cultures into deep- 17
well culture blocks. Essentially, a single positive culture for each clone was grown and used to prepare plasmid DNA according to the Quia-Prep Turbo protocol.
CHO cells were transfected with the prepared plasmid DNA using the Pfx-6 PerFect Lipid system (Invitrogen, Cat #T930-16). Yeast cells (INVScl) were transfected using the S.C. EasyComp Transformation kit (Invitrogen, Cat #K5050- 01). Expression was verified by Western blot using anti-V5 antibody to detect the epitope tag. A total of 558 clones expressing a correct protein were obtained after a single pass.
Example 2 - High-throughput Expression of Human Gene Sequences
The following example illustrates the construction of a library of expressible human gene sequences using the method of the invention. Primers were constructed based on sequences of human genes available from GenBank.
Fetal human heart tissue was obtained from the International Institute for the Advancement of Medicine (HAM). Poly A+ mRNA was isolated using the FastTrack™ 2.0 Kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The mRNA was converted to first-strand cDNA using a cDNA Cycle® Kit (Invitrogen) using the oligo dT primer provided and the protocols suggested. A single cDNA synthesis reaction was split into 12 separate wells of a 96-well PCR amplification plate, and PCR amplifications were performed using specific primer sets, essentially as described above, with the exception that the ratio of Taq to Pfu was 50:1 in the initial amplification (final cone. 2 U Taq:0.04 U Pfu/well).
Primers were synthesized using a Primerstation 960 (Intelligent Automation Systems, Inc.) used according to the manufacturer's instructions and were designed from sequences downloaded from Unigene and sent directly to the synthesizer. Approximately 15 nMoles of each primer, having an average length of 25 basepairs, was synthesized in a 96-well format. After synthesis, the primers were cleaved from the supports, deprotected and dried in the same 96-well format (see manufacturer's instructions). 18
The amplified gene sequences were purified and inserted into the pcDNA3.1/GS expression vector essentially as described above. The expression vectors containing sequences verified to be in the correct orientation were transfected into CHO cells in 96-well deep-well blocks using the Pfx-6 PerFect Lipid system (Invitrogen, Cat #T930-16). Cell lysates were made 48 hours after transfection, and the lysates were separated by SDS-PAGE and analyzed by Western blot according to standard protocols using an anti-V5 epitope tag Mab/horseradish peroxidase conjugate. Table 1 lists the human proteins successfully expressed using this methodology. A total of 66 clones expressing a correct protein, out of 118, were obtained after a single pass.
Table 1 - Human ORFs
Plate Number Accession Number Description Predicted Actual Size Size
M235 C7 H-A06977 albumin 67.1 67.0kDa
El H-AB002391 Human mRNA for KIAA0393 68.09 68 gene, complete eds
H3 H-AB006969 Homo sapiens hGAAl mRNA, 68.42 70 complete eds
E2 H-AB007875 Homo sapiens KIAA0415 mRNA, 51.48 51 complete eds
Dl H-AB007887 Homo sapiens KIAA0427 mRNA, 66.55 70 complete eds
M421 D6 H-AB010710 Homo sapiens mRNA for lectin- 30.14 45.0kDa like oxidized LDL receptor, complete eds
G3 H-AD001528 Homo sapiens spermidine 40.37 40 aminopropyltransferase mRNA, complete eds
B5 H-AE000659 Homo sapiens T-cell receptor 12.39 16 alpha delta locus from bases 250472 to 501670 (section 2 of 5) of the C
E2 H-AF004022 Homo sapiens protein kinase 38.28 44 mRNA, complete eds
M428 Cl H-AF004231 Homo sapiens 65.78 70.0kDa monocyte/macrophage Ig-related receptor MIR-10 (MIR cl-10) mRNA, complete eds
A5 H-AF004327 Homo sapiens angiopoietin-2 54.67 60 mRNA, complete eds
Cl H-AF006501 Homo sapiens chromosome 22 14.08 24 cosmid clone cl 155, RNA polymerase II subunit 14.4 kDa (POLRF) gene, complete eds 19
H4 H-AF008936 Homo sapiens syntaxin-16B 35.75 47 mRNA, complete eds
H5 H-AF009243 Homo sapiens proline-rich Gla 22.33 36 protein 2 (PRGP2) mRNA, complete eds
M462 D6 H-AFO 13249 Homo sapiens leukocyte- 31.68 40.0kDa associated Ig-like receptor- 1 (LAIR-1) mRNA, complete eds
Al H-AF013512 untitled 53.02 53
A3 H-AFO 13970 Homo sapiens MTG8-like protein 66.55 70 (MTGR1) mRNA, complete eds
M467 A7 H-AFO 14807 Homo sapiens 23.54 29.0kDa phosphatidylinositol synthase (PIS) mRNA, complete eds
D2 H-AFO 15257 Homo sapiens flow-induced 41.36 40 endothelial G protein-coupled receptor (FEG-1) mRNA, complete eds
M422 B5 H-AFO 17307 Homo sapiens Ets-related 40.92 49.0kDa transcription factor (ERT) mRNA, complete eds
A6 H-AFO 17656 Homo sapiens G protein beta 5 38.94 48 subunit mRNA, complete eds
El H-AFO 17995 Homo sapiens 3-phosρhoinositide 61.27 52 dependent protein kinase- 1 (PDK1) mRNA, complete eds
GI H-AF019612 Homo sapiens S2P mRNA, 57.2 57 complete eds
D3 H-AF020591 Homo sapiens zinc finger protein 78.76 74 mRNA, complete eds
A7 H-AF022385 Homo sapiens apoptosis-related 23.43 33 protein TFAR15 (TFAR15) mRNA, complete eds
H6 H-AF024714 Homo sapiens interferon- 37.84 48 inducible protein (AIM2) mRNA, complete eds
Bl H-AF025527 Homo sapiens leucocyte 48.4 47 immunoglobulin-like receptor-4 (LIR-4) mRNA, complete eds
M424 B4 H-AF025532 Homo sapiens leucocyte 49.39 59.0kDa immunoglobulin-like receptor-5 (LIR-5) mRNA, complete eds
H5 H-AF026071 Homo sapiens soluble death 30.58 50 receptor 3 beta (DR3) mRNA, complete eds
M428 Al H-AF026273 Homo sapiens interleukin-1 65.01 68.0kDa receptor-associated kinase-2 mRNA, complete eds
B6 H-AF026293 Homo sapiens chaperonin 58.96 58 containing t-complex polypeptide 1, beta subunit (Cctb) mRNA, complete eds 20
B5 H-AF026548 Homo sapiens branched chain 45.43 50 alpha-ketoacid dehydrogenase kinase precursor, mRNA, nuclear gene encoding mitochondrial protein, complete eds
B2 H-AF027204 Homo sapiens putative tetraspan 21.78 27 transmembrane protein L6H (TM4SF5) mRNA, complete eds
M426 D3 H-AF028008 Homo sapiens SPl-like zinc 56.43 64.0kDa finger transcription factor SLP mRNA, complete eds
Bl H-AF029232 Homo sapiens calpamodulin 70.62 70 (CalpM) mRNA, complete eds
M422 A7 H-AF029761 Homo sapiens decoy receptor 2 42.57 50.0kDa mRNA, complete eds
M477 F3 H-AF029893 Homo sapiens i-beta-l,3-N- 45.76 50.0kDa acetylglucosaminyltransferase mRNA, complete eds
C5 H-AF032437 Homo sapiens mitogen activated 51.92 50 protein kinase activated protein kinase gene, complete eds
M416 F3 H-AF035824 Homo sapiens vesicle soluble 25.63 36.0kDa NSF attachment protein receptor (VTI1) mRNA, complete eds
F3 H-AF037335 Homo sapiens carbonic anhydrase 39.05 39 precursor (CA 12) mRNA, complete eds
GI H-AF039019 Homo sapiens zinc finger DNA 87.45 87 binding protein 89 kDa (ZBP-89) mRNA, complete eds
GI H-AF039136 Homo sapiens Fas binding protein 81.51 98 (hDaxx) mRNA, complete eds
A7 H-AF040705 Homo sapiens putative tumor 31.57 41 suppressor protein unspliced form (Fus-2) mRNA, complete eds
M469 F1 H-AF040958 Homo sapiens lysosomal 45.76 46.0kDa neuraminidase precursor, mRNA, complete eds
G2 H-AF043472 Homo sapiens Shab-related 54.12 64 delayed-rectifier K+ channel alpha subunit (Kv9.3) mRNA, complete eds
E2 H-AJ001340 Homo sapiens mRNA for U3 52.36 60 snoRNP associated 55 kDa protein
GI H-D00096 Transtyretin (prealbumin) 16.28 20
C4 H-D00408 Cytochrome P450 IIIA7 (P450- 55.44 64 HFLa)
M302 E7 H-D00682 cofilin 18.37 30
M383 G2 H-D00726 ferrochelatase 46.64 50.0kDa
M383 C3 H-D00760 proteasome, subunit HC3 25.85 34.0kDa
M305 B4 H-D00761 proteasome, subunit HC5 26.62 33 M266 F7 H-D00763 proteasome, subunit HC9 28.82 33 21
E2 H-D00860 Phosphoribosyl pyrophosphate 35.09 47 synthetase subunit I
215-13 H-D 10522 human mRNA for 80K-L protein 35 36.59
M423 F5 H-D 11086 Interleukin 2 receptor gamma 40.7 45.0kDa chain
M248 D2 H-D 11094 positive modulator of HIV tat- 47.74 40.0kDa mediated transactivation
G3 H-D11428 Peripheral myelin protein 22 17.71 17
M424 D3 H-D13168 Human gene for endothelin-B 48.73 48.0kDa receptor (hET-BR)
M271 B8 H-D13315 glyoxalase I, 20.35 34.0kDa
LACTOYLGLUTATΗIONE LYASE. CATALYZES THE CONVERSION OF HEMIMERCAPTAL, FORMED FROM METHYLGLYOXAL AND GLUTATHIONE, TO S- LACTOYLGLUTATHIONE.
M306 FI H-D13627 hypothetical protein 60.39 90 (GB:D 13627)
M248 Dl H-D13630 hypothetical protein 46.2 49
(GB:D 13630) , Human mRNA for
KIAA0005 gene, complete eds
M270 D5 H-D13634 hypothetical protein 34.65 42.0kDa (GB:D13634)
M250 D2 H-D13642 hypothetical protein 44 48.0kDa (GB:D13642),Human mRNA for KIAA0017 gene, complete eds
M250 E6 H-D13748 translation initiation factor 4A 44.77 49.0kDa
M305 C3 H-D 13892 carboxyl methyltransferase, 25.19 34 aspartate
Dl H-D13900 enoyl-Coenzyme A hydratase, 32.01 58 short chain, mitochondrial
El H-D14446 Human HFREP-1 mRNA for 34.43 40 unknown protein, complete eds
167-14 H-D14497 H.sapiens (Ewing's sarcoma cell 51.44 64 line) mRNA encoding open reading frame
M266 D2 H-D 14520 basic transcription element- 24.2 33.0kDa binding protein 2
M318 D2 H-D14658 hypothetical protein 13.64 17 (GB:D14658)
D2 H-D 14661 Human mRNA for KIAA0105 16.72 28 gene, complete eds
M236 E2 H-D14662 HYPOTHETICAL 29.5 KD 24.75 36.0kDa PROTEIN IN UBP13-KIP1 INTERGENIC REGION [Saccharomyces cerevisiae]
M271 G6 H-D14695 hypothetical protein 43.12 50.0kDa (GB:D14695), Human mRNA for KIAA0025 gene, complete eds.
M311 A3 H-D 14696 hypothetical protein 25.74 30.0kDa (GB:D14696) 22
H3 H-D 14697 Farnesyl diphosphate synthase 46.2 55 (farnesyl pyrophosphate synthetase, dimethylallyltranstransferase, geranyltranstransferase)
M271 E7 H-D 14705 catenin, alpha 2(E). Catenin 99.77 110 (cadherin-associated protein), alpha 1 (102kD). ASSOCIATES WITH THE CYTOPLASMIC DOMAIN OF A VARIETY OF CADHERINS.
M236 A6 H-D14811 hypothetical protein 30.25 42 (GB:D14811)
M250 A3 H-D14812 hypothetical protein (GB:D14812), Human mRNA for KIAA0026 gene, complete eds
A5 H-D 14874 Human mRNA for 20.46 33 adrenomedullin, complete eds
F3 H-D 14887 Human mRNA for TFIIA-42, 41.47 50 complete eds
M250 H6 H-D 16234 phospholipase C, alpha , 55.66 56.0kDa PROBABLE PROTEIN DISULFIDE ISOMERASE ER- 60 PRECURSOR [Homo sapiens]
M305 Bl H-D 16480 enoyl-CoA hydratase/3- 84.04 84 hydroxyacyl-CoA dehydrogenase trifunctional protein, alpha- subunit, mitochobdrial
M271 G2 H-D 16481 3-ketoacyl-CoA thiolase, beta subunit, mitochodrial, Hydroxyacyl-Coenzyme A dehydrogenase/3 -ketoacy 1- Coenzyme Athiolase/enoyl- Coenzyme A hydratase (trifunctional protein), beta subunit
HI H-D 16626 Histidine ammonia-lyase 72.38 64
A2 H-D17532 Human mRNA for RCK, 52.03 53 complete eds
M266 F4 H-D17554 DNA-binding protein TAX 31.79 38
M248 A3 H-D21235 xeroderma pigmentosum group C 40.04 55 repair complementing protein HHR23A
M235 E1 H-D21261 SM22-ALPHA HOMOLOG, 22 31 hypothetical protein (GB:D21261)
M311 E1 H-D21262 hypothetical protein 77.950 63 (GB:D21262)
M466 B4 H-D21853 Human mRNA for KI A A0111 45.32 49.0kDa gene, complete eds
M311 H3 H-D23660 ribosomal protein L4 47.08 47
M419 E1 H-D26309 human mRNA for LIMK (LIM 71.240 75.0kDa kinase) 23
M271 B9 H-D26362 hypothetical protein 79.97 70 (GB:D26362), Human mRNA for KIAA0043 gene, complete eds
M361 H2 H-D26598 proteasome, subunit HsC 10-11 22.66 33.0kDa
M302 G4 H-D26599 proteasome, subunit HsC7-I 22.22 34
GI H-D26600 Human mRNA for proteasome 29.15 36 subunit HsN3, complete eds
G9 H-D28540 hypothetical protein, CDC 10 44.77 60 homolog
M266 A5 H-D29011 proteasome, subunit X 22.99 23
M236 F3 H-D29012 Proteasome (prosome, macropain) 26.4 32.0kDa delta subunit, beta type, 6
Cl H-D30037 Human mRNA for 29.92 38 phosphatidylinositol transfer protein (PI-TPbeta), complete eds
M250 H4 H-D30655 translation initiation factor 4AII, 44.88 45.0kDa and ribosomal binding protein
167-26 H-D30742 human mRNA for calmodulin- 52.10 55 dependent protein kinase IV
M236 A4 H-D31767 hypothetical protein 18.59 30
(GB:D31767), Human mRNA for
KIAA0058 gene, complete eds
El H-D31883 Human mRNA for KIAA0059 50.93 64 gene, complete eds
G2 H-D32129 MHC class I protein HLA-A 40.26 50
M422 A6 H-D37965 Human mRNA for PDGF receptor 41.36 45.0kDa beta-like tumor suppressor (PRLTS), complete eds
M305 H4 H-D38047 26S proteasome regulatory 28.340 34.0kDa subunit P31
M423 B2 H-D38081 Thromboxane A2 receptor 37.84 45.0kDa
M317 D3 H-D38305 ErbB-2 transducer 38.06 49
M270 A8 H-D38583 calgizzarin, Human mRNA for 11.66 12 calgizzarin, complete eds
M270 A6 H-D42038 hypothetical protein 15.29 27 (GB:D42038), Human mRNA for KIAA0087 gene, complete eds
M318 F3 H-D42085 hypothetical protein 90.2 100 (GB:D42085)
M311 C2 H-D43642 YL-1 protein homolog 40.15 36
El H-D45213 Human mRNA for zinc finger 12.87 20 protein, complete eds
M236 B2 H-D45248 proteasome activator hPA28, 26.4 38 subunit bet , may be cell adhesion protein
H3 H-D45887 Human mRNA for calmodulin, 16.5 20 complete eds
166-3 H-D45906 human mRNA for LIMK-2 70 70.25
A7 H-D49357 Human mRNA for S- 43.56 51 adenosylmethionine synthetase, complete eds
C5 H-D49489 Human mRNA for protein 48.51 54 disulfide isomerase-related protein P5, complete eds 24
M482 E2 H-D49958 Human fetus brain mRNA for 30.69 32.0kDa membrane glycoprotein M6, complete eds
M305 G5 H-D50063 proteasome, subunit p40 35.75 39
M250 B6 H-D50310 cyelin I, Human mRNA for cyelin 41.58 47 I, complete eds
E3 H-D50419 Homo sapiens mRNA for OTK18, 78.32 64 complete eds
M298 B1 H-D50495 transcription elongation factor h- 33 33.0kDa SII-T1 (GB:D50495)
M302 A3 H-D50840 ceramide glucosyltransferase 43.45 44
167-40 H-D50863 human mRNA for TESK1 68.9 3 70
166-28 H-D50927 human myeloblast mRNA for 60.46 64 KIAA0137 gene
Dl H-D63521 Homo sapiens mRNA for LECT2 16.72 16 precursor, complete eds
M302 A5 H-D78134 glycine-rich binding protein CIRP 19.03 30.0kDa
M313 E5 H-D78275 proteasome subunit p42 42.9 48.0kDa
B3 H-D79205 Human mRNA for ribosomal 5.72 10 protein L39, complete eds
A4 H-D79206 Human gene for ryudocan core 21.89 33 protein, exonl-5, complete eds
Al H-D80008 Human mRNA for KIAA0186 21.67 32 gene, complete eds
M298 H4 H-D83004 ubiquitin-conjugating enzyme E2 16.83 32.0kDa similar to Drosophila bendless gene product
C3 H-D83702 Human brain mRNA for 64.57 64 photolyase homolog, complete eds
M306 Al H-D83735 neutral calponin 34.1 34.0kDa
H2 H-D86322 Homo sapiens mRNA for 67.21 64 calmegin, complete eds
Bl H-D86979 Human mRNA for KIAA0226 82.72 82 gene, complete eds
169-16 H-D87116 dual specificity mitogen-activated 38.24 42 protein kinase kinase 3
166-27 H-D87119 human cancellous bone osteoblast 37.80 40 mRNA for GS3955
E2 H-D88308 Homo sapiens mRNA for very- 68.31 64 long-chain acyl-CoA synthetase, complete eds
166-26 H-D89077 human mRNA for Src-like 30.43 38 adapter protein
M440 H2 H-D89479 Homo sapiens mRNA for ST1B2, 32.67 38.0kDa complete eds
HI H-D90086 Human pyruvate dehydrogenase 39.6 35 (EC 1.2.4.1) beta subunit gene, exons 1-10
M362 FI H-D90209 DNA-binding protein 38.72 48.0kDa TAXREB67
M316 B2 H-J00068 actin, alpha 1, skeletal muscle 41.58 50
M250 B2 H-J00194 major histocompatibihty complex, 28.05 36.0kDa MHC class II, DR alpha 25
G2 H-J00212 Interferon, alpha 21 20.9 30
GI H-J00287 Human pepsinogen gene 42.79 48
M298 C2 H-J02611 apolipoprotein D 20.9 31.0kDa
M266 C4 H-J02683 ADP/ATP carrier protein 32.89 36
M383 H2 H-J02685 plasminogen activator inhibitor, 45.76 50.0kDa placenta
167-3 H-J02853 "casein kinase II, alpha chain" 43.08 50
E3 H-J02854 Human 20-kDa myosin light 19.03 31 chain (MLC-2) mRNA, complete eds
M248 F3 H-J02874 fatty-acid-binding protein 4, 14.63 17 adipocyte, LIPID TRANSPORT PROTEIN IN ADIPOCYTES
M235 D5 H-J02939 antigen 4F2, heavy chain 58.3 58
C3 H-J02943 Corticosteroid binding globulin 44.66 50
M248 F2 H-J02966 adenine nucleotide translocator 1 32.78 33 (skeletal muscle) [ANT1], CATALYZES THE EXCHANGE OF ADP AND ATP ACROSS THE MITOCHONDRIAL INNER MEMBRANE.
El H-J02982 Glycophorin B 10.12 20
167-91 H-J03075 "protein kinase c substrate, 80 kD 58.04 98 protein heavy chain"
M266 A3 H-J03191 profilin 1 15.51 17.0kDa
M248 H4 H-J03231 glucose-6-phosphate 56.76 51 dehydrogenase [G6PD]
M266 F2 H-J03459 LEUKOTRIENE A-4 67.32 64 HYDROLASE [Homo sapiens]
A2 H-J03460 Prolactin-induced protein 16.17 26
M271 E5 H-J03799 laminin receptor 1, Laminin 32.56 receptor (2H5 epitope). 40S RIBOSOMAL PROTEIN SA [Homo sapiens].
M440 A4 H-J03890 Human pulmonary surfactant 21.78 30.0kDa protein C (SP-C) and pulmonary surfactant protein Cl (SP-C1) genes, complete eds
M271 D8 H-J03934 NAD(P)H menadione 30.25 38 oxidoreductase 1, dioxin- inducible. INVOLVED IN DETOXICATION PATHWAYS.
M271 A8 H-J04031 trifunctional enzyme 102.96 117.0kDa (GB:J04031). C-l- TETRAHYDROFOLATE SYNTHASE, CYTOPLASMIC [Homo sapiens]
M305 F6 H-J04046 calmodulin 3 [CALM3] 16.5 20
M305 G7 H-J04071 cytotoxic T-lymphocyte- 27.28 38 associated serine esterase 1 (cathepsin G-like 1, granzyme B) [CTLA1]
M311 D2 H-J04183 lysosomal-associated membrane 44.99 47 protein 2 26
M300 F4 H-J04205 Sjogren syndrome antigen B 44.99 51.0kDa
M416 G8 H-J04430 Acid phosphatase 5, tartrate 35.64 45.0kDa resistant
Bl H-J04501 Glycogen synthase 1 (muscle) 81.18 81
M313 B5 H-J04543 synexin 51.37 51
Bl H-J04605 Peptidase D 54.34 55
M250 C6 H-J04615 small nuclear ribonucleoprotein 26.51 34.0kDa SM-D, ROLE IN THE PRE- mRNA SPLICING OR IN SNRNPSTRUCTURE.
M248 E2 H-J04964 steroid sulfatase (microsomal) 64.24 60.0kDa [STS]
M250 A7 H-J05249 replication protein A, 32 kDa 29.81 36.0kDa subunit , REQUIRED FOR SV 40 DNA REPLICATION IN VITRO, RP-A IS SINGLE- STRANDED DNA-BINDING PROTEIN.
FI H-J05272 IMP (inosine monophosphate) 56.65 51 dehydrogenase 1
169-15 H-J05401 "creatine kinase, sarcomeric 50 46.16 mitochondrial precursor"
M266 E4 H-J05448 RNA polymerase II, subunit B33 30.36 35.0kDa
M305 C2 H-K00558 tubulin, alpha kl [TUBA*] 49.72 52.0kDa
M416 H7 H-K01571 Human T-cell receptor active 34.43 36.0kDa beta-chain, mRNA from cell line MOLT-3, complete eds
M311 E4 H-K01763 haptoglobin 38.28 47.0kDa
G5 H-K02100 Human ornithine 39.05 47 transcarbamylase (OTC) mRNA, complete coding sequence
M302 D5 H-K02574 purine nucleoside phosphorylase 31.9 36.0kDa
169-39 H-K02581 "thymidine kinase, cytosolic" 34 25.81
M248 E4 H-K03020 phenylalanine hydroxylase [PAH] 49.83 50
M556 B3 H-K03191 Cytochrome P450, subfamily I 56.43 53.0kDa (aromatic compound-inducible), polypeptide 1
H2 H-L00190 Antithrombin III 51.15 55
169-62 H-L01087 "protein kinase c, theta type" 80 77.7
M318 C2 H-L01124 ribosomal protein S13 16.72 28
M313 F1 H-L02321 glutathione S-transferase M5 24.09 28
M305 E5 H-L02426 protease 26S, regulatory subunit 4 48.51 53
M302 D4 H-L02547 cleavage stimulation factor, 50 47.52 50.0kDa kDa subunit
M266 H7 H-L02648 transcobalamin II 47.08 48.0kDa
E2 H-L02932 Human peroxisome proliferator 51.59 59 activated receptor mRNA, complete eds
M270 A1 H-L03380 gonadotropin-releasing hormone 36.19 36 receptor [GRHR], THIS RECEPTOR MEDIATES ITS ACTION BY ASSOCIATION WITH G PROTEINS 27
M270 H1 H-L03411 RD protein [RDBP], Radin blood 41.91 59.0kDa group
D3 H-L03426 Human XE7 mRNA, complete 42.46 45 alternate coding regions
Bl H-L03785 Myosin, light polypeptide 5, 19.14 32 regulatory
A7 H-L04483 ribosomal protein S21 9.24 34
M416 B2 H-L05147 Human dual specificity 20.46 30.0kDa phosphatase tyrosine/serine mRNA, complete eds
215-38 H-L05624 dual specificity mitogen-activated 50 43.30 protein kinase kinase 1
M271 D4 H-L06132 anion channel, voltage-gated, 31.24 37 isoform 1. FORMS A CHANNEL THROUGH THE CELL MEMBRANE, THAT ALLOWS DIFFUSION FROM SMALL HYDROPHYLIC MOLECULES.
169-27 H-L06139 tyrosine-protein kinase receptor 125 123.7 TIE-2 precursor
HI H-L06147 Human (clone SY11) golgin-95 68.31 68 mRNA, complete eds
M250 A1 H-L06419 procollagen-lysine, 2-oxoglutarate 80.08 80.0kDa 5-dioxygenase (lysine hydroxylase) [PLOD]
M236 F6 H-L06498 ribosomal protein S20 13.2 23.0kDa
M318 D1 H-L06499 ribosomal protein L37a 10.23 27
M270 D1 H-L07414 CD40 antigen ligand [CD40LG], 28.82 36 NVOLVED IN
IMMUNOGLOBULIN CLASS SWITCHING.
M298 A6 H-L07548 aminoacylase 1 44.99 52.0kDa
M424 C3 H-L07592 Human peroxisome proliferator 48.62 48.0kDa activated receptor mRNA, complete eds
M298 G6 H-L07633 proteasome (prosome, macropain) 27.5 33.0kDa activator subunit 1 (PA28 alpha) [PSME1]
M318 B1 H-L08096 CD70 antigen (CD27 ligand) 21.34 28 [CD70]
D2 H-L08187 cytokine receptor EBI3 25.3 42
M313 F4 H-L08850 amyloid, non-A beta component, 15.51 31.0kDa Alzheimer's disease
M426 El H-L08895 MADS box transcription enhancer 52.14 60.0kDa factor 2, polypeptide C (myocyte enhancer factor 2C)
M266 A8 H-L09235 ATPase, vacuolar 67.98 64.0kDa
M266 D1 H-L09604 differentiation-dependent 16.83 17.0kDa intestinal membrane A4 protein (Homo sapiens)
M317 C1 H-L10338 sodium channel, voltage-gated, 24.09 24 type I, beta polypeptide [SCN1B]
M317 E1 H-L10717 tyrosine-protein kinase ITK/TSK 68.270 68.0kDa M300 B5 H-L 10820 formyl peptide receptor 1 [FPR1] 38.61 37 28
M312 A4 H-L 10838 pre-mRNA splicing factor SRp20 18.15 31.0kDa
M300 A5 H-L10918 chemokine (C-C) receptor 1 39.16 30 [CMKBR1]
M311 F2 H-L11245 complement component 4-binding 27.83 30 protein, beta
M266 B7 H-L11353 neurofibromatosis 2 (bilateral 65.56 63.0kDa acoustic neuroma) [NF2]
M311 B3 H-L 11667 cyclophilin 40 40.81 50.0kDa
215-49 H-L 11695 serine/threonine-protein kinase 64 55.40 receptor R4 precursor
M466 C2 H-L11931 Human cytosolic serine 53.24 56.0kDa hydroxymethyltransferase (SHMT) mRNA, complete eds
M271 B7 H-L12168 ADENYLYL CYCLASE- 52.36 60.0kDa ASSOCIATED PROTEIN 1 [Homo sapiens]
M416 D4 H-L 12964 Interleukin-activated receptor, 28.16 38.0kDa homolog of mouse Ly63
B3 H-L 13203 Human HNF-3/fork-head 38.72 49 homolog-3 HFH-3 mRNA, complete eds
D2 H-L13744 Human AF-9 mRNA, complete 62.59 63 eds
167-8 H-L13943 glycerol kinase 60 57.71
M311 G3 H-L13974 leucine zipper protein 41.14 51 (GB:L13974)
M271 H5 H-L 13977 LYSOSOMAL PRO-X 54.67 57 CARBOXYPEPTIDASE PRECURSOR [Homo sapiens].
M270 G2 H-L 14283 protein kinase C, zeta [PRKCZ], 65.23 98 SERINE- AND THREONINE- SPECIFIC ENZYME.
M235 A3 H-L 14286 antioxidant protein, thiol-specific 21.89 32.0kDa
M426 H3 H-L 14778 Protein phosphatase 3 (formerly 57.42 60.0kDa 2B), catalytic subunit, alpha isoform (calcineurin A alpha) {alternative products}
B4 H-L 15702 complement factor B 84.15 100
M426 A4 H-L 16794 Human transcription factor 57.42 60.0kDa (MEF2) mRNA, complete eds
215-25 H-L16862 g protein-coupled receptor kinase 70 63.4 GRK6
167-74 H-L16991 thymidylate kinase 36 23.39
169-3 H-L 18964 "protein kinase c, iota type" 80 64.64
M305 E2 H-L 18972 hypothetical protein (GB:L 18972) 75.24 78
M426 D4 H-L 19067 Human NF-kappa-B transcription 59.18 63.0kDa factor p65 subunit mRNA, complete eds
215-26 H-L 19268 Homo sapiens myotonic 70 68.71 dystrophy associated protein kinase mRNA 29
M271 E1 H-L 19297 carbonic anhydrase V [CA5], 33.66 42 Mitochondrial carbonic anhydrase. REVERSIBLE HYDRATATION OF CARBON DIOXIDE.
M298 G4 H-L19437 transaldolase 37.18 39.0kDa
M423 C4 H-L 19593 Interleukin 8 receptor, beta 39.71 41.0kDa
GI H-L 19686 Homo sapiens macrophage 12.76 13 migration inhibitory factor (MIF) gene, complete eds
G2 H-L 19739 metallopanstimulin 1 9.35 32
M302 E3 H-L 19871 activating transcription factor 3 20.02 36.0kDa
167-86 H-L20422 14-3-3 protein eta 34 27.1 3
M440 B2 H-L20492 Human gamma-glutamyl 24.86 35.0kDa transpeptidase mRNA, complete eds
M315 B1 H-L20688 GDP-dissociation inhibitor 22.22 32 protein rhoA
M271 H3 H-L20941 ferritin, heavy polypeptide. 20.24 32 FERRITIN IS AN INTRACELLULAR MOLECULE THAT STORES IRON IN A SOLUBLE, NONTOXIC, READILY AVAILABLE FORM.
M235 B7 H-L21893 Na+/taurocholate cotransporter, STRICTLY DEPENDENT ON THE
FI H-L21934 Sterol O-acyltransferase (acyl- 60.61 60 Coenzyme A: cholesterol acyltransferase)
C2 H-L22075 Human guanine nucleotide 41.58 50 regulatory protein (G13) mRNA, complete eds
169-18 H-L22206 vasopressin v2 receptor 60 58.00
M421 A10 H-L22214 Human adenosine Al receptor 35.97 38.0kDa (ADORA1) mRNA exons 1-6, complete eds
M424 FI H-L23959 Homo sapiens E2F-related 45.21 53.0kDa transcription factor (DP-1) mRNA, complete eds
C2 H-L24498 Human gadd45 gene, complete 18.26 28 eds
M302 E2 H-L25080 proto-oncogene rhoA, multidrug 21.34 31 resistance protein
M270 B8 H-L25081 guanine nucleotide-binding and 21.34 30 transforming protein rhoC, Aplysia ras-related homolog 9
M236 E3 H-L25085 Secόl complex, beta subunit, 10.67 19 PROTEIN TRANSLOCATION IN THE ENDOPLASMIC RETICULUM
167-85 H-L25610 cyclin-dependent kinase inhibitor 32 18.11 1 30
B2 H-L25610 cyclin-dependent kinase inhibitor 18.110 40 1
M297 H2 H-L26232 cathepsin A/phospholipid transfer 54.34 64.0kDa protein
167-4 H-L26318 stress-activated protein kinase 52 42.31 JNK1
M428 FI H-L27586 Human TR4 orphan receptor 67.76 67.0kDa mRNA, complete eds
M302 E5 H-L27711 protein phosphatase KAP1 23.43 28
M250 A6 H-L28010 Homo sapiens HnRNP F protein mRNA, complete eds,
FI H-L28821 Alpha mannosidase II isozyme 87.67 87
167-89 H-L28824 tyrosine-protein kinase SYK 70 69.92
M298 E6 H-L28997 ADP-ribosylation factor-like gene 20.02 33.0kDa 1
D4 H-L29219 Homo sapiens clkl mRNA, 53.35 60 complete eds
169-63 H-L29222 Homo sapiens clkl mRNA 25 15.0 3
M429 B3 H-L29277 Signal transducer and activator of 84.81 88.0kDa transcription 3 (acute-phase response factor)
Cl H-L29433 Human factor X (blood 53.79 64 coagulation factor) gene
G3 H-L31860 Glycophorin A 16.61 26
Dl H-L31881 Nuclear factor I/X (CCAAT- 48.62 48 binding transcription factor)
169-13 H-L31951 human protein kinase (JNK2) 55 46.71 mRNA
Al H-L32179 Arylacetamide deacetylase 44 50 (esterase)
B2 H-L33404 Human stratum corneum 27.94 36 chymotryptic enzyme mRNA, complete eds
M312 D3 H-L33799 procollagen C-proteinase 49.5 51.0kDa enhancer
169-77 H-L33801 human protein kinase mRNA 55 46.27 GSK-3
M305 D6 H-L34041 L-glycerol-3-phosphate:NAD+ 38.5 42.0kDa oxidoreductase
B4 H-L34355 Homo sapiens (clone p4) 50 kD 42.68 47 dystrophin-associated glycoprotein mRNA, complete eds
M297 B3 H-L35013 spliceosomal protein SAP 49 46.75 52.0kDa
167-32 H-L35253 human CSaids binding protein 52 39.67 (CSBPl) mRNA
M266 D6 H-L35545 C/activated protein C receptor, 26.29 38.0kDa endothelial
M300 FI H-L35594 autotaxin 100.76 91.0kDa
M318 E2 H-L36720 bystin 33.77 29
M305 H2 H-L37127 RNA polymerase II 12.98 16
M300 D1 H-L38490 ADP-ribosylation factor 22.22 32 (GB:L38490) 31
M318 E1 H-L38941 ribosomal protein L34 12.98 18
C2 H-L38969 Homo sapiens thrombospondin 3 105.27 110 (THBS3) gene, complete eds
M476 F4 H-L39060 Homo sapiens transcription factor 49.61 53.0kDa SL1 mRNA, complete eds
M300 E4 H-L40399 hypothetical protein (GB:L40399) 29.26 36
E3 H-L40802 Homo sapiens 17-beta- 42.68 60 hydroxysteroid dehydrogenase (17-HSD) gene
M478 FI H-L40904 H. sapiens peroxisome 52.69 60.0kDa proliferator activated receptor gamma, complete eds
M306 C2 H-L41268 natural killer associated transcript 37.62 40 2 [NKAT2*]
M306 E2 H-L41270 natural killer associated transcript 50.16 65.0kDa 4 [NKAT4*]
M306 F2 H-L41347 natural killer associated transcript 33.55 40 5 [NKAT5*]
M468 C3 H-L41351 Homo sapiens prostasin mRNA, 37.84 45.0kDa complete eds
169-53 H-L41816 Homo sapiens cam kinase I 48 40.77 mRNA
167-25 H-L41939 tyrosine-protein kinase receptor 108 108.6 EPH-3 precursor
C3 H-L42374 Homo sapiens protein 54.78 64 phosphatase 2A B56-beta (PP2A) mRNA, complete eds
M306 Bl H-L42531 glutathione synthetase 52.25 54.0kDa
M302 F6 H-L42856 RNA polymerase II transcription 13.09 20.0kDa factor SIII, pi 8 subunit
M313 C7 H-L76200 guanylate kinase (GUK1) 21.78 32.0kDa
M428 El H-L76702 Homo sapiens protein 66.33 68.0kDa phosphatase 2A B56-delta (PP2A) mRNA, complete eds
M478 Al H-L76703 Homo sapiens protein 51.48 60.0kDa phosphatase 2A B56-epsilon (PP2A) mRNA, complete eds
166-52 H-L77213 H.sapiens phosphomevalonate 34 21.19 kinase mRNA
169-64 H-L77964 H.sapiens ERK3 mRNA 100 79.38
M360 C3 H-M10050 fatty-acid-binding protein 2, 14.08 20.0kDa intestinal
D5 H-M 10050 fatty-acid-binding protein 2, 14.08 36 intestinal
M421 E7 H-M10058 Asialoglycoprotein receptor 1 32.12 48.0kDa
M429 D3 H-M 10901 Glucocorticoid receptor 85.58 85.0kDa
M312 G1 H-M 11025 asialoglycoprotein receptor 2 34.32 34.0kDa
167-44 H-M 11026 interferon alpha-4 precursor 33 20.86
F2 H-M11321 Human group-specific component 52.25 56 vitamin D-binding protein mRNA, complete eds
M236 B5 H-M11354 histone H3.2, CENTRAL ROLE 15.07 24 IN NUCLEOSOME FORMATION. 32
M236 G2 H-M11433 retinol-binding protein 1, cellular 14.96 28 transport protein
M270 G7 H-M 11560 aldolase A , FRUCTOSE- 40.15 40 BISPHOSPHATE ALDOLASE A [Homo sapiens]
H3 H-M11717 Human heat shock protein (hsp 70.51 60 70) gene, complete eds
El H-M12523 Human serum albumin (ALB) 67.1 70 gene, complete eds
B5 H-M 12963 Alcohol dehydrogenase 1 (class 41.36 48 I), alpha polypeptide
D6 H-M13228 51.15 50
D4 H-M13981 Inhibin, alpha 40.37 50
M236 G4 H-M 13982 interleukin 4 [IL4] precursor, B- 16.94 30 cell activator
M271 B6 H-M 14043 lipocortin II, Annexin II 37.4 45.0kDa (lipocortin II). CALCIUM- REGULATED MEMBRANE- BINDING PROTEIN
M271 F4 H-M14218 argininosuccinate lyase 51.04 56
M297 A3 H-M 14221 cathepsin B 37.4 32.0kDa
M305 B2 H-M 14328 enolase, alpha 47.85 50
167-54 H-M14333 human c-syn protooncogene 60 59.14
167-51 H-M 14505 H.sapiens mRNA (open reading 36 33.40 frame; patient SK29(AV))
215-74 H-M 14676 human src-like kinase (slk) 60 59.14 mRNA
167-55 H-M 14780 "creatine kinase, m chain" 52 41.98
M416 F8 H-M 15059 Fc fragment of IgE, low affinity 35.42 45.0kDa II, receptor for (CD23A)
M271 F1 H-M15182 glucuronidase, beta [GUSB], 71.72 72 PLAYS AN IMPORTANT ROLE IN THE DEGRADATION OF DERMATAN AND KERATAN SULFATES.
215-37 H-M 15465 human pyruvate kinase type L 64 59.80 mRNA
M298 A4 H-M 15796 cyelin 28.82 43.0kDa
C3 H-M 15800 Mai, T-cell differentiation protein 16.94 17
M440 El H-M15841 Human U2 small nuclear RNA- 24.86 34.0kDa associated B" antigen mRNA, complete eds
M248 C3 H-M15887 endozepine 9.68 15.0kDa
M463 A2 H-M 15990 human c-yes-1 mRNA 59.800 65.0kDa
M418 E2 H-M 16038 tyrosine-protein kinase LYN 56.390 64.0kDa
M266 D3 H-M 16342 HETEROGENEOUS NUCLEAR 32.01 49 RIBONUCLEOPROTEINS C1/C2 [Homo sapiens]; small nuclear ribonucleoprotein, polypeptide C
167-20 H-M 16591 tyrosine-protein kinase HCK 60 55.62 C7 H-M16591 tyrosine-protein kinase HCK 55.620 70 33
M305 E7 H-M 16660 heat shock 90kD protein 1, beta 79.75 80 [HSPCB]
167-65 H-M 16750 PIM-1 proto-oncogene 38 34.50 serine/threonine-protein kinase
M311 Al H-M 16827 acyl-Coenzyme A dehydrogenase, 46.42 50.0kDa C-4 to C-12 straight-chain
D3 H-M 16961 Alpha-2-HS-glycoprotein alpha 40.48 50 and beta chain
D3 H-M 16974 Complement component 8, alpha 64.35 55 polypeptide
M248 C2 H-M17017 INTERLEUKIN-8 PRECURSOR 11 11 [Homo sapiens]
M305 E4 H-M17885 ribosomal phosphoprotein P0, 34.98 37.0kDa acidic
M339 E2 H-M 17887 ribosomal phosphoprotein P2 12.76 19.0kDa
M248 D5 H-M 18731 galactose- 1 -phosphate 41.91 42 uridylyltransferase [GALT]
F2 H-M19309 Troponin Tl, skeletal, slow 30.69 40
M385 E2 H-M19713 tropomyosin, alpha, muscle 31.35 41.0kDa
167-79 H-M 19722 proto-oncogene tyrosine-protein 64 58.26 kinase FGR
M248 HI H-M20560 Annexin III (lipocortin III), 35.64 37 INHIBITOR OF PHOSPHOLIPASE A2
M235 HI H-M20681 GLUCOSE TRANSPORTER 54.67 50 TYPE 3, BRAIN
167-29 H-M21616 beta platelet-derived growth 121 121.7 factor receptor precursor
M305 A3 H-M21812 myosin light chain 2 18.81 30
167-30 H-M22146 "40S ribosomal protein S4, x 34 26.91 isoform"
M302 D6 H-M22430 phospholipase A2 RASF-A 15.95 31.0kDa
E2 H-M22491 Bone morphogenetic protein 3 52.03 55 (osteogenic)
M340 A2 H-M22538 NADH-ubiquinone reductase, 24 27.5 33 kDa subunit, mitochondrial
B2 H-M22632 Glutamic-oxaloacetic 47.41 47 transaminase 2, mitochondrial (aspartate aminotransferase 2)
B4 H-M22960 Protective protein for beta- 52.91 60 galactosidase (galactosialidosis)
M250 C4 H-M22995 ras-related protein RAP1A, member of RAS oneogene family
B3 H-M23254 Calpain, large polypeptide L2 77.11 77
M266 B4 H-M23613 Nucleophosmin (nucleolar 32.45 42 phosphoprotein B23, numatrin), BELIEVED TO BIND SINGLE- STRANDED NUCLEIC ACIDS
M469 D2 H-M23668 Homo sapiens adrenodoxin gene 20.35 25.0kDa
M478 H3 H-M24439 Human liver/bone/kidney-type 57.75 64.0kDa alkaline phosphatase (ALPL) gene
F5 H-M24470 Glucose-6-phosphate 38.06 44 dehydrogenase 34
M270 E5 H-M24898 thyroid hormone triiodothyronine 67.65 85 receptor c-erbA, ear-1, Thyroid hormone receptor, alpha (avian erythroblastic leukemia viral (v- erb-a) oneogene homolog)
D3 H-M24902 Acid phosphatase, prostate 42.57 54
D6 H-M25809 ATPase, H+ transporting, 56.32 57 lysosomal (vacuolar proton pump), beta polypeptide, 56/58kD, isoform 1
167-77 H-M26252 "pyruvate kinase, M2 isozyme" 60 58.48
M271 F8 H-M26326 keratin 18 47.41 50.0kDa
Bl H-M26901 Human renin gene 44.44 50
M271 G4 H-M27396 asparagine synthetase 61.82 62
M338 B3 H-M27542 globulin, sex hormone-binding 39.200 40
M512 B6 H-M27602 Protease, serine, 2 (trypsin 2) 27.28 36.0kDa
M270 B6 H-M27691 DNA-binding protein CREB, 36.08 50 cAMP-responsive
Cl H-M27878 Zinc finger protein 84 (HPF2) 81.29 81
M270 F6 H-M28209 guanine nucleotide-binding 22.66 30.0kDa protein rabl
M512 H5 H-M28210 RAB3A, member RAS oneogene 24.31 36.0kDa family
B3 H-M28214 Homo sapiens GTP-binding 24.2 34 protein (RAB3B) mRNA, complete eds
M300 C5 H-M28249 integrin, alpha 2 (CD49B, alpha 2 130.02 130.0kDa subunit of VLA-2 receptor) [ITGA2]
M248 B6 H-M28372 zinc finger protein 9 (a cellular 19.58 28.0kDa retroviral nucleic acid binding protein) [ZNF9]
M248 C5 H-M28983 interleukin 1, alpha [ILIA] 29.92 42
M298 C1 H-M29536 translation initiation factor 2, beta 36.74 50.0kDa subunit
M425 A5 H-M29696 Interleukin 7 receptor 50.6 63.0kDa
El H-M29960 Human steroid receptor (TR2-11) 66.44 65 mRNA, complete eds
M361 D3 H-M29971 6-O-methylguanine-DNA 22.88 33.0kDa methyltransferase [MGMT]
167-67 H-M30448 "casein kinase II, beta chain" 34 23.72
M250 E2 H-M31211 MYOSIN LIGHT CHAIN 1, 22.99 30.0kDa SLOW-TWITCH MUSCLE A ISOFORM [Homo sapiens]
M311 C4 H-M31452 proline-rich protein 65.78 68
M312 H3 H-M31469 ras-like protein TC4 23.87 32.0kDa
167-41 H-M31606 "phosphorylase B kinase gamma 50 44.7 catalytic chain, testis isoform"
B4 H-M31642 Hypoxanthine 24.09 36 phosphoribosyltransferase 1 (Lesch-Nyhan syndrome)
M416 D8 H-M31932 Fc fragment of IgG, low affinity 34.98 45.0kDa Ila, receptor for (CD32) 35
M305 A8 H-M32011 neutrophil cytosolic factor 2 57.97 58 (65kD, chronic granulomatous disease, autosomal 2) [NCF2]
B2 H-M32315 Human tumor necrosis factor 50.82 60 receptor mRNA, complete eds
M266 C2 H-M33374 cell adhesion protein SQM1 14.96 18.0kDa
M431 F1 H-M33375 dihydrodiol dehydrogenase 4 33.99 40.0kDa
G6 H-M33680 Human 26-kDa cell surface 26.07 24 protein TAPA-1 mRNA, complete eds
FI H-M33772 Human fast skeletal muscle 17.71 29 troponin C gene
167-15 H-M34065 m-phase inducer phosphatase 3 55 52.10
F4 H-M34079 Human immunodeficiency virus 44.55 52 tat transactivator binding protein- 1 (tbp-1) mRNA, complete eds
169-86 H-M34181 "cAMP-dependent protein kinase, 50 38.68 beta-catalytic subunit"
Dl H-M34379 Elastatse 2, neutrophil 29.48 35
M314 E1 H-M34671 CD59 glycoprotein precursor 14.150 20
M266 C3 H-M35252 CO-029 (GB:M35252) 26.18 30
M315 A4 H-M36035 benzodiazapine receptor 18.7 19 (peripheral) [BZRP]
M300 Cl H-M36340 ADP-ribosylation factor 1 20.02 30
M312 C3 H-M36341 ADP-ribosylation factor 2 19.91 29
D6 H-M36634 Vasoactive intestinal peptide 18.81 28
169-26 H-M36881 proto-oncogene tyrosine-protein 60 56.06 kinase LCK
167-76 H-M36981 nucleoside diphosphate kinase B 26 16.79
M298 D6 H-M37400 aspartate aminotransferase, 45.54 50.0kDa cytosolic
167-88 H-M37712 galactosyltransferase associated 55 48.36 protein kinase P58/GTA
M424 F4 H-M38258 Retinoic acid receptor, gamma 1 50.05 58.0kDa
M266 H3 H-M38690 CD9 antigen, INVOLVED IN 25.19 26.0kDa PLATELET ACTIVATION AND AGGREGATION.
M270 A5 H-M55265 casein kinase II, alpha catalytic 43.12 50 subunit
169-74 H-M55284 human protein kinase C-L 80 75.09 (PRKCL) mRNA
M512 B3 H-M55514 Potassium voltage-gated channel, 71.94 lOO.OkDa shaker-related subfamily, member 4
M271 F5 H-M57567 ADP-ribosylation factor 5 [AR5]. 19.91 32.0kDa INVOLVED IN PROTEIN TRAFFICKING AND ACTS AS AN ALLOSTERIC ACTIVATOR OF CHOLERA TOXIN.
M250 D1 H-M57627 interleukin 10 [IL10], 19.69 27 SUPPRESSOR FACTOR FOR TH1 IMMUNE RESPONSES (BY SIMILARITY). 36
M302 D3 H-M57730 EPH-related receptor tyrosine 22.620 36.0kDa kinase ligand 1 precursor
M248 B5 H-M58458 ribosomal protein S4, X-linked 29.04 36.0kDa [RPS4X]
M248 A5 H-M58459 ribosomal protein S4, Y-linked 29.04 36 [RPS4Y]
M248 G5 H-M58525 CATECHOL 0- 29.92 36 METHYLTRANSFERASE, MEMBRANE-BOUND FORM [Homo sapiens], COMT
M482 B2 H-M59916 Sphingomyelin phosphodiesterase 69.3 69.0kDa 1, acid lysosomal (acid sphingomyelinase)
M390 C1 H-M60091 galactose- 1 -phosphate 41.8 50.0kDa uridylyltransferase
M316 B1 H-M60314 bone morphogenetic protein 5 50.05 55 [BMP5]
B4 H-M60459 Erythropoietin receptor 55.99 60
C7 H-M60483 Human protein phosphatase 2A 34.1 56 catalytic subunit-alpha gene, complete eds
M462 D7 H-M60484 Human protein phosphatase 2A 34.1 44.0kDa catalytic subunit-beta gene, complete eds
A12 H-M60527 deoxycytidine kinase 28.670 50
167-5 H-M60724 human p70 ribosomal S6 kinase 66 57.82 alpha-I mRNA
167-17 H-M60725 human p70 ribosomal S6 kinase 62 55.29 alpha-II mRNA
M271 A4 H-M61199 cleavage signal 1, ESTs, Highly 27.5 36.0kDa similar to CLEAVAGE SIGNAL- 1 PROTEIN [Homo sapiens]
Bl H-M61733 Homo sapiens erythroid 70.62 71 membrane protein 4.1 mRNA, complete eds
M298 A1 H-M61764 tubulin, gamma 49.72 55.0kDa
M422 E2 H-M62505 Complement component 5 38.61 38.0kDa receptor 1 (C5a ligand)
M313 G5 H-M62810 transcription factor 1, 27.17 35.0kDa mitochondrial
C9 H-M62839 apolipoprotein H 38.06 60
G5 H-M63154 Gastric intrinsic factor (vitamin B 45.98 52 synthesis)
167-6 H-M63167 RAC-alpha serine/threonine 64 52.87 kinase
Bl H-M63573 Peptidylprolyl isomerase B 23.87 33 (cyclophilin B)
M302 H2 H-M63603 phospholamban 5.83 6
M306 Dl H-M63838 interferon, gamma-inducible 80.3 108 protein 16
M423 H3 H-M63959 Low density lipoprotein-related 39.38 48.0kDa protein-associated protein 1 (alpha-2-macroglobulin receptor- associated protein 1 37
G3 H-M64099 Human gamma-glutmyl 64.57 52 transpeptidase-related protein (GGT-Rel) mRNA, complete eds
M475 B8 H-M64673 Human heat shock factor 1 58.3 65.0kDa (TCF5) mRNA, complete eds
M266 D5 H-M64716 ribosomal protein S25 13.86 17.0kDa
M248 C6 H-M64752 glutamate receptor, ionotropic, 99.88 100 AMPA 1 [GRIA1]
M312 G3 H-M64925 palmitoylated membrane protein, 51.37 51.0kDa erythrocyte, 55 kDa
M302 C7 H-M65292 complement factor H-related 36.41 50 protein (GB:M65292)
D3 H-M68516 Human protein C inhibitor gene, 44.77 54 complete eds
167-27 H-M68520 cell division protein kinase 2 38 32.85
M236 D5 H-M68867 Cellular retinoic acid-binding 15.29 19.0kDa protein 2, MAY REGULATE THE ACCESS OF RETINOIC ACID TO THE NUCLEAR RETINOIC ACID RECEPTORS.
M441 El H-M69226 monoamine oxidase A [MAOA] 58.08 64.0kDa
M298 D5 H-M72393 calcium-dependent phospholipid- 82.5 117.0kDa binding protein [PLA2*]
M422 D5 H-M73238 Ciliary neurotrophic factor 41.03 51.0kDa receptor
Cl H-M73255 Human vascular cell adhesion 81.4 81 molecule- 1 (VCAMl) gene, complete CDS
M422 G6 H-M73481 Human gastrin releasing peptide 42.35 45.0kDa receptor (GRPR) mRNA, complete eds
M235 G6 H-M73499 carboxylesterase, INVOLVED IN 62.48 90.0kDa THE DETOXIFICATION OF XENOBIOTICS AND THE ACTIVATION OF ESTER AND AMIDE PRODRUGS.
M302 D1 H-M73547 polyposis locus DPI 20.46 28
M300 H4 H-M73969 interleukin 8 receptor, beta 39.71 36 [IL8RB]
GI H-M74491 ADP-ribosylation factor 3 20.02 31
B4 H-M74816 49.5 50
B2 H-M75110 H,K-ATPase, beta subunit 32.12 37
M416 B8 H-M76766 General transcription factor IIB 34.87 44.0kDa
167-18 H-M77198 RAC-beta serine/threonine kinase 64 57.27
167-87 H-M77348 PMEL 17 protein precursor 74 73.55
C4 H-M77698 YY1 transcription factor 45.65 48
M248 G6 H-M80261 apurinic/apyrimidinic (abasic) 35.09 37.0kDa endonuclease [APE], REPAIRS OXIDATIVE DNA DAMAGES IN VITRO
169-50 H-M80359 putative serine/threonine-protein 80 78.50 kinase P78
M330 H1 H-M80461 immunoglobulin-associated beta 25.370 27.0kDa (B29) [IGB] 38
169-1 H-M80613 ring3 protein 100 83.01
M298 A2 H-M80783 B 12 protein 34.87 43.0kDa
217-1 H-M81457 calpactin I light chain 10 10.74
M422 C6 H-M81589 Homo sapiens serotonin ID 41.58 41.0kDa receptor (5-HTlD%) mRNA, complete eds
M424 Al H-M81590 Homo sapiens serotonin ID 43.01 48.0kDa receptor (5-HT1D-) mRNA, complete eds
M250 H1 H-M81592 gamma-glutamyl carboxylase 83.49 85 [GGCX], CONVERTS GLUTAMATE RESIDUES TO GAMMA- CARBOXYGLUTAMATE
M250 F2 H-M81601 TRANSCRIPTION 33.22 36.0kDa ELONGATION FACTOR S-II [Homo sapiens]
C2 H-M81650 Human semenogelin I (SEMGI) 50.93 52 gene, complete eds
M266 A4 H-M81757 ribosomal protein S19 16.06 18
169-61 H-M81933 m-phase inducer phosphatase 1 57 57.60
M302 HI H-M82809 annexin IV 35.42 38.0kDa
M300 C4 H-M83653 cytoplasmic phosphotyrosyl 17.49 28.0kDa protein phosphatase, type 1
169-14 H-M83941 tyrosne-protein kinase receptor 108 108.2 ETK1 precursor
FI H-M84443 Galactokinase 2 50.49 52
M305 H6 H-M84747 interleukin 9 receptor [IL9R] 57.53 58
167-53 H-M86400 14-3-3 protein zeta/delta 33 27.02
M271 C8 H-M86521 transketolase 68.64 68.0kDa
169-51 H-M86699 human kinase (TTK) mRNA 92 92.58
M316 F2 H-M86752 transformation-sensitive protein 59.84 60.0kDa
M270 C8 H-M86921 membrane glycoprotein mb-1, 24.97 34 Immunoglobulin-associated alpha, ASSOCIATED TO SURFACE IGM-RECEPTOR; MAY BE INVOLVED IN SIGNAL TRANSDUCTION
A5 H-M87507 Homo sapien interleukin- 1 beta 44.55 50 convertase (IL1BCE) mRNA, complete eds
M305 B7 H-M88011 glucokinase [GCK] 51.26 60
M305 HI H-M88279 immunophilin FKBP52 50.6 64.0kDa
M420 FI H-M88468 mevalonate kinase 43.600 47.0kDa
M305 A7 H-M89913 dUTP pyrophosphatase 15.62 19 (dUTPase) [DUT*]
M316 E2 H-M90657 tumor-associated antigen L6 22.33 28
167-31 H-M90813 human D-type cyelin (CCND2) 36 31.86 mRNA
Al H-M91036 H.sapiens G-gamma globin and 16.28 18 A-gamma globin genes, complete cds's 39
G2 H-M91463 Human glucose transporter 55.66 52 (GLUT4) gene, complete eds
Al H-M91670 Human ubiquitin carrier protein 24.86 36 (E2-EPF) mRNA, complete eds
E4 H-M92444 Homo sapiens 35.09 45 apurinic/apyrimidinic endonuclease (HAP1) gene, complete eds
M305 C4 H-M94556 single-stranded DNA-binding 16.39 20 protein, mitochondrial
G12 H-M94856 fatty-acid-binding protein 14.96 36 homolog
M453 C3 H-M95623 Homo sapiens 39.82 50.0kDa hydroxymethylbilane synthase gene, complete eds
M302 F2 H-M95787 smooth muscle protein SM22 22.22 33.0kDa
Al H-M95809 Human basic transcription factor 60.39 64 62kD subunit (BTF2), complete eds
M271 E8 H-M96982 small nuclear ribonucleoprotein 26.51 39.0kDa U2 auxiliary factor, 35 kDa , SPLICING FACTOR U2AF 35 KD SUBUNIT. NECESSARY FOR THE SPLICING OF PRE- mRNA.
M416 B3 H-M96995 Growth factor receptor-bound 23.98 32.0kDa protein 2
G2 H-M96995 Growth factor receptor-bound 23.98 49 protein 2
H4 H-M97016 Bone morphogenetic protein 8 44.33 61 (osteogenic protein 2)
M271 D1 H-M97190 Sp2 transcription factor [SP2], 54.56 60 BINDS TO GC BOX PROMOTERS ELEMENTS AND SELECTIVELY ACTIVATES mRNA SYNTHESIS FROM GENES THAT CONTAIN FUNCTIONAL RECOGNITION SITES.
M271 Cl H-M97191 Sp3 transcription factor [SP3], 71.94 72 BINDS TO GT AND GC BOXES PROMOTERS ELEMENTS. PROBABLE TRANSCTRIPTIONAL ACTIVATOR.
M305 C7 H-M97388 transcription repressor (interacting 19.47 30 with the TATA-binding protein) [DR1*]
217-13 H-M97675 human transmembrane receptor 100 103.1 (rorl) mRNA
B3 H-M97856 Nuclear autoantigenic sperm 86.68 87 protein (histone-binding)
M429 G2 H-M97935 Homo sapiens transcription factor 82.61 89.0kDa ISGF-3 mRNA, complete eds 40
Dl H-M99487 Human prostate-specific 82.61 92 membrane antigen (PSM) mRNA, complete eds
M363 Al H-P0002 riboflavin synthase beta chain 17.27 (ribE)
M363 Bl H-P0004 carbonic anhydrase (icfA) 24.42
M363 Cl H-P0005 orotidine 5'-phosphate 25.08 decarboxylase (pyrF)
M363 Dl H-P0006 pantoate-beta-alanine ligase 30.47 (panC)
M379 Al H-P0010-2 chaperone and heat shock protein 60.17 (groEL)
M363 El H-P0011 co-chaperone (groES) 13.09
M363 FI H-P0012 DNA primase (dnaG) 61.6
M363 GI H-P0013 hypothetical protein 38.61
M363 HI H-P0014 hypothetical protein 30.36
M363 A2 H-P0015 hypothetical protein 10.34
M363 B2 H-P0016 hypothetical protein 9.68
M363 C2 H-P0017 virB4 homolog (virB4) 86.68
M363 D2 H-P0018 hypothetical protein 51.7
M363 E2 H-P0021 hypothetical protein 21.01
M363 F2 H-P0022 conserved hypothetical integral 57.42 membrane protein
M363 G2 H-P0026 citrate synthase (gltA) 46.97
M363 H2 H-P0027 isocitrate dehydrogenase (icd) 46.86
M363 A3 H-P0028 conserved hypothetical secreted 19.58 protein
M363 B3 H-P0030 hypothetical protein 65.34
M363 C3 H-P0031 hypothetical protein 15.18
M363 D3 H-P0034 aspartate 1-decarboxylase (panD) 12.98
M363 E3 H-P0035 conserved hypothetical protein 10.78
M363 F3 H-P0037 NADH-ubiquinone 38.72 oxidoreductase subunit
M363 G3 H-P0044 GDP-D-mannose dehydratase 42.02 (rfbD)
M363 H3 H-P0047 hydrogenase expression/formation 36.63 protein (hypE)
M363 A4 H-P0048 transcriptional regulator (hypF) 84.7
M363 B4 H-P0052 hypothetical protein 36.41
M363 C4 H-P0055 proline permease (putP) 54.67
M363 D4 H-P0056 delta- 1 -pyrroline-5-carboxylate 130.46 dehydrogenase
M363 E4 H-P0057 hypothetical protein 7.7
M363 F4 H-P0063 hypothetical protein 54.67
M363 G4 H-P0064 hypothetical protein 15.4
M363 H4 H-P0066 conserved hypothetical ATP- 91.52 binding protein
M363 A5 H-P0067 urease accessory protein (ureH) 29.26
M363 B5 H-P0068 urease accessory protein (ureG) 22
M363 C5 H-P0075 urease protein (ureC) 49.06
M363 D5 H-P0077 peptide chain release factor RF-1 38.83 (prfA) 41
M363 E5 H-P0082 methyl-accepting chemotaxis 74.14 transducer (tlpC)
M363 F5 H-P0086 conserved hypothetical protein 49.61
M363 G5 H-P0087 hypothetical protein 50.38
M363 H5 H-P0088 RNA polymerase sigma-70 factor 73.92 (rpoD)
M363 A6 H-P0089 pfs protein (pfs) 25.52
M363 B6 H-P0090 malonyl coenzyme A-acyl carrier 34.1 protein transacylase (fabD)
M363 C6 H-P0093 hypothetical protein 12.21
M363 D6 H-P0096 phosphoglycerate dehydrogenase 34.65
M304 A1 H-P0099 methyl-accepting chemotaxis 74.36 protein (tlpA)
M304 B1 H-P0100 conserved hypothetical protein 40.59
M304 Cl H-P0101 hypothetical protein 27.94
M304 Dl H-P0104 2',3'-cyclic-nucleotide 2'- 64.02 phosphodiesterase (cpdB)
M304 El H-P0105 conserved hypothetical protein 17.16
M304 FI H-P0106 cystathionine gamma-synthase 41.91 (metB)
M304 GI H-P0107 cysteine synthetase (cysK) 33.77
M304 HI H-P0108 hypothetical protein 20.57
M304 A2 H-P0109 chaperone and heat shock protein 68.31 70 (dnaK)
M304 B2 H-P0110 co-chaperone and heat shock 20.9 protein (grpE)
M304 C2 H-P0111 hypothetical protein 30.47
M304 D2 H-P0113 hypothetical protein 10.89
M304 E2 H-P0114 hypothetical protein 69.19
M304 F2 H-P0115 flagellin B (flaB) 56.65
M304 G2 H-P0116 DNA topoisomerase I (topA) 81.07
M304 H2 H-P0117 conserved hypothetical protein 33.99
M304 A3 H-P0118 hypothetical protein 43.56
M304 B3 H-P0119 hypothetical protein 50.82
M304 C3 H-P0120 hypothetical protein 43.89
M304 D3 H-P0121 phosphoenolpyruvate synthase 89.43 (ppsA)
M304 E3 H-P0122 hypothetical protein 4.84
M304 F3 H-P0123 threonyl-tRNA synthetase (thrS) 67.43
M304 G3 H-P0124 translation initiation factor IF-3 22.44 (infC)
M304 H3 H-P0125 ribosomal protein L35 (rpl35) 7.15
M304 A4 H-P0126 ribosomal protein L20 (rpl20) 12.87
M304 B4 H-P0127 outer membrane protein (omp4) 31.57
M304 C4 H-P0128 hypothetical protein 4.62
M304 D4 H-P0129 hypothetical protein 15.62
M304 E4 H-P0130 hypothetical protein 31.57
M304 F4 H-P0131 hypothetical protein 3.74
M304 G4 H-P0132 L-serine deaminase (sdaA) 50.16
M304 H4 H-P0133 serine transporter (sdaC) 45.54
M304 A5 H-P0134 3-deoxy-D-arabino-heptulosonate 49.5 7-phosphate synthase (dhsl) M304 B5 H-P0135 hypothetical protein 4.95 42
M304 C5 H-P0136 bacterioferritin comigratory 16.83 protein (bcp)
M304 D5 H-P0137 hypothetical protein 23.32
M304 E5 H-P0138 conserved hypothetical iron-sulfur 53.02 protein
M304 F5 H-P0139 conserved hypothetical secreted 26.73 protein
M304 G5 H-P0140 L-lactate permease (lctP) 60.5
M304 H5 H-P0141 L-lactate permease (lctP) 60.72
M304 A6 H-P0142 A/G-specific adenine glycosylase 36.19 (mutY)
M304 B6 H-P0144 cytochrome c oxidase, heme b 53.79 and copper-binding subunit, membrane-bound (fixN)
M304 C6 H-P0145 cytochrome c oxidase, monoheme 25.63 subunit, membrane-bound (fixO)
M304 D6 H-P0146 cbb3-type cytochrome c oxidase 8.14 subunit Q (CcoQ)
M304 E6 H-P0147 cytochrome c oxidase, diheme 31.57 subunit, membrane-bound (fixP)
M304 F6 H-P0148 hypothetical protein 7.59
M304 G6 H-P0150 hypothetical protein 21.67
M304 H6 H-P0152 hypothetical protein 31.68
M304 A7 H-P0153 recombinase (recA) 38.28
M304 B7 H-P0154 enolase (eno) 46.97
M304 C7 H-P0155 hypothetical protein 10.12
M304 D7 H-P0157 shikimic acid kinase I (aroK) 17.93
M304 E7 H-P0158 hypothetical protein 35.09
M304 F7 H-P0159 lipopolysaccharide 1,2- 41.03 glucosyltransferase (rfaJ)
M304 G7 H-P0161 hypothetical protein 4.07
M304 H7 H-P0162 conserved hypothetical protein 26.51
M304 A8 H-P0163 delta-aminolevulinic acid 35.64 dehydratase (hemB)
M304 B8 H-P0164 signal-transducing protein, 28.05 histidine kinase
M304 C8 H-P0165 hypothetical protein 19.14
M304 D8 H-P0166 response regulator (ompR) 24.86
M304 E8 H-P0167 hypothetical protein 17.38
M304 F8 H-P0168 hypothetical protein 9.68
M304 G8 H-P0170 hypothetical protein 27.94
M304 H8 H-P0171 peptide chain release factor RF-2 40.04 (prffi)
M304 A9 H-P0172 molybdopterin biosynthesis 43.12 protein (moeA)
M304 B9 H-P0173 flagellar biosynthetic protein 28.16 (fliR)
M304 C9 H-P0174 hypothetical protein 28.49
M304 D9 H-P0175 cell binding factor 2 33
M304 E9 H-P0176 fructose-bisphosphate aldolase 33.88 (tsr)
M304 F9 H-P0177 translation elongation factor EF-P 20.68 (efp) 43
M304 G9 H-P0178 spore coat polysaccharide 37.51 biosynthesis protein E
M304 H9 H-P0179 ABC transporter, ATP-binding 23.54 protein
M304 A10 H-P0180 apolipoprotein N-acyltransferase 46.86 (cute)
M304 BIO H-P0182 lysyl-tRNA synthetase (lysS) 55.22
M304 C10 H-P0183 serine hydroxymethyltransferase 45.87
(giyA)
M304 D10 H-P0184 hypothetical protein 19.91
M304 E10 H-P0185 hypothetical protein 29.48
M304 F10 H-P0186 hypothetical protein 44.55
M304 GIO H-P0187 hypothetical protein 10.56
M304 H10 H-P0188 hypothetical protein 3.74
M304 A11 H-P0189 conserved hypothetical integral 19.58 membrane protein
M304 B11 H-P0190 conserved hypothetical secreted 55.33 protein
M304 C11 H-P0191 fumarate reductase, iron-sulfur 27.06 subunit (frdB)
M304 D11 H-P0192 fumarate reductase, flavoprotein 78.65 subunit (frdA)
M304 El 1 H-P0193 fumarate reductase, cytochrome b 28.16 subunit (frdC)
M304 FI 1 H-P0194 triosephosphate isomerase (tpi) 25.85
M304 G11 H-P0195 enoyl-(acyl-carrier-protein) 30.36 reductase (NADH) (fabl)
M365 Al H-P0197 S-adenosylmethionine synthetase 42.46 2 (metX)
M365 Bl H-P0203 hypothetical protein 10.12
M365 Cl H-P0209 hypothetical protein 49.61
M365 Dl H-P0213 glucose inhibited division protein 68.42 (gidA)
M381 El H-P0218 hypothetical protein 20.24
M365 El H-P0221 nifU-like protein 35.97
M365 FI H-P0227 outer membrane protein (omp5) 76.12
M365 GI H-P0228 conserved hypothetical integral 43.01 membrane protein
M365 HI H-P0230 CTP:CMP-3-deoxy-D-manno- 26.84 octulosonate-cytidylyl-transferase
(kdsB)
M365 A2 H-P0233 conserved hypothetical protein 43.01
M365 B2 H-P0235 conserved hypothetical secreted 39.16 protein
M365 C2 H-P0236 hypothetical protein 13.64
M365 D2 H-P0238 prolyl-tRNA synthetase (proS) 63.58
M381 E2 H-P0243 neutrophil activating protein 15.95 (napA) (bacterioferritin)
M365 E2 H-P0244 signal-transducing protein, 42.02 histidine kinase (atoS)
M365 F2 H-P0246 flagellar basal-body P-ring protein 37.73 (flgi) 44
M365 G2 H-P0247 ATP-dependent RNA helicase, 54.23 DEAD-box family (deaD)
M365 H2 H-P0248 conserved hypothetical protein 39.93
M379 B1 H-P0249-2 hypothetical protein 19.8
M379 Cl H-P0250-2 oligopeptide ABC transporter, 56.87 ATP-binding protein (oppD)
M381 A3 H-P0251 oligopeptide ABC transporter, 37.29 permease protein (oppC)
M379 El H-P0252-2 outer membrane protein (omp7) 53.68
M365 A3 H-P0254 outer membrane protein (omp8) 47.52
M365 B3 H-P0255 adenylosuccinate synthetase 45.32 (purA)
M365 C3 H-P0257 conserved hypothetical secreted 24.2 protein
M365 D3 H-P0259 exonuclease VII, large subunit 46.31 (xseA)
M381 D3 H-P0260 adenine specific DNA 42.35 methyltransferase (mod)
M365 E3 H-P0263 adenine specific DNA 27.83 methyltransferase (hpaim)
M365 F3 H-P0264 ATP-dependent protease binding 94.27 subunit (clpB)
M365 G3 H-P0266 dihydroorotase (pyrC) 41.69
M365 H3 H-P0267 chlorohydrolase 45.1
M365 A4 H-P0271 hypothetical protein 36.08
M365 B4 H-P0275 ATP-dependent nuclease (addB) 47.41
M381 G3 H-P0276 hypothetical protein 20.46
M365 C4 H-P0278 guanosine pentaphosphate 53.35 phosphohydrolase (gppA)
M365 D4 H-P0279 lipopolysaccharide 37.51 heptosyltransferase-1 (rfaC)
M365 E4 H-P0280 heat shock protein B (ibpB) 36.19
M365 F4 H-P0282 hypothetical protein 52.91
M365 G4 H-P0283 3-dehydroquinate synthase (aroB) 37.84
M365 H4 H-P0284 conserved hypothetical integral 57.64 membrane protein
M365 A5 H-P0285 conserved hypothetical protein 46.09
M381 A4 H-P0287 hypothetical protein 19.03
M381 C4 H-P0288 hypothetical protein 17.38
M366 Al H-P0389 superoxide dismutase (sodB) 23.54
M366 Bl H-P0390 adhesin-thiol peroxidase (tagD) 18.37
M366 Cl H-P0391 purine-binding chemotaxis 18.26 protein (cheW)
M366 Dl H-P0392 histidine kinase (cheA) 88.44
M366 El H-P0393 chemotaxis protein (cheV) 34.32
M366 FI H-P0394 hypothetical protein 27.83
M366 GI H-P0395 conserved hypothetical protein 24.53
M366 HI H-P0396 conserved hypothetical protein 67.87
M366 A2 H-P0397 phosphoglycerate dehydrogenase 57.75 (serA)
M366 B2 H-P0398 hypothetical protein 20.13 M366 C2 H-P0399 ribosomal protein SI (rpsl) 61.27 45
M366 D2 H-P0403 phenylalanyl-tRNA synthetase, 36.19 alpha subunit (pheS)
M366 E2 H-P0404 protein kinase C inhibitor 11.55 (SP:P16436)
M366 F2 H-P0405 nifS-like protein 48.51
M366 G2 H-P0406 hypothetical protein 21.67
M366 H2 H-P0407 biotin sulfoxide reductase (bisC) 87.67
M381 Dl H-P0409 GMP synthase (guaA) 55.99
M381 FI H-P0410 putative neuraminyllactose- 27.5 binding hemagglutinin homolog (hpaA)
M366 A3 H-P0411 hypothetical protein 11.66
M366 B3 H-P0412 hypothetical protein 3.63
M366 C3 H-P0413 transposase-like protein, PS3IS 29.59
M366 D3 H-P0414 IS200 insertion sequence from 15.29 SARA 17
M366 E3 H-P0415 conserved hypothetical integral 68.64 membrane protein
M366 F3 H-P0416 cyclopropane fatty acid synthase 42.9 (cfa)
M366 G3 H-P0417 methionyl-tRNA synthetase 71.61 (metS)
M366 H3 H-P0418 hypothetical protein 36.96
M366 A4 H-P0419 conserved hypothetical protein 28.82
M366 B4 H-P0420 hypothetical protein 15.73
M366 C4 H-P0421 type 1 capsular polysaccharide 42.9 biosynthesis protein J (capJ)
M366 D4 H-P0422 arginine decarboxylase (speA) 67.76
M366 E4 H-P0424 hypothetical protein 68.2
M366 F4 H-P0425 hypothetical protein 45.98
M366 G4 H-P0427 hypothetical protein 12.32
M366 H4 H-P0433 hypothetical protein 16.28
M366 A5 H-P0436 hypothetical protein 13.42
M366 B5 H-P0437 IS605 transposase (tnpA) 15.73
M366 C5 H-P0438 IS605 transposase (tnpB) 47.08
M366 D5 H-P0442 hypothetical protein 9.79
M366 E5 H-P0445 hypothetical protein 6.82
M366 F5 H-P0452 hypothetical protein 57.09
M366 G5 H-P0455 hypothetical protein 11.44
M366 H5 H-P0457 hypothetical protein 9.68
M366 A6 H-P0463 type I restriction enzyme M 53.68 protein (hsdM)
M366 B6 H-P0464 type I restriction enzyme R 116.16 protein (hsdR)
M366 C6 H-P0465 conserved hypothetical protein 69.52
M366 D6 H-P0466 conserved hypothetical protein 28.16
M366 E6 H-P0467 conserved hypothetical integral 12.76 membrane protein
M366 F6 H-P0468 conserved hypothetical protein 54.56
M366 G6 H-P0469 conserved hypothetical protein 17.93
M366 H6 H-P0471 glutathione-regulated potassium- 45.87 efflux system protein (kefB) M366 A7 H-P0472 outer membrane protein (ompl 1) 20.57 46
M366 B7 H-P0473 molybdenum ABC transporter, 27.17 periplasmic molybdate-binding protein (modA)
M366 C7 H-P0474 molybdenum ABC transporter, 24.75 permease protein (modB)
M366 D7 H-P0475 molybdenum ABC transporter, 29.26 ATP-binding protein (modD)
M366 E7 H-P0476 glutamyl-tRNA synthetase (gltX) 51.04
M366 F7 H-P0477 outer membrane protein (ompl 2) 40.48
M366 G7 H-P0478 adenine specific DNA 60.06 methyltransferase (VSPIM)
M366 H7 H-P0479 hypothetical protein 31.13
M366 A8 H-P0481 adenine specific DNA 23.32 methyltransferase (MFOKI)
M366 B8 H-P0482 hypothetical protein 18.81
M366 C8 H-P0483 cytosine specific DNA 36.3 methyltransferase (H-PHIMC)
M367 Al H-P0486 hypothetical protein 58.19
M367 B1 H-P0487 hypothetical protein 52.91
M367 Cl H-P0489 hypothetical protein 32.56
M367 Dl H-P0490 putative potassium channel 41.69 protein, putative
M367 El H-P0491 ribosomal protein L28 (rpL28) 6.93
M367 FI H-P0492 hypothetical protein 30.69
M367 G1 H-P0494 UDP-N-acetylmuramoylalanine- 46.53 D-glutamate ligase (murD)
M367 HI H-P0495 hypothetical protein 9.57
M367 A2 H-P0496 conserved hypothetical protein 14.74
M367 B2 H-P0498 sodium- and chloride-dependent 48.73 transporter
M367 C2 H-P0499 phospholipase Al precursor (DR- 39.16 phospholipase A)
M367 D2 H-P0500 DNA polymerase III beta-subunit 41.25 (dnaN)
M367 E2 H-P0501 DNA gyrase, sub B (gyrB) 85.14
M367 F2 H-P0503 hypothetical protein 27.17
M367 G2 H-P0504 hypothetical protein 5.5
M367 H2 H-P0505 hypothetical protein 17.05
M367 A3 H-P0507 conserved hypothetical protein 23.43
M367 B3 H-P0509 glycolate oxidase subunit (glcD) 50.6
M367 C3 H-P0510 dihydrodipicolinate reductase 28.05 (dapB)
M367 D3 H-P0512 glutamine synthetase (glnA) 53.02
M367 E3 H-P0514 ribosomal protein L9 (rpl9) 16.61
M367 F3 H-P0515 heat shock protein (hslV) 19.91
M367 G3 H-P0516 heat shock protein (hslU) ORF1 48.84
M367 H3 H-P0517 GTP-binding protein (era) 33.33
M367 A4 H-P0519 conserved hypothetical protein 30.47
M367 B4 H-P0520 cag pathogenicity island protein 12.76 (cagl)
M367 C4 H-P0522 cag pathogenicity island protein 53.02 (cag3) 47
M367 D4 H-P0523 cag pathogenicity island protein 18.7 (cag4)
M367 E4 H-P0525 virBl l homolog 36.41
M367 F4 H-P0526 cag pathogenicity island protein 22 (cag6)
M367 G4 H-P0528 cag pathogenicity island protein 57.53 (cag8)
M379 HI H-P0531-2 cag pathogenicity island protein 24.09 (cagl l)
M367 H4 H-P0532 cag pathogenicity island protein 30.91 (cagl2)
M367 A5 H-P0534 cag pathogenicity island protein 21.67 (cagl3)
M367 B5 H-P0541 cag pathogenicity island protein 40.81 (cag20)
M367 C5 H-P0542 cag pathogenicity island protein 15.73 (cag21)
M367 D5 H-P0545 cag pathogenicity island protein 22.88 (cag24)
M367 E5 H-P0549 glutamate racemase (glr) 28.16
M367 F5 H-P0550 transcription termination factor 48.29 Rho (rho)
M367 G5 H-P0551 ribosomal protein L31 (rpl31) 7.48
M367 H5 H-P0552 conserved hypothetical protein 31.68
M367 A6 H-P0553 conserved hypothetical protein 25.08
M367 B6 H-P0554 hypothetical protein 35.42
M367 C6 H-P0555 hypothetical protein 30.14
M367 D6 H-P0556 hypothetical protein 16.06
M367 E6 H-P0557 acetyl-coenzyme A carboxylase 34.43 (accA)
M367 F6 H-P0558 beta ketoacyl-acyl carrier protein 45.43 synthase II (fabF)
M367 G6 H-P0561 3-ketoacyl-acyl carrier protein 27.28 reductase (fabG)
M367 H6 H-P0562 ribosomal protein S21 (rps21) 7.81
M367 A7 H-P0563 hypothetical protein 45.87
M367 B7 H-P0566 diaminopimelate epimerase 30.14 (dapF)
M367 C7 H-P0568 hypothetical protein 28.16
M367 D7 H-P0570 aminopeptidase a/i (pepA) 54.67
M367 E7 H-P0571 conserved hypothetical integral 21.23 membrane protein
M379 A2 H-P0572-2 adenine 19.8 phosphoribosyltransferase (apt)
M379 B2 H-P0573-2 hypothetical protein 12.21
M379 C2 H-P0574-2 galactosidase acetyltransferase 16.72 (lacA)
M379 D2 H-P0575-2 conserved hypothetical membrane 25.63 protein
M379 E2 H-P0576-2 signal peptidase I (lepB) 32.01
M367 F7 H-P0577 methylene-tetrahydrofolate 32.23 dehydrogenase (folD) M367 G7 H-P0579 hypothetical protein 20.35 48
M367 H7 H-P0580 hypothetical protein 41.03
M367 A8 H-P0581 dihydroorotase (pyrC) 37.4
M367 B8 H-P0582 hypothetical protein 35.75
M367 C8 H-P0583 hypothetical protein 32.34
M368 Al H-P0584 flagellar switch protein (fliN) 13.64
M368 Bl H-P0585 endonuclease III (nth) 24.09
M368 Cl H-P0587 aminodeoxychorismate lyase 36.3 (pabC)
M368 Dl H-P0591 ferredoxin oxidoreductase, 20.57 gamma subunit
M368 El H-P0593 adenine specific DNA 65.89 methyltransferase (mod)
M368 FI H-P0594 hypothetical protein 6.05
M368 GI H-P0596 hypothetical protein 21.23
M368 HI H-P0597 penicillin-binding protein 1A 72.6 (PBP-1A)
M368 A2 H-P0599 hemolysin secretion protein 47.74 precursor (hylB)
M368 B2 H-P0601 flagellin A (flaA) 56.21
M368 C2 H-P0602 endonuclease III 24.09
M368 D2 H-P0603 hypothetical protein 20.9
M379 F2 H-P0608-2 hypothetical protein 17.71
M368 E2 H-P0614 hypothetical protein 12.32
M368 F2 H-P0616 chemotaxis protein (cheV) 34.54
M368 G2 H-P0617 aspartyl-tRNA synthetase (aspS) 63.58
M368 H2 H-P0621 DNA mismatch repair protein 83.93 (MutS)
M368 A3 H-P0622 hypothetical protein 13.31
M368 B3 H-P0623 UDP-N-acetylmuramate-alanine 49.5 ligase (murC)
M368 C3 H-P0625 protein E (gcpE) 39.6
M368 D3 H-P0626 tetrahydrodipicolinate N- 44.22 succinyltransferase (dapD)
M368 E3 H-P0627 hypothetical protein 12.21
M368 F3 H-P0629 hypothetical protein 75.02
M368 G3 H-P0630 modulator of drug activity 21.45 (mda66)
M368 H3 H-P0631 quinone-reactive Ni/Fe 42.35 hydrogenase, small subunit (hydA)
M368 A4 H-P0632 quinone-reactive Ni/Fe 63.69 hydrogenase, large subunit (hydB)
M368 B4 H-P0633 quinone-reactive Ni/Fe 24.75 hydrogenase, cytochrome b subunit (hydC)
M368 C4 H-P0634 quinone-reactive Ni/Fe 19.69 hydrogenase (hydD)
M368 D4 H-P0635 hypothetical protein 56.43
M368 E4 H-P0636 hypothetical protein 10.23
M368 F4 H-P0637 hypothetical protein 16.61
M368 G4 H-P0638 outer membrane protein (ompl 3) 33.66 M368 H4 H-P0643 glutamyl-tRNA synthetase (gltX) 48.4 49
M368 A5 H-P0644 conserved hypothetical integral 10.78 membrane protein
M368 B5 H-P0645 soluble lytic murein 61.71 transglycosylase (sit)
M368 C5 H-P0646 UDP-glucose pyrophosphorylase 30.14 (galU)
M368 D5 H-P0647 hypothetical protein 14.96
M368 E5 H-P0648 UDP-N-acetylglucosamine 46.53 enolpyruvyl transferase (murZ)
M368 F5 H-P0649 aspartate ammonia-lyase (aspA) 51.59
M368 G5 H-P0650 hypothetical protein 21.67
M379 A3 H-P0651-2 fucosyltransferase 52.47
M381 E3 H-P0652 phosphoserine phosphatase (serB) 22.88
M368 H5 H-P0653 nonheme iron-containing ferritin 18.48
(pfr)
M379 G2 H-P0654-2 conserved hypothetical protein 39.71
M379 H2 H-P0655-2 protective surface antigen D15 100.87
M368 A6 H-P0656 conserved hypothetical protein 42.24
M368 B6 H-P0657 processing protease (ymxG) 47.63
M368 C6 H-P0658 PET112-like protein 52.36
M368 D6 H-P0659 hypothetical protein 45.65
M368 E6 H-P0660 hypothetical protein 37.29
M368 F6 H-P0661 ribonuclease H (rnhA) 15.84
M368 G6 H-P0662 ribonuclease III (rnc) 26.51
M368 H6 H-P0663 chorismate synthase (aroC) 40.26
M368 A7 H-P0665 oxygen-independent 50.38 coproporphyrinogen III oxidase (hemN)
M368 B7 H-P0667 hypothetical protein 9.46
M368 C7 H-P0668 hypothetical protein 66.88
M368 D7 H-P0671 outer membrane protein (ompl 4) 29.81
M368 E7 H-P0672 solute-binding signature and 43.01 mitochondrial signature protein (aspB)
M379 B3 H-P0673-2 hypothetical protein 46.97
M381 H3 H-P0674 hypothetical protein 25.19
M368 F7 H-P0676 methylated-DNA~protein- 18.59 cysteine methyltransferase (datl)
M368 G7 H-P0677 conserved hypothetical integral 28.16 membrane protein
M368 H7 H-P0679 lipopolysaccharide biosynthesis 31.9 protein (wbpB)
M369 Al H-P0681 hypothetical protein 18.59
M369 B1 H-P0682 hypothetical protein 13.97
M369 Cl H-P0683 UDP-N-acetylglucosamine 47.74 pyrophosphorylase (glmU)
M369 Dl H-P0685 flagellar biosynthetic protein 19.03 (fliP)
M369 El H-P0687 iron(II) transport protein (feoB) 70.73
M369 FI H-P0688 hypothetical protein 18.37
M369 GI H-P0690 acetyl coenzyme A 43.12 acetyltransferase (thiolase) (fadA) 50
M381 Al H-P0691 3-oxoadipate coA-transferase 25.63 subunit A (yxjD)
M381 B1 H-P0692 3-oxoadipate coA-transferase 22.88 subunit B (yxjE)
M369 HI H-P0694 hypothetical protein 28.38
M369 A2 H-P0695 hydantoin utilization protein A 78.54 (hyuA)
M369 B2 H-P0697 hypothetical protein 18.59
M369 C2 H-P0699 hypothetical protein 37.73
M369 D2 H-P0700 diacylglycerol kinase (dgkA) 14.19
M369 E2 H-P0701 DNA gyrase, sub A (gyrA) 91.08
M369 F2 H-P0703 response regulator 42.02
M369 G2 H-P0707 conserved hypothetical protein 33.99
M369 H2 H-P0711 hypothetical protein 44.77
M369 A3 H-P0715 ABC transporter, ATP-binding 26.51 protein
M369 B3 H-P0716 conserved hypothetical protein 14.74
M369 C3 H-P0718 conserved hypothetical integral 23.21 membrane protein
M369 D3 H-P0719 hypothetical protein 12.1
M369 E3 H-P0723 L-asparaginase II (ansB) 36.41
M369 F3 H-P0724 anaerobic C4-dicarboxylate 48.84 transport protein (dcuA)
M369 G3 H-P0727 transcriptional regulator, putative 36.19
M369 H3 H-P0728 conserved hypothetical protein 37.07
M369 A4 H-P0730 hypothetical protein 11.22
M369 B4 H-P0732 hypothetical protein 13.09
M369 C4 H-P0734 conserved hypothetical protein 48.4
M369 D4 H-P0735 xanthine guanine phosphoribosyl 16.94 transferase (gpt)
M369 E4 H-P0737 conserved hypothetical integral 17.49 membrane protein
M381 H2 H-P0738 D-alanine:D-alanine ligase A 38.28 (ddlA)
M369 F4 H-P0739 2-hydroxy-6-oxohepta-2,4- 26.62 dienoate hydrolase
M369 G4 H-P0741 conserved hypothetical protein 17.82
M369 H4 H-P0745 conserved hypothetical protein 36.08
M369 A5 H-P0747 conserved hypothetical protein 43.34
M369 B5 H-P0748 cell division protein (ftsE) 24.64
M369 C5 H-P0749 cell division membrane protein 29.59 (ftsX)
M369 D5 H-P0750 hypothetical protein 44.11
M369 E5 H-P0752 flagellar hook-associated protein 74.25 2 (fliD)
M381 F3 H-P0755 molybdopterin biosynthesis 23.21 protein (moeB)
M379 C3 H-P0757-2 beta-alanine synthetase homolog 32.23
M369 F5 H-P0758 conserved hypothetical integral 48.18 membrane protein
M369 G5 H-P0759 conserved hypothetical integral 45.98 membrane protein M369 H5 H-P0761 hypothetical protein 22.11 51
M369 A6 H-P0762 hypothetical protein 20.46
M369 B6 H-P0767 hypothetical protein 2.75
M369 C6 H-P0768 molybdenum cofactor 35.42 biosynthesis protein A (moaA)
M369 D6 H-P0769 molybdopterin-guanine 22.22 dinucleotide biosynthesis protein A (mobA)
M369 E6 H-P0771 hypothetical protein 27.06
M369 F6 H-P0772 N-acetylmuramoyl-L-alanine 48.51 amidase (amiA)
M369 G6 H-P0773 hypothetical protein 40.04
M369 H6 H-P0777 uridine 5'-monophosphate (UMP) 26.51 kinase (pyrH)
M370 Al H-P0782 hypothetical protein 50.16
M370 B1 H-P0783 hypothetical protein 18.26
M370 C1 H-P0792 sigma-54 interacting protein 55.77
M370 D1 H-P0793 polypeptide deformylase (def) 19.25
M370 El H-P0794 ATP-dependent clp protease 21.67 proteolytic component (clpP)
M370 FI H-P0796 outer membrane protein (ompl 8) 30.69
M379 G3 H-P0797-2 flagellar sheath adhesin hpaA 28.71
M379 H3 H-P0798-2 molybdenum cofactor 17.49 biosynthesis protein C (moaC)
M370 GI H-P0799 molybdopterin biosynthesis 19.47 protein (mog)
M370 HI H-P0800 molybdopterin converting factor, 16.06 subunit 2 (moaE)
M379 A4 H-P0801-2 molybdopterin converting factor, 8.25 subunit 1 (moaD)
M379 B4 H-P0802-2 GTP cyclohydrolase II (ribA) 21.23
M379 D3 H-P0803-2 hypothetical protein 30.8
M379 E3 H-P0804-2 GTP cyclohydrolase 11/3,4- 37.95 dihydroxy-2-butanone 4- phosphate synthase (ribA, ribB)
M379 F3 H-P0805-2 lipooligosaccharide 5G8 epitope 31.35 biosynthesis-associated protein (lex2B)
M370 A2 H-P0806 hypothetical protein 22.77
M379 C4 H-P0807-2 iron(III) dicitrate transport protein 86.68 (fecA)
M370 B2 H-P0808 holo-acp synthase (acpS) 13.2
M370 C2 H-P0809 hypothetical protein 20.24
M370 D2 H-P0810 conserved hypothetical protein 22.11
M370 E2 H-P0811 hypothetical protein 11.99
M370 F2 H-P0812 hypothetical protein 37.07
M370 G2 H-P0813 conserved hypothetical protein 22.66
M370 H2 H-P0814 thiamin biosynthesis protein 28.16 (thiF)
M370 A3 H-P0815 flagellar motor rotation protein 28.38 (motA)
M370 B3 H-P0831 conserved hypothetical ATP 21.67 binding protein M379 D4 H-P0832-2 spermidine synthase (speE) 28.93 52
M379 E4 H-P0833-2 hypothetical protein 32.23
M370 C3 H-P0834 GTP-binding protein homologue 50.49 (yphC)
M370 D3 H-P0835 histone-like DNA-binding protein 10.45 HU (hup)
M370 E3 H-P0836 hypothetical protein 13.2
M370 F3 H-P0837 hypothetical protein 11.33
M370 G3 H-P0838 hypothetical protein 22.66
M370 H3 H-P0839 outer membrane protein PI 64.68 (ompPl)
M370 A4 H-P0840 flaAl protein 36.74
M370 B4 H-P0841 pantothenate metabolism 46.86 flavoprotein (dfp)
M370 C4 H-P0843 thiamin phosphate 24.2 pyrophosphorylase/hyroxyethylthi azole kinase (thiB)
M370 D4 H-P0845 thiamin phosphate 30.14 pyrophosphorylase/hyroxyethylthi azole kinase (thiM)
M370 E4 H-P0850 type I restriction enzyme M 58.08 protein (hsdM)
M370 F4 H-P0851 conserved hypothetical integral 25.08 membrane protein
M370 G4 H-P0854 GMP reductase (guaC) 36.08
M370 H4 H-P0858 ADP-heptose synthase (rfaE) 50.82
M370 A5 H-P0859 ADP-L-glycero-D-mannoheptose- 36.41 6-epimerase (rfaD)
M370 B5 H-P0861 hypothetical protein 27.17
M370 C5 H-P0862 hypothetical protein 24.64
M379 F4 H-P0863-2 hypothetical protein 59.73
M370 D5 H-P0865 deoxyuridine 5'-triphosphate 16.06 nucleotidohydrolase (dut)
M370 E5 H-P0866 transcription elongation factor 18.15 GreA (greA)
M379 G4 H-P0867-2 lipid A disaccharide synthetase 39.71 (lpxB)
M379 H4 H-P0870-2 flagellar hook (flgE) 79.09
M370 F5 H-P0871 CDP-diglyceride hydrolase (cdh) 26.95
M370 G5 H-P0872 alkylphosphonate uptake protein 12.1 (phnA)
M370 H5 H-P0873 hypothetical protein 7.92
M371 Al H-P0879 hypothetical protein 22.33
M371 Bl H-P0883 Holliday junction DNA helicase 20.24 (ruvA)
M371 Cl H-P0885 virulence factor mviN protein 50.82 (mviN)
M371 Dl H-P0886 cysteinyl-tRNA synthetase (cysS) 51.26
M371 El H-P0889 iron(III) dicitrate ABC 35.97 transporter, permease protein (fecD)
M371 FI H-P0890 conserved hypothetical protein 28.27
M371 GI H-P0891 conserved hypothetical protein 19.25 M371 HI H-P0892 conserved hypothetical protein 10.01 53
M371 A2 H-P0894 conserved hypothetical protein 9.79
M371 B2 H-P0895 hypothetical protein 13.86
M371 C2 H-P0896 outer membrane protein (ompl 9) 77.99
M371 D2 H-P0897 hypothetical protein 22.99
M371 E2 H-P0898 hydrogenase expression formation 40.81 protein (hypD)
M371 F2 H-P0899 hydrogenase expression/formation 8.58 protein (hypC)
M371 G2 H-P0900 hydrogenase expression/formation 26.73 protein (hypB)
M371 H2 H-P0905 phosphotransacetylase (pta) 24.64
M371 A3 H-P0906 hypothetical protein 58.08
M371 B3 H-P0907 hook assembly protein, flagella 33.22 (AgD)
M371 C3 H-P0909 hypothetical protein 22.22
M371 D3 H-P0912 outer membrane protein (omp20) 56.76
M371 E3 H-P0913 outer membrane protein (omp21) 58.3
M371 F3 H-P0914 hypothetical protein 56.65
M371 G3 H-P0915 iron-regulated outer membrane 61.93 protein (frpB)
M371 H3 H-P0916 iron-regulated outer membrane 27.5 protein (frpB)
M380 Al H-P0917-2 hypothetical protein 2.64
M371 A4 H-P0918 hypothetical protein 15.84
M371 B4 H-P0920 conserved hypothetical integral 25.41 membrane protein
M371 C4 H-P0921 glyceraldehyde-3-phosphate 36.63 dehydrogenase (gap)
M371 D4 H-P0923 outer membrane protein (omp22) 40.7
M371 E4 H-P0925 recombinational DNA repair 21.34 protein (recR)
M371 F4 H-P0927 heat shock protein (htpX) 35.97
M371 G4 H-P0928 GTP cyclohydrolase I (folE) 19.91
M371 H4 H-P0929 geranyltranstransferase (ispA) 33.44
M371 A5 H-P0930 stationary-phase survival protein 29.48 (surE)
M371 B5 H-P0931 hypothetical protein 16.17
M371 C5 H-P0932 hypothetical protein 11.11
M371 D5 H-P0933 hypothetical protein 22.11
M371 E5 H-P0934 conserved hypothetical protein 27.72
M371 F5 H-P0935 hypothetical protein 17.82
M371 G5 H-P0936 proline/betaine transporter (proP) 42.9
M371 H5 H-P0938 hypothetical protein 12.76
M371 A6 H-P0939 amino acid ABC transporter, 26.18 permease protein (yckJ)
M371 B6 H-P0940 amino acid ABC transporter, 28.27 periplasmic binding protein (yckK)
M371 C6 H-P0941 alanine racemase, biosynthetic 41.58 (air)
M371 D6 H-P0942 D-alanine glycine permease 49.61 (dagA) 54
M371 E6 H-P0943 D-amino acid dehydrogenase 45.21 (dadA)
M371 F6 H-P0944 translation initiation inhibitor, 13.86 putative
M371 G6 H-P0946 conserved hypothetical integral 54.67 membrane protein
M371 H6 H-P0947 hypothetical protein 13.31
M371 A7 H-P0949 conserved hypothetical secreted 16.61 protein
M371 B7 H-P0950 acetyl-CoA carboxylase beta 31.9 subunit (accD)
M371 C7 H-P0951 hypothetical protein 22.66
M371 D7 H-P0952 conserved hypothetical integral 24.09 membrane protein
M371 E7 H-P0953 hypothetical protein 20.79
M371 F7 H-P0955 prolipoprotein diacylglyceryl 31.35 transferase (lgt)
M371 G7 H-P0956 conserved hypothetical protein 26.73
M371 H7 H-P0957 3 -deoxy-d-manno-octulosonic- 43.34 acid transferase (kdtA)
M371 A8 H-P0958 hypothetical protein 28.05
M371 B8 H-P0960 glycyl-tRNA synthetase, alpha 33.44 subunit (glyQ)
M371 C8 H-P0961 glycerol-3-phosphate 34.43 dehydrogenase (NAD(P)+)
M380 B1 H-P0965-2 hypothetical protein 48.84
M371 D8 H-P0966 conserved hypothetical protein 60.5
M380 FI H-P0968-2 hypothetical protein 2.42
M371 E8 H-P0969 cation efflux system protein 112.31 (czcA)
M371 F8 H-P0970 nickel-cobalt-cadmium resistance 39.6 protein (nccB)
M371 G8 H-P0971 hypothetical protein 45.54
M371 H8 H-P0972 glycyl-tRNA synthetase, beta 77.22 subunit (glyS)
M371 A9 H-P0973 hypothetical protein 38.94
M380 Cl H-P0974-2 phosphoglycerate mutase (pgm) 54.12
M380 D1 H-P0975-2 conserved hypothetical protein 10.34
M380 E1 H-P0976-2 adenosylmethionine-8-amino-7- 48.07 oxononanoate aminotransferase (bioA)
M380 H1 H-P0994-2 hypothetical protein 29.48
M380 G1 H-P 1000-2 PARA protein 24.09
M380 A2 H-P1001-2 hypothetical protein 10.45
M380 B2 H-P1002-2 hypothetical protein 43.45
M380 C2 H-Pl 003-2 hypothetical protein 40.81
M380 D2 H-P 1004-2 hypothetical protein 30.14
M380 E2 H-P 1005-2 hypothetical protein 11.55
M380 F2 H-P 1006-2 conjugal transfer protein (traG) 19.58
M380 G2 H-P1017-2 amino acid permease (rocE) 57.2
M380 H2 H-P 1042-2 hypothetical protein 38.39 M380 A3 H-P 1056-2 hypothetical protein 31.35 55
M380 B3 H-P 1075-2 conserved hypothetical secreted 48.29 protein
M373 Al H-P 1076 hypothetical protein 18.92
M373 Bl H-P 1077 nickel transport protein (nixA) 36.52
M373 Cl H-P 1080 conserved hypothetical integral 20.9 membrane protein
M373 Dl H-P 1081 hypothetical protein 22.88
M373 El H-P1082 multidrug resistance protein 60.72 (msbA)
M373 FI H-P 1083 hypothetical protein 52.8
M373 GI H-P 1084 aspartate transcarbamoylase 33.88 (pyrB)
M373 HI H-P 1085 hypothetical protein 18.92
M373 A2 H-P 1086 hemolysin (tly) 25.96
M373 B2 H-P1087 riboflavin biosynthesis regulatory 30.91 protein (ribC)
M373 C2 H-P1088 transketolase A (tktA) 70.62
M373 D2 H-P 1091 alpha-ketoglutarate permease 46.97 (kgtP)
M373 E2 H-P 1092 flagellar basal-body rod protein 29.7 (AgG)
M373 F2 H-P 1096 IS605 transposase (tnpA) 15.73
M373 G2 H-P1098 conserved hypothetical secreted 32.01 protein
M373 H2 H-P1101 glucose-6-phosphate 46.86 dehydrogenase (g6pD)
M373 A3 H-P 1102 glucose-6-phosphate 1- 25.08 dehydrogenase (devB)
M373 B3 H-P 1103 glucokinase (glk) 37.07
M373 C3 H-P 1108 pyruvate ferredoxin 20.57 oxidoreductase, gamma subunit
M373 D3 H-P 1109 pyruvate ferredoxin 14.41 oxidoreductase, delta subunit
M373 E3 H-P1110 pyruvate ferredoxin 44.88 oxidoreductase, alpha subunit
M373 F3 H-Pl l l l pyruvate ferredoxin 34.65 oxidoreductase, beta subunit
M373 G3 H-P1 112 adenylosuccinate lyase (purB) 48.51
M380 C3 H-Pl 113-2 outer membrane protein (omp24) 30.58
M373 H3 H-P1117 conserved hypothetical secreted 28.27 protein
M373 A4 H-P 1120 hypothetical protein 15.95
M373 B4 H-P1121 cytosine specific DNA 34.43 methyltransferase (BSP6IM)
M380 D3 H-Pl 122-2 hypothetical protein 8.47
M373 C4 H-P 1123 peptidyl-prolyl cis-trans 20.46 isomerase, FKBP-type rotamase (slyD)
M373 D4 H-Pl 124 hypothetical protein 36.52
M373 E4 H-Pl 125 peptidoglycan associated 19.8 lipoprotein precursor (ompl 8)
M373 F4 H-Pl 126 colicin tolerance-like protein 45.98 (tolB) 56
M373 G4 H-Pl 128 hypothetical protein 9.35
M373 H4 H-Pl 129 biopolymer transport protein 14.74 (exbD)
M373 A5 H-Pl 131 ATP synthase FI, subunit epsilon 13.75 (atpC)
M373 B5 H-Pl 134 ATP synthase FI, subunit alpha 55.44 (atpA)
M373 C5 H-Pl 135 ATP synthase FI, subunit delta 19.91 (atpH)
M373 D5 H-Pl 137 ATP synthase F0, subunit b' 15.95 (atpF)
M373 E5 H-Pl 138 plasmid replication-partition 32.01 related protein
M373 F5 H-Pl 139 SpoOJ regulator (soj) 29.15
M373 G5 H-Pl 140 biotin operon repressor/biotin 23.43 acetyl coenzyme A carboxylase synthetase (birA)
M373 H5 H-Pl 141 methionyl-tRNA 33.44 formyltransferase (frnt)
M373 A6 H-Pl 144 hypothetical protein 9.46
M373 B6 H-Pl 145 hypothetical protein 11.44
M373 C6 H-Pl 147 ribosomal protein L19 (rpll9) 13.09
M373 D6 H-Pl 148 tRNA (guanine-Nl)- 25.3 methyltransferase (trmD)
M373 E6 H-Pl 149 conserved hypothetical protein 20.35
M380 F3 H-Pl 150-2 hypothetical protein 12.76
M373 F6 H-Pl 152 signal recognition particle protein 49.39 (ffh)
M380 G3 H-Pl 153-2 valyl-tRNA synthetase (valS) 96.25
M380 E3 H-Pl 157-2 outer membrane protein (omp26) 135.41
M373 G6 H-Pl 158 pyrroline-5-carboxylate reductase 28.38 (proC)
M373 H6 H-Pl 159 cell filamentation protein (fie) 19.58
M373 A7 H-Pl 160 conserved hypothetical protein 15.51
M380 A4 H-Pl 163-2 hypothetical protein 7.04
M373 B7 H-Pl 165 tetracycline resistance protein 42.57 tetA(P), putative
M373 C7 H-Pl 168 carbon starvation protein (cstA) 75.68
M373 D7 H-Pl 169 glutamine ABC transporter, 23.98 permease protein (glnP)
M380 H3 H-Pl 169-2 glutamine ABC transporter, 23.98 permease protein (glnP)
M374 A1 H-Pl 170 glutamine ABC transporter, 24.64 permease protein (glnP)
M374 B1 H-Pl 171 glutamine ABC transporter, ATP- 27.39 binding protein (glnQ)
M374 Cl H-P 1172 glutamine ABC transporter, 30.58 periplasmic glutamine-binding protein (glnH)
M374 Dl H-Pl 173 hypothetical protein 20.24
M374 El H-Pl 174 glucose/galactose transporter 44.88 (gluP) 57
M374 FI H-Pl 175 conserved hypothetical integral 47.96 membrane protein
M374 GI H-Pl 177 outer membrane protein (omp27) 70.62
M374 HI H-Pl 178 purine-nucleoside phosphorylase 25.74 (deoD)
M374 A2 H-Pl 179 phosphopentomutase (deoB) 45.54
M374 B2 H-Pl 180 pyrimidine nucleoside transport 46.09 protein (nupC)
M374 C2 H-Pl 183 NA+/H+ antiporter (napA) 42.24
M374 D2 H-Pl 184 conserved hypothetical integral 50.6 membrane protein
M374 E2 H-Pl 185 conserved hypothetical integral 43.12 membrane protein
M374 F2 H-Pl 186 carbonic anhydrase 22.33
M374 G2 H-Pl 187 hypothetical protein 42.46
M374 H2 H-Pl 188 hypothetical protein 29.7
M374 A3 H-Pl 189 aspartate-semialdehyde 38.17 dehydrogenase (asd)
M374 B3 H-Pl 191 ADP-heptose-lps 38.5 heptosyltransferase II (rfaF)
M374 C3 H-Pl 196 ribosomal protein S7 (rps7) 17.16
M374 D3 H-P1200 ribosomal protein L10 (rpllO) 18.15
M374 E3 H-P 1201 ribosomal protein LI (rpll) 25.85
M374 F3 H-P 1202 ribosomal protein LI 1 (rpll 1) 15.62
M374 G3 H-P 1203 transcription termination factor 19.47 NusG (nusG)
M380 B4 H-P1205-2 translation elongation factor EF- 44 Tu (tufB)
M374 H3 H-P 1206 multidrug resistance protein 63.69 (hetA)
M374 A4 H-P1207 hypothetical protein 24.53
M374 B4 H-P1210 serine acetyltransferase (cysE) 18.92
M380 F4 H-P1213-2 polynucleotide phosphorylase 75.79 (pnp)
M380 G4 H-P1214-2 conserved hypothetical protein 26.51
M380 C4 H-P1215-2 hypothetical protein 8.91
M380 D4 H-P1216-2 conserved hypothetical secreted 72.71 protein
M380 E4 H-P1217-2 hypothetical protein 17.6
M374 C4 H-P1220 ABC transporter, ATP-binding 25.19 protein (yhcG)
M374 D4 H-P1221 conserved hypothetical protein 25.85
M374 E4 H-P 1222 D-lactate dehydrogenase (did) 104.39
M374 F4 H-P 1224 uroporphyrinogen III eosynthase 24.97 (hemD)
M374 G4 H-P 1225 conserved hypothetical integral 14.41 membrane protein
M374 H4 H-P1226 oxygen-independent 38.83 coproporphyrinogen III oxidase (hemN)
M380 H4 H-P1227-2 cytochrome c553 10.67
M380 A5 H-P1228-2 invasion protein (invA) 17.16 M380 B5 H-P1229-2 aspartokinase (lysC) 44.66 58
M374 A5 H-P1230 hypothetical protein 19.91
M374 B5 H-P 1231 DNA polymerase III delta prime 24.09 subunit (holB)
M374 C5 H-P1232 dihydropteroate synthase (folP) 41.91
M380 D5 H-P1233-2 hypothetical protein 16.94
M374 D5 H-P 1234 conserved hypothetical integral 32.89 membrane protein
M374 E5 H-P1235 conserved hypothetical integral 45.76 membrane protein
M374 F5 H-P1236 hypothetical protein 20.24
M374 G5 H-P1237 carbamoyl-phosphate synthetase 41.36 (pyrAa)
M374 H5 H-P1240 conserved hypothetical protein 21.01
M380 C5 H-P1241-2 alanyl-tRNA synthetase (alaS) 93.28
M374 A6 H-P1242 conserved hypothetical protein 8.47
M380 H5 H-P 1243-2 outer membrane protein (omp28) 80.74
M374 B6 H-P1244 ribosomal protein SI 8 (rpslδ) 9.46
M374 C6 H-P1245 single-strand DNA-binding 19.8 protein (ssb)
M374 D6 H-P 1246 ribosomal protein S6 (rps6) 15.73
M380 A6 H-P1247-2 hypothetical protein 37.51
M374 E6 H-P 1248 virulence associated protein 70.95 homolog (vacB)
M380 B6 H-P 1249-2 shikimate 5-dehydrogenase (aroE) 29.04
M380 E5 H-P 1251-2 oligopeptide ABC transporter, 38.39 permease protein (oppB)
M380 F5 H-P 1252-2 oligopeptide ABC transporter, 65.45 periplasmic oligopeptide-binding protein (oppA)
M380 G5 H-P1253-2 tryptophanyl-tRNA synthetase 37.4 (trpS)
M374 F6 H-P1254 biotin synthesis protein (bioC) 26.51
M374 G6 H-P1255 protein translocation protein, low 22.22 temperature (secG)
M374 H6 H-P1256 ribosome releasing factor (frr) 20.46
M374 A7 H-P1257 orotate phosphonbosyltransferase 22.22 (pyrE)
M374 B7 H-P1258 conserved hypothetical 17.05 mitochondrial protein 4
M374 C7 H-P1260 NADH-ubiquinone 14.74 oxidoreductase, NQ07 subunit (NQ07)
M374 D7 H-P1262 NADH-ubiquinone 29.37 oxidoreductase, NQ05 subunit (NQ05)
M374 E7 H-P 1263 NADH-ubiquinone 45.1 oxidoreductase, NQ04 subunit (NQ04)
M380 C6 H-P1264-2 hypothetical protein 8.47
M374 F7 H-P 1265 hypothetical protein 36.19
M375 Al H-P 1268 NADH-ubiquinone 24.31 oxidoreductase, NQ09 subunit (NQ09) 59
M375 Bl H-P1275 phosphomannomutase (algC) 50.6
M375 Cl H-P1277 tryptophan synthase, alpha 28.93 subunit (trpA)
M375 Dl H-P1278 tryptophan synthase, beta subunit 43.34 (trpB)
M375 El H-P1279 anthranilate isomerase (trpC) 49.83
M375 FI H-P1282 anthranilate synthase component I 55.11 (trpE)
M375 GI H-P 1285 conserved hypothetical secreted 25.41 protein
M375 HI H-P1286 conserved hypothetical secreted 20.13 protein
M375 A2 H-P1287 transcriptional regulator (tenA) 23.98
M375 B2 H-P1288 hypothetical protein 14.63
M375 C2 H-P 1289 hypothetical protein 17.82
M375 D2 H-P1290 nicotinamide mononucleotide 24.31 transporter (pnuC)
M375 E2 H-P1291 conserved hypothetical protein 22.55
M375 F2 H-P1292 ribosomal protein LI 7 (rpll 7) 12.87
M375 G2 H-P1293 DNA-directed RNA polymerase, 37.95 alpha subunit (rpoA)
M375 H2 H-P1294 ribosomal protein S4 (rps4) 22.99
M375 A3 H-P1295 ribosomal protein SI 1 (rpsl 1) 14.52
M375 B3 H-P1296 ribosomal protein S13 (rpsl 3) 13.31
M380 D6 H-P1298-2 translation initiation factor EF-1 8.03 (infA)
M375 C3 H-P1299 methionine amino peptidase 27.94 (map)
M375 D3 H-P 1302 ribosomal protein S5 (rps5) 16.94
M375 E3 H-P1303 ribosomal protein LI 8 (rpll 8) 13.2
M375 F3 H-P1305 ribosomal protein S8 (rps8) 14.52
M375 G3 H-P1307 ribosomal protein L5 (rpl5) 20.02
M375 H3 H-P1308 ribosomal protein L24 (rpl24) 8.14
M375 A4 H-P1309 ribosomal protein L14 (rpll 4) 13.53
M375 B4 H-P1310 ribosomal protein S17 (rpsl 7) 9.57
M375 C4 H-P1312 ribosomal protein LI 6 (rpll 6) 15.62
M375 D4 H-P1314 ribosomal protein L22 (rpl22) 13.53
M375 E4 H-P1315 ribosomal protein S19 (rpsl 9) 10.34
M375 F4 H-P1318 ribosomal protein L4 (rpl4) 23.76
M375 G4 H-P1319 ribosomal protein L3 (rpl3) 21.12
M375 H4 H-P 1320 ribosomal protein S10 (rpslO) 11.55
M375 A5 H-P 1321 conserved hypothetical ATP- 41.58 binding protein
M375 B5 H-P 1322 hypothetical protein 22.22
M375 C5 H-P 1323 ribonuclease HII (rnhB) 23.1
M375 D5 H-Pl 324 hypothetical protein 9.24
M375 E5 H-Pl 326 hypothetical protein 13.86
M375 F5 H-P 1327 hypothetical protein 45.43
M375 G5 H-P 1328 cation efflux system protein 37.29 (czcA)
M375 H5 H-P1330 conserved hypothetical integral 12.76 membrane protein 60
M375 A6 H-P1331 conserved hypothetical integral 25.19 membrane protein
M375 B6 H-P 1332 co-chaperone and heat shock 40.7 protein (dnaJ)
M375 C6 H-P1333 hypothetical protein 42.13
M375 D6 H-P1335 conserved hypothetical protein 39.71
M375 E6 H-P1336 hypothetical protein 27.94
M375 F6 H-P1337 conserved hypothetical protein 19.25
M375 G6 H-P1338 conserved hypothetical protein 16.39
M375 H6 H-P 1340 biopolymer transport protein 14.3 (exbD)
M375 A7 H-P 1341 siderophore-mediated iron 31.46 transport protein (tonB)
M375 B7 H-P 1342 outer membrane protein (omp29) 76.12
M375 C7 H-P 1343 conserved hypothetical integral 26.73 membrane protein
M375 D7 H-P 1344 magnesium and cobalt transport 35.09 protein (corA)
M375 E7 H-P1345 phosphoglycerate kinase 44.33
M375 F7 H-P 1346 glyceraldehyde-3 -phosphate 36.41 dehydrogenase (gap)
M375 G7 H-Pl 347 uracil-DNA glycosylase (ung) 25.74
M375 H7 H-P 1349 hypothetical protein 42.68
M375 A8 H-P1350 protease 50.6
M375 B8 H-P1355 nicotinate-nucleotide 30.14 pyrophosphorylase (nadC)
M375 C8 H-P1356 quinolinate synthetase A (nadA) 37.07
M375 D8 H-P1357 phosphatidylserine decarboxylase 29.48 proenzyme (psd)
M375 E8 H-P1358 hypothetical protein 18.59
M375 F8 H-P 1360 4-hydroxybenzoate 32.45 octaprenyltransferase (ubiA)
M375 G8 H-P1361 competence locus E (comE3) 45.98
M375 H8 H-P1362 replicative DNA helicase (dnaB) 53.79
M375 A9 H-P1363 conserved hypothetical integral 51.37 membrane protein
M376 A1 H-P1364 signal-transducing protein, 43.78 histidine kinase
M376 B1 H-Pl 365 response regulator 23.54
M376 Cl H-Pl 371 type III restriction enzyme R 106.59 protein
M376 D1 H-Pl 372 rod shape-determining protein 27.39 (mreC)
M376 El H-P1373 rod shape-determining protein 38.28 (mreB)
M376 FI H-P1374 ATP-dependent protease ATPase 49.17 subunit (clpX)
M376 G1 H-P1375 UDP-N-acetylglucosamine 29.81 acyltransferase (lpxA)
M376 HI H-P 1376 (3R)-hydroxymyristoyl-(acyl 17.6 carrier protein) dehydratase (fabZ) M376 A2 H-P 1377 hypothetical protein 16.17 61
M376 B2 H-P 1378 competence lipoprotein (comL) 24.31
M376 C2 H-P1379 ATP-dependent protease (Ion) 91.96
M376 D2 H-Pl 380 prephenate dehydrogenase (tyrA) 29.26
M381 C1 H-P1381 hypothetical protein 8.58
M376 E2 H-P 1382 hypothetical protein 14.41
M376 F2 H-P 1383 restriction modification system S 17.71 subunit
M376 G2 H-P1384 hypothetical protein 7.59
M376 H2 H-P1385 fructose- 1 ,6-bisphosphatase 32.01
M376 A3 H-P1386 D-ribulose-5-phosphate 3 23.98 epimerase (rpe)
M376 B3 H-P 1388 hypothetical protein 16.5
M376 C3 H-P 1389 hypothetical protein 6.71
M376 D3 H-P 1390 hypothetical protein 18.37
M376 E3 H-P 1391 hypothetical protein 10.89
M376 F3 H-P 1392 fibronectin/fibrinogen-binding 47.96 protein
M376 G3 H-P1393 DNA repair protein (recN) 57.75
M376 H3 H-Pl 394 conserved hypothetical protein 31.35
M376 A4 H-P1395 outer membrane protein (omp30) 26.73
M376 B4 H-P1396 hypothetical protein 31.79
M376 C4 H-P1398 alanine dehydrogenase (aid) 41.91
M376 D4 H-P1399 arginase (rocF) 35.53
M376 E4 H-P1400 iron(III) dicitrate transport protein 92.73 (fecA)
M376 F4 H-P1401 conserved hypothetical protein 25.96
M381 A2 H-P 1402 type I restriction enzyme R 109.34 protein (hsdR)
M381 B2 H-P 1403 type I restriction enzyme M 89.98 protein (hsdM)
M376 G4 H-P1405 hypothetical protein 3.85
M376 H4 H-P 1406 biotin synthetase (bioB) 31.13
M376 A5 H-P 1407 conserved hypothetical integral 32.23 membrane protein
M381 C2 H-P1408 hypothetical protein 12.32
M381 D2 H-P1409 hypothetical protein 63.69
M376 B5 H-P1410 hypothetical protein 43.45
M376 C5 H-P1411 hypothetical protein 68.2
M376 D5 H-P1412 hypothetical protein 33.99
M376 E5 H-P1413 conserved hypothetical protein 16.39
M376 F5 H-P1414 conserved hypothetical protein 12.54
M376 G5 H-P1415 tRNA delta(2)- 29.37 isopentenylpyrophosphate transferase (miaA)
M376 H5 H-P1418 UDP-N- 28.6 acetylenolpyruvoylglucosamine reductase (murB)
M376 A6 H-P1419 flagellar biosynthetic protein 9.79 (fliQ)
M376 B6 H-P1420 flagellar export protein ATP 47.85 synthase (flil)
M376 C6 H-P1421 conjugative transfer regulon 33.55 protein (trbB) 62
M376 D6 H-P 1423 conserved hypothetical protein 9.35
M376 E6 H-P 1424 hypothetical protein 22.77
M376 F6 H-P 1425 hypothetical protein 8.36
M376 G6 H-P1427 histidine-rich, metal binding 6.71 polypeptide (hpn)
M376 H6 H-P1428 conserved hypothetical protein 39.38
M376 A7 H-P 1429 polysialic acid capsule expression 36.3 protein (kpsF)
M376 B7 H-P1430 conserved hypothetical ATP- 75.9 binding protein
M376 C7 H-P 1431 16S rRNA (adenosine-N6,N6-> 29.92 dimethyltransferase (ksgA)
M376 D7 H-P1432 histidine and glutamine-rich 8.03 protein
M376 E7 H-P1433 hypothetical protein 94.27
M376 F7 H-P 1434 formyltetrahydrofolate hydrolase 32.34 (purU)
M376 G7 H-P 1435 protease IV (PspA) 32.23
M376 H7 H-P1436 hypothetical protein 9.13
M376 A8 H-P 1438 conserved hypothetical 37.29 lipoprotein
M376 B8 H-P1439 hypothetical protein 9.02
M376 C8 H-P 1440 hypothetical protein 28.6
M376 D8 H-P 1441 peptidyl-prolyl cis-trans 18.04 isomerase B, cyclosporin-type rotamase (ppi)
M376 E8 H-P 1442 carbon storage regulator (csrA) 8.47
M376 F8 H-P 1443 conserved hypothetical protein 29.59
M376 G8 H-P 1444 small protem (smpB) 16.83
M376 H8 H-P 1445 biopolymer transport protein 16.61 (exbB)
M376 A9 H-P 1446 biopolymer transport protein 14.74 (exbD)
M376 B9 H-P1447 ribosomal protein L34 (rpl34) 4.95
M376 C9 H-P1448 ribonuclease P, protein 17.82 component (rnpA)
M376 D9 H-P1449 conserved hypothetical protein 12.98
M376 E9 H-P 1450 60 kDa inner-membrane protein 60.28
M376 F9 H-P1451 hypothetical protein 29.15
M376 G9 H-P1452 thiophene and furan oxidizer 50.82 (tdhF)
M376 H9 H-P1453 conserved hypothetical protein 82.17
M376 A10 H-P1454 hypothetical protein 33.44
M376 BIO H-P1455 hypothetical protein 14.41
M376 C10 H-P1456 membrane-associated lipoprotein 19.36 (lpP20)
M376 D10 H-P 1457 hypothetical protein 23.21
M376 E10 H-P1458 thioredoxin 11.55
M376 F10 H-P 1461 cytochrome c551 peroxidase 38.61
M377 Al H-P 1462 secreted protein involved in 19.03 flagellar motility M377 B1 H-P 1463 hypothetical protein 24.86 63
M377 C1 H-P1464 conserved hypothetical secreted 29.92 protein
M377 D1 H-P 1465 ABC transporter, ATP-binding 28.82 protein (HI 1087)
M377 El H-P 1466 conserved hypothetical integral 41.58 membrane protein
M377 F1 H-P1467 hypothetical protein 25.52
M377 GI H-P 1468 branched-chain-amino-acid 37.51 aminotransferase (ilvE)
M377 HI H-P1469 outer membrane protein (omp31) 27.39
M377 A2 H-P1473 hypothetical protein 21.12
M377 B2 H-P 1474 thymidylate kinase (tmk) 21.12
M377 C2 H-P 1475 lipopolysaccharide core 17.38 biosynthesis protein (kdtB)
M377 D2 H-P 1476 phenylacrylic acid decarboxylase 20.68
M377 E2 H-P 1479 hypothetical protein 92.95
M377 F2 H-P 1480 seryl-tRNA synthetase (serS) 45.76
M377 G2 H-P 1481 hypothetical protein 29.26
M377 H2 H-P 1482 hypothetical protein 9.57
M377 A3 H-P1483 gerC2 protein (gerC2) 27.17
M377 B3 H-P1484 conserved hypothetical integral 16.39 membrane protein
M377 C3 H-P 1485 proline dipeptidase (pepQ) 21.01
M377 D3 H-P1486 conserved hypothetical integral 41.47 membrane protein
M377 E3 H-P1487 conserved hypothetical integral 40.26 membrane protein
M377 F3 H-P1488 conserved hypothetical secreted 36.3 protein
M377 G3 H-P1489 lipase-like protein 56.21
M381 GI H-P 1490 hemolysin 49.5
M377 H3 H-P 1491 phosphate permease 58.74
M377 A4 H-P 1492 conserved hypothetical nifU-like 9.9 protein
M377 B4 H-P1493 hypothetical protein 22.44
M377 C4 H-P 1494 UDP-MurNac-tripeptide 49.28 synthetase (murE)
M377 D4 H-P 1495 transaldolase (tal) 34.87
M377 E4 H-P 1496 general stress protein (etc) 19.69
M377 F4 H-P 1497 peptidyl-tRNA hydrolase (pth) 20.57
M377 G4 H-P 1499 hypothetical protein 30.03
M377 H4 H-P 1501 outer membrane protein (omp32) 42.79
M377 A5 H-Pl 502 hypothetical protein 16.06
M377 B5 H-P 1503 cation-transporting ATPase, P- 86.79 type (copA)
M377 C5 H-P 1504 conserved hypothetical protein 26.29
M377 D5 H-P 1505 riboflavin biosynthesis protein 37.95 (ribG)
M377 E5 H-P 1506 glutamate permease (gltS) 44.99
M377 F5 H-P 1507 conserved hypothetical ATP- 42.46 binding protein M381 F2 H-P 1508 ferrodoxin-like protein 50.49 64
M377 G5 H-P 1509 conserved hypothetical integral 28.93 membrane protein
M377 H5 H-P1510 conserved hypothetical protein 12.98
M377 A6 H-P1511 hypothetical protein 11.99
M377 B6 H-P1512 iron-regulated outer membrane 96.58 protein (frpB)
M377 C6 H-P1513 selenocystein synthase (selA) 42.57
M377 D6 H-Pl 514 transcription termination factor 43.56 NusA (nusA)
M377 E6 H-P1518 hypothetical protein 10.56
M381 B3 H-P1521 type III restriction enzyme R 106.48 protein (res)
M381 C3 H-P 1523 DNA recombinase (recG) 68.64
M377 F6 H-P 1524 hypothetical protein 12.76
M377 G6 H-Pl 525 hypothetical protein 23.32
M377 H6 H-Pl 526 exodeoxyribonuclease (lexA) 27.61
M377 A7 H-P 1527 hypothetical protein 52.8
M377 B7 H-P1530 purine nucleoside phosphorylase 19.91 (punB)
M377 C7 H-P 1531 hypothetical protein 8.8
M377 D7 H-P 1532 glucosamine fructose-6-phosphate 65.78 aminotransferase (isomerizing) (glmS)
M377 E7 H-P1533 conserved hypothetical protein 25.52
M377 F7 H-Pl 534 IS605 transposase (tnpB) 47.08
M377 G7 H-P1535 IS605 transposase (tnpA) 15.73
M377 H7 H-Pl 541 transcription-repair coupling 110 factor (trcF)
M377 A8 H-P 1548 conserved hypothetical integral 12.43 membrane protein
M377 B8 H-P 1551 conserved hypothetical secreted 14.08 protein
M377 C8 H-P 1552 Na+/H+ antiporter (nhaA) 48.29
M381 B4 H-P1554 ribosomal protein S2 (rps2) 29.15
M381 D4 H-P1555 translation elongation factor EF- 39.16 Ts (tsf)
M377 D8 H-P 1556 cell division protein (ftsl) 67.76
M381 E4 H-P1557 flagellar basal-body protein (fliE) 12.1
M381 F4 H-P1558 flagellar basal-body rod protein 17.82 (flgC) (proximal rod protein)
M381 G4 H-Pl 559 flagellar basal-body rod protein 15.51 (flgB) (proximal rod protein)
M378 A1 H-Pl 560 cell division protein (ftsW) 42.79
M378 B1 H-Pl 561 iron(III) ABC transporter, 36.96 periplasmic iron-binding protein (ceuE)
M378 Cl H-Pl 562 iron(III) ABC transporter, 36.74 periplasmic iron-binding protein (ceuE)
M378 Dl H-P 1563 alkyl hydroperoxide reductase 21.89 (tsaA) M378 El H-P 1564 outer membrane protein 29.92 65
M378 F1 H-P1565 penicillin-binding protein 2 64.79 (pbp2)
M378 GI H-P 1566 hypothetical protein 16.28
M378 HI H-P 1567 conserved hypothetical ATP- 22.99 binding protein
M378 A2 H-Pl 568 hypothetical protein 20.24
M378 B2 H-P 1569 hypothetical protein 21.78
M378 C2 H-Pl 570 conserved hypothetical protein 18.15
M378 D2 H-Pl 571 rare lipoprotein A (rlpA) 34.76
M378 E2 H-P 1572 regulatory protein DniR 41.03
M378 F2 H-P1573 conserved hypothetical protein 28.05
M378 G2 H-P 1576 ABC transporter, ATP-binding 36.08 protein (abc)
M378 H2 H-Pl 577 ABC transporter, permease 23.76 protein (yaeE)
M378 A3 H-Pl 580 hypothetical protein 24.31
M378 B3 H-P1581 methicillin resistance protein 37.07 (11m)
M378 C3 H-P1582 pyridoxal phosphate biosynthetic 28.93 protein J (pdxJ)
M378 D3 H-P 1583 pyridoxal phosphate biosynthetic 33.88 protein A (pdxA)
M378 E3 H-P 1584 sialoglycoprotease (gcp) 37.51
M378 F3 H-P1585 flagellar basal-body rod protein 28.93 (AgG)
M378 G3 H-P1587 conserved hypothetical protein 17.16
M378 H3 H-P1588 conserved hypothetical protein 27.94
M381 HI H-P 1590 hypothetical protein 4.4
M318 G2 H-S38729 autoimmune antigen Ku, p70 67.1 67 subunit
HI H-S39329 Kallikrein 1 24.64 30
(renal/pancreas/salivary) {alternative products}
M270 G4 H-S43855 Recoverin, photoreceptor protein 22.11 32.0kDa
M300 C2 H-S56151 milk fat globule protein HMFG 24.09 30
M318 C1 H-S57153 retinoblastoma-binding protein 1, 101.31 101 isoform I [RBBP1]
M271 B2 H-S57162 retinoblastoma-binding protein 1, 93.72 110 isoform III [RBBP1], INTERACTS WITH THE VIRAL PROTEIN-BINDING DOMAIN OF THE RETINOBLASTOMA PROTEIN.
M317 H3 H-S62027 transducin, gamma subunit 8.25 11
M270 G6 H-S66793 arrestin, X-arrestin=S-antigen 42.79 50.0kDa homolog [human, retina, mRNA, 1314 nt], MAY PLAY A ROLE IN AN AS YET UNDEFINED RETINA-SPECIFIC SIGNAL TRANSDUCTION.
M419 C2 H-S67859 "transcription initiation factor He, 48.360 64.0kDa alpha subunit" 66
M302 D7 H-S69022 myosin, light polypeptide 2, 18.26 31 ventricular
H5 H-S69272 cytoplasmic antiproteinase=38 41.47 50 kda intracellular serine proteinase inhibitor [human, placenta, mRNA, 1465 nt]
Dl H-S72043 GIF=growth inhibitory factor 7.59 19 [human, brain, Genomic, 2015 nt]
M266 B3 H-S74221 cytokine IK factor 17.93 36.0kDa
Dl H-S74445 cellular retinoic acid-binding 15.18 23 protein [human, skin, mRNA, 735 nt]
E3 H-S74728 antiquitin=26g turgor protein 56.32 53 homolog [human, kidney, mRNA, 1809 nt]
D4 H-S75174 E2F transcription factor 4, 45.87 58 pl07/pl30-binding
166-61 H-S76474 "trkB {alternately spliced} 55 52.54 [human, brain, mRNA]"
169-40 H-S76617 "Blk=protein tyrosine kinase 60 55.62 [human, B lymphocytes, mRNA, 2608 nt]"
M250 D3 H-S79522 ubiquitin carboxyl-terminal 17.27 17.0kDa extension protein, Ubiquitin A-52 residue ribosomal protein fusion product 1
M236 B4 H-S80562 calponin, acidic 36.3 49
GI H-S82470 BBl=malignant cell expression- 37.73 34 enhanced gene/tumor progression-enhanced gene [human, UM-UC-9 bladder carcinoma cell line, mRNA, 1897 nt]
M313 E1 H-S85655 prohibitin [PHB] 30.03 40.0kDa
M465 A6 H-S87759 protein phosphatase 2C alpha 42.13 52.0kDa [human, teratocarcinoma, mRNA, 2346 nt]
M472 B1 H-U00803 tyrosine-protein kinase FRK 55.620 64.0kDa
B2 H-U02390 Human adenylyl cyclase- 52.58 55 associated protein homolog CAP2 (CAP2) mRNA, complete eds
167-2 H-U02680 human protein tyrosine kinase 36 38.57 mRNA
G2 H-U03056 Human tumor suppressor (LUCA- 47.96 47 1) mRNA, complete eds
M512 E3 H-U03100 Human alpha2(E)-catenin mRNA, 102.52 102.0kDa complete eds
M306 G3 H-U03187 72.93 95.0kDa
H3 H-U03398 Human receptor 4- IBB ligand 28.05 51 mRNA, complete eds
D3 H-U03486 Human connexin40 gene, 39.49 40 complete eds M300 C3 H-U03643 leukophysin 25.96 34 67
F5 H-U03749 Human chromogranin A (CHGA) 50.38 50 gene, promoter and
M314 C3 H-U03886 GS2 (GB:U03886) 27.94 32.0kDa
M306 E3 H-U04343 CD86 antigen (CD28 antigen 35.64 47 ligand 2, B7-2 antigen) [CD86]
167-61 H-U05012 TrkC 92 90.82
M302 G5 H-U05340 cell division cycle protein p55 55 55
A4 H-U05659 Hydroxysteroid (17-beta) 34.21 36 dehydrogenase 3
FI H-U05861 Human hepatic dihydrodiol 35.64 40 dehydrogenase gene
M302 B2 H-U06452 antigen MART-1, melanoma 13.09 20.0kDa
169-52 H-U06454 human AMP-activated protein 70 60.79 kinase (hAMPK) mRNA
M315 A3 H-U06643 lectin, epidermal 15.07 18
HI H-U06715 Cytochrome B561 27.06 25
M476 E5 H-U07132 Human steroid hormone receptor 50.82 55.0kDa Ner-I mRNA, complete eds
M236 D3 H-U07151 guanine nucleotide-binding 20.13 34 protein ADP-ribosylation factor like gene 3
M317 G3 H-U07559 homeotic protein Islet- 1 38.17 38
M266 H1 H-U07681 Human NAD(H)-specific 40.37 40 isocitrate dehydrogenase alpha subunit precursor mRNA, complete eds
E3 H-U07919 Aldehyde dehydrogenase 6 56.43 53
M298 A3 H-U08021 nicotinamide N-methyltransferase 29.15 36.0kDa
M297 B1 H-U08024 alcohol/hydroxysteroid 31.46 50.0kDa sulfotransferase
A2 H-U08336 Human basic helix-loop-helix 21.89 42 transcription factor mRNA, complete eds
E2 H-U09303 Human T cell leukemia LERK-2 38.17 40 (EPLG2) mRNA, complete eds
M250 H5 H-U09559 RCH1, RAG (recombination 58.3 58.0kDa activating gene) cohort 1
167-50 H-U09564 human serine kinase mRNA 72 72.12
166-74 H-U09578 human MAPKAP kinase (3pK) 50 42.09 mRNA
M302 C4 H-U09813 ATP synthase, subunit 9, 15.73 30 mitochondrial
Al H-U09850 Zinc finger protein 143 (clone 68.97 68 pHZ-1)
M423 El H-U09937 Human urokinase-type 36.96 49.0kDa plasminogen receptor
M450 H4 H-U10117 Human endothelial-monocyte 34.43 38.0kDa activating polypeptide II mRNA, complete eds
M314 G1 H-U 10248 ribosomal protein L29 17.6 27
M298 H1 H-U 10323 nuclear factor 45 44.77 45
El H-U 10492 Human Moxl protein (MOX1) 28.05 37 mRNA, complete eds 68
F3 H-U 10686 Human MAGE-11 antigen 35.2 35 (MAGE11) gene, complete eds
167-38 H-U 11050 human NIMA-like protein kinase 55 49.02 1 (NLK1) mRNA
M266 B2 H-U11292 Human Ki nuclear autoantigen 29.48 32 mRNA, complete eds, may play a rol in cell adhesion
167-62 H-U11791 human cyelin H m RNA 40 35.60
M423 D5 H-U 12255 immunoglobulin gamma heavy 40.26 48.0kDa chain Fc receptor RI, high affinity
M302 F7 H-U 12404 Csa-19 23.98 32
M236 A2 H-U12465 ribosomal protein L35 13.64 24
169-4 H-U12535 human epidermal growth factor 100 90.49 receptor kinase substrate (Eps8) mRNA
F3 H-U12597 Human tumor necrosis factor type 55.22 64 2 receptor associated protein (TRAP3) mRNA, complete eds
M314 D1 H-U12979 transcriptional coactivator PC4 14.08 23
M476 G4 H-U 13044 GA-binding protein transcription 50.05 53.0kDa factor, alpha subunit (60kD)
M302 F3 H-U13665 cathepsin O (GB:U13665) 36.3 50.0kDa
M311 G4 H-U13831 cellular retinol binding protein II 14.85 20.0kDa
A2 H-U13991 Human TATA-binding protein 24.09 34 associated factor 30 kDa subunit (tafII30) mRNA, complete eds
M416 A4 H-U14187 Human receptor tyrosine kinase 26.29 29.0kDa ligand LERK-3 (EPLG3) mRNA, complete eds
M250 A2 H-U14188 eph-related receptor tyrosine 22.22 27 kinase ligand 4 [EPLG4]
M302 D2 H-U14193 human TFIIA gamma subunit 12.060 28.0kDa mRNA
M416 G1 H-U14603 Human protein-tyrosine 18.48 30.0kDa phosphatase (HU-PP-1) mRNA, partial sequence
E2 H-U14747 Visinin-like 1 21.12 25
M266 D4 H-U14966 ribosomal protein L5 32.78 38
M314 E2 H-U14967 ribosomal protein L21 17.71 29
M266 F5 H-U14968 ribosomal protein L27a 16.39 19.0kDa
M248 E3 H-U14969 ribosomal protein L28 15.18 27
M266 E1 H-U 14971 ribosomal protein S9 21.45 30
M250 C2 H-U 15009 small nuclear ribonucleoprotein, 13.97 17.0kDa Sm D3
M311 D4 H-U 16660 enoyl-Coenzyme A hydratase-like 36.19 38 protein, peroxisomal
M302 H4 H-U 17074 cyclin-dependent kinase 6 18.59 29 inhibitor pi 8
M306 A2 H-U17195 A-kinase anchor protein 100 72.05 100 [AKAP100*]
Dl H-U 17280 Steroidogenic acute regulatory 31.46 35 protein M316 F1 H-U18291 cell division cycle protein 16 68.2 71.0kDa 69
C5 H-U 18420 Human ras-related small GTP 23.87 33 binding protein Rab5 (rab5) mRNA, complete eds
M311 A2 H-U 18423 spinal muscular atrophy gene 32.45 41
M248 D4 H-U18914 hypothetical protein, (Human 20.35 32 19.8 kDa protein mRNA, complete eds)
M302 B5 H-U19718 microfibril-associated 20.24 34.0kDa glycoprotein 2
M305 E3 H-U20240 CCAAT/enhancer-binding protein 16.61 29 gamma
M302 A8 H-U20352 malate dehydrogenase 36.85 40
M416 F4 H-U20391 Human folate receptor (FOLR1) 28.38 34.0kDa gene, complete eds
M311 D1 H-U20536 apoptotic cysteine protease Mch2 32.34 38.0kDa
M431 G2 H-U20659 RNA polymerase II, subunit B7 19.03 31.0kDa
M499 Cl H-U20938 Human lymphocyte 112.86 lOO.OkDa dihydropyrimidine dehydrogenase mRNA, complete eds
M305 F2 H-U20972 14-3-3 protein, epsilon 28.16 36
M271 D3 H-U21049 hypothetical protein 12.65 16 (GB:U21049), ESTs, Highly similar to DD96 [H.sapiens].
M421 G5 H-U21858 Human transcriptional activation 29.15 38.0kDa factor TAFII32 mRNA, complete eds
M424 H3 H-U22662 Human nuclear orphan receptor 49.28 49.0kDa LXR-alpha mRNA, complete eds
M271 D2 H-U24074 killer cell inhibitory receptor 37.62 43 [KIR], Homo sapiens natural killer-associated transcript 3 (NKAT3), complete eds. RECEPTOR ON NATURAL KILLER (NK) CELLS FOR HLA-C ALLELES.
169-29 H-U24153 human p21 -activated protein 60 57.82 kinase (Pak2) gene
M385 H2 H-U24166 EB1 29.59 36.0kDa
GI H-U24169 Human JTV-1 (JTV-1) mRNA, 34.43 40 complete eds
El H-U24576 Human breast tumor autoantigen 18.26 27 mRNA, complete sequence
G4 H-U24577 Human LDL-phospholipase A2 48.62 52 mRNA, complete eds
HI H-U25789 Human ribosomal protein L21 17.71 32 mRNA, complete eds
M416 D1 H-U25849 Human red cell-type low 17.49 28.0kDa molecular weight acid phosphatase (ACPI) gene, 5' flanking region and
M300 A3 H-U26312 heterochromatin protein H-PlHs- 19.14 30 gamma 70
M416 D3 H-U26403 Human receptor tyrosine kinase 25.19 30.0kDa ligand LERK-7 precursor (EPLG7) mRNA, complete eds
M317 E2 H-U27143 human protein kinase C inhibitor- 13.900 17.0kDa I cDNA
E5 H-U28249 Human 1 lkd protein mRNA, 12.32 12 complete eds
F4 H-U28386 Human nuclear localization 58.3 54 sequence receptor hSRPl alpha mRNA, complete eds
M423 E3 H-U28694 Chemokine (C-C) receptor 3 39.16 39.0kDa
M266 G6 H-U28963 Gρs2 36.08 36
M306 D3 H-U30610 CD94 antigen (NK T-cell C-type 19.8 27 lectin receptor) [CD94]
Bl H-U31116 Human beta-sarcoglycan A3b 35.09 33 mRNA, complete eds
M297 C2 H-U31278 mitotic feedback control protein 22.66 31.0kDa Madp2 homolog
M302 G2 H-U31384 guanine nucleotide-binding 8.14 10 protein, gamma 11 subunit
F4 H-U31986 Human cartilage-specific 35.97 47 homeodomain protein Cart-1 mRNA, complete eds
M390 F3 H-U32114 caveolin 2 17.93 18.0kDa
E4 H-U32324 Human interleukin- 11 receptor 46.53 54 alpha chain mRNA, complete eds
FI H-U32576 Apolipoprotein C-IV 14.08 16
M298 C4 H-U32907 p37NB protein 34.54 39
M300 D3 H-U32944 dynein, light chain 1, cytoplasmic 9.9 15
M297 D1 H-U32989 tryptophan 2,3-dioxygenase 44.77 50.0kDa
166-51 H-U33052 "protein kinase PRK2 [human, 110 108.3 DX3 B-cell myeloma cell line, mRNA]"
166-64 H-U33054 "human G protein-coupled 52 63.65 receptor kinase GRK4 mRNA, alpha splice variant"
166-88 H-U33055 "human G protein-coupled 60 60.1 receptor kinase GRK4 mRNA, beta splice variant"
166-76 H-U33056 "human G protein-coupled 58 58.59 receptor kinase GRK4 mRNA, gamma splice variant"
A2 H-U34584 17.71 31
169-87 H-U34820 human MAP kinase mRNA 55 46.49
215-2 H-U34822 human JNK1 alpha2 protein 55 47.04 kinase (JNK1A2) mRNA
169-37 H-U35002 human JNK2 betal protein kinase 50 42.09 (JNK2Bl) mRNA
169-25 H-U35003 human JNK2 beta2 protein kinase 55 46.71 (JNK2B2) mRNA
167-16 H-U35004 human JNK1 betal protein kinase 52 42.31 (JNKlBl) mRNA M300 B2 H-U35048 TSC-22 protein 15.95 27 71
M423 E5 H-U35398 Human G protein-coupled 40.26 48.0kDa receptor mRNA, complete eds
A3 H-U35735 Human RACH1 (RACH1) 42.9 78 mRNA, complete eds
M250 E5 H-U36764 Eukaryotic translation initiation 35.86 36.0kDa factor 3 (eIF-3) p36 subunit, transforming growth factor-beta receptor II interacting protein 1
M270 E4 H-U37283 microfibril-associated 19.14 32 glycoprotein-2 (GB:U37283)
M426 F3 H-U37352 Protein phosphatase 2A, 56.65 55.0kDa regulatory subunit B' alpha- 1
El H-U37529 Human substance P beta-PPT-A 14.3 22 mRNA, complete eds
M305 H5 H-U37547 apoptosis inhibitor 68.09 64
M424 D5 H-U38480 Human retinoid X receptor- 51.04 61.0kDa gamma mRNA, complete eds
M270 F4 H-U38810 Human mab-21 cell fate- determining protein homolog (CAGRl) mRNA,
M467 F6 H-U38904 Human zinc finger protein C2H2- 40.48 47.0kDa 25 mRNA, complete eds
E2 H-U39318 Human E2 ubiquitin conjugating 16.28 22 enzyme UbcH5C (UBCH5C) mRNA, complete eds
166-75 H-U39657 human MAP kinase kinase 6 40 36.81 (MKK6) mRNA
M298 E4 H-U39945 human adenylate kinase 2 (adk2) 26.3633 38.0kDa mRNA
166-38 H-U40282 human integrin-linked kinase 55 49.68 (ILK) mRNA
169-65 H-U40343 human CDK inhibitor pl9INK4d 18 18. 33 mRNA
E2 H-U40705 Homo sapiens telomeric repeat 48.4 52 binding factor (TRF1) mRNA, complete eds
166-50 H-U40989 human tat interactive protein 60 53.09 mRNA
M266 H6 H-U41767 metargidin precursor 89.65 90
M270 F3 H-U41804 Human putative T1/ST2 receptor 25.08 35.0kDa binding protein precursor mRNA, complete eds
D5 H-U42360 Human N33 gene 38.28 38
Al H-U43368 Vascular endothelial growth 22.88 33 factor B
M421 G7 H-U43901 Human 37 kD laminin receptor 32.56 58.0kDa precursor/p40 ribosome associated protein gene, complete eds
M392 C2 H-U43923 transcription factor SUPTH4 12.98 16.0kDa
E2 H-U46024 Myotubular myopathy 1 66.44 58
M330 A1 H-U46838 pl05MCM 90.42 97
M476 E2 H-U47677 Human transcription factor E2F1 48.18 53.0kDa (E2F1) gene, promoter and 72
M421 HI H-U48707 Human protein phosphatase- 1 18.92 36.0kDa inhibitor mRNA, complete eds
M302 B7 H-U49070 peptidyl-prolyl isomerase PIN1 18.04 28.0kDa
Cl H-U49188 Human placenta (Difβ3) mRNA, 54.45 70 complete eds
M485 H2 H-U49837 Human LIM protein MLP mRNA, 21.45 34.0kDa complete eds
D2 H-U49897 Homo sapiens phenylalanine 49.83 64 hydroxylase (PAH) mRNA, complete eds
B2 H-U49957 Human LIM protein (LPP) 67.43 67 mRNA, partial eds
166-16 H-U50196 human adenosine kinase mRNA 50 38.02
A4 H-U50939 Human amyloid precursor 58.85 60 protein-binding protein 1 mRNA, complete eds
G3 H-U51224 Human U2AFBPL gene, complete 52.8 55 eds
M486 E3 H-U51333 Hexokinase 3 (white cell) 101.64 lOO.OkDa
M305 Dl H-U51478 ATPase, Na+/K+ transporting, 30.8 36 beta 3 subunit
M416 H3 H-U52112 Homo sapiens Xq28 genomic 25.96 36.0kDa DNA in the region of the LI CAM locus containing the genes for neural cell adhesion molecule LI (LI CAM), arginine-vasopressin receptor (AVPR2), Cl pi 15 (Cl), ARD1 N-acetyltransferase related protein (TE2), renin-binding protein (RbP), host cell factor 1 (HCF1), and interleukin- 1 receptor-associated kinase (IRAK) genes, complete eds, and Xq281u2 gene
M463 El H-U53442 human p38Beta MAP kinase 40.99 49.0kDa mRNA
G3 H-U53446 Human mitogen-responsive 84.81 98 phosphoprotein DOC-2 mRNA, complete eds
M463 Cl H-U54617 human pyruvate dehydrogenase 45.28 52.0kDa kinase isoform 4 mRNA
169-38 H-U54645 methylmalonyl-coA mutase 38 25.59 precursor
M300 H3 H-U56255 t-complex sterility protein 12.54 16 homolog CW-1
C4 H-U56417 Human lysophosphatidic acid 31.24 46 acyltransferase-alpha mRNA, complete eds
M305 A2 H-U56637 actin-capping protein alpha 31.57 31 subunit isoform 1 73
M235 E6 H-U56814 Human DNasel-Like III protein 33.66 40.0kDa (DNAS1L3) mRNA, complete eds, involved in apoptosis Binds specifically to G-ACTIN AND BLOCKS ACT1N POLYMERIZATION.
D5 H-U57059 31.02 36
B3 H-U57093 Human small GTP-binding 24.09 34 protein rab27b mRNA, complete eds
D3 H-U57099 Human APEG-1 mRNA, 12.54 20 complete eds
FI H-U58331 Sarcoglycan, delta (35kD 28.27 24 dystrophin-associated glycoprotein)
M512 F4 H-U58334 Human Bcl2, p53 binding protein 110.66 108.0kDa Bbp/53BP2 (BBP/53BP2) mRNA, complete eds
B3 H-U58516 Human breast epithelial antigen 42.68 50 BA46 mRNA, complete eds
M250 E4 H-U58522 Human huntingtin interacting 22.11 30 protein (HIP2) mRNA, complete eds
M419 G2 H-U60207 human stress responsive 53.640 63.0kDa serine/threonine protein kinase Krs-2 mRNA
M298 B2 H-U60276 arsA homolog (hASNA-I) 36.63 47.0kDa
B2 H-U60521 Human protease proMchό (Mch6) 45.87 52 mRNA, complete eds
F3 H-U61166 Human SH3 domain-containing 57.31 57 protein SH3P17 mRNA, complete eds
M250 B5 H-U61232 cofactor E (tubulin-folding protein), REQUIRED FOR VIABILITY IN THE ABSENCE OF THE KINESIN-RELATED CIN8
A5 H-U62392 Homo sapiens zinc finger protein 43.45 52 mRNA, complete eds
GI H-U62801 Human protease M mRNA, 26.95 33 complete eds
M266 B1 H-U62962 Int-6 , Human Int-6 mRNA, 49.06 52.0kDa complete eds
M300 GI H-U63295 seven in absentia homolog 31.13 36
M306 H3 H-U64198 94.93 98
H3 H-U64863 Human hPD-1 (hPD-l) mRNA, 31.79 37 complete eds
B3 H-U65581 Human ribosomal protein L3-like 44.88 52 mRNA, complete eds
M341 Dl H-U65918 DAZ homologue [DAZLA] 32.56 36.0kDa
M302 El H-U65928 Jun activation domain binding 36.85 48.0kDa protein 74
M512 D3 H-U66347 Homo sapiens cAMP 46.97 60.0kDa phosphodiesterase (PDE4C) mRNA, 4C-426 isoform, complete eds
M306 F3 H-U66867 ubiquitin-conjugating enzyme E2I 17.49 28 [UBE2I]
M416 E2 H-U68111 Human protein phosphatase 22.66 37.0kDa inhibitor 2 (PPP1R2) gene
F2 H-U68382 Mannosidase, alpha B, lysosomal 35.64 36
G2 H-U69141 Glutaryl-Coenzyme A 48.29 56 dehydrogenase
B2 H-U70660 Human copper transport protein 7.59 16 HAHl (HAHl) mRNA, complete eds
M297 B2 H-U71374 peroxisomal membrane protein 40.15 40.0kDa (Pexl3p)
M306 A3 H-U75272 progastricsin [PGC] 42.79 49.0kDa
A2 H-U75285 Homo sapiens apoptosis inhibitor 15.73 25 survivin gene, complete eds
B2 H-U77456 Human nucleosome assembly 41.36 50 protein 2 mRNA, complete eds
C2 H-U78294 Homo sapiens 15S-lipoxygenase 74.47 74 mRNA, complete eds
F6 H-U78302 Human 2,4-dienoyl-CoA 36.96 40 reductase gene
M478 G3 H-U78798 Human TNF receptor associated 57.53 65.0kDa factor 6 (TRAF6) mRNA, complete eds
G3 H-U80982 Human myeloid-specific C/EBP- 27.5 51 epsilon transcription factor (CEBPE) gene, complete eds
M468 B7 H-U82256 Homo sapiens arginase type II 39.05 45.0kDa mRNA, complete eds
M465 B2 H-U82812 Human scavenger receptor 38.28 48.0kDa cysteine rich Sp alpha mRNA, complete eds
M484 D7 H-U83410 Human CUL-2 (cul-2) mRNA, 82.06 85.0kDa complete eds
M467 E6 H-U83460 Human high-affinity copper 21.01 32.0kDa uptake protein (hCTRl) mRNA, complete eds
D2 H-U84763 Homo sapiens UCP3 mRNA, 34.43 42 complete eds
B2 H-U86070 Homo sapiens 28.93 36 phosphomannomutase mRNA, complete eds
C2 H-U90441 Human prolyl 4-hydroxylase 58.96 64 alpha (II) subunit mRNA, complete eds
B2 H-U90543 Human butyrophilin (BTF1) 58.08 54 mRNA, complete eds
H2 H-U90545 Human sodium phosphate 44.22 36 transporter (NPT4) mRNA, complete eds 75
G2 H-U90552 Human butyrophilin (BTF5) 56.54 48 mRNA, complete eds
C3 H-U91521 Peroxisomal biogenesis factor 12 39.6 48
HI H-U91641 Human alpha2,8-sialyltransferase 41.47 45 mRNA, complete eds
Cl H-U93869 Human RNA polymerase III 34.98 36 subunit (RPC39) mRNA, complete eds
F2 H-U94346 Human calpain-like protease 70.4 65 (htra-3) mRNA, complete eds
C2 H-U94855 Human translation initiation 39.38 36 factor 3 47 kDa subunit mRNA, complete eds
M271 F7 H-U95089 Epidermal growth factor receptor. 44.66 47
M424 A5 H-U95847 Human GDNF receptor alpha 50.71 52.0kDa mRNA, complete eds
D2 H-U96094 Human sarcolipin (SLN) mRNA, 3.52 10 complete eds
B3 H-U96769 Homo sapiens chondroadherin 39.6 43 gene, 5'flanking region and
M298 G2 H-V00566 prolactin 25.08 35
M298 H2 H-V00571 corticotropin-releasing factor 21.67 49
217-61 H-V00572 phosphoglycerate kinase 1 50 45.94
M314 B3 H-V00597 parathyroid hormone 12.76 14
M305 B8 H-X00129 retinol-binding protein 4, 22 51 interstitial [RBP4]
F2 H-X00351 Human mRNA for beta-actin 41.36 41
A4 H-X00570 apolipoprotein C-I 9.24 35
M362 El H-X01057 interleukin 2 receptor, alpha 30.03 40.0kDa [IL2RA]
A4 H-X01677P Human liver mRNA for 10.45 10 glyceraldehyde-3 -phosphate dehydrogenase (G3PD, EC 1.2.1.12)
M271 D6 H-X02152 lactate dehydrogenase A [LDHA], 36.63 45.0kDa L-LACTATE DEHYDROGENASE M CHAIN
Al H-X02158 Human gene for erythropoietin 21.34 32
H4 H-X02415 Human gene for fibrinogen 48.18 50 gamma chain
A5 H-X02750 Protein C (inactivator of 50.82 53 coagulation factors Va and Villa)
M302 B3 H-X02751 proto-oncogene N-ras 20.9 25.0kDa
D3 H-X02812 Human mRNA for transforming 43.12 50 growth factor-beta (TGF-beta)
M302 C1 H-X03124 tissue inhibitor of 22.88 36.0kDa metalloproteinase 1
M362 B1 H-X03342 ribosomal protein L32 14.96 24.0kDa
M235 A2 H-X03484 human mRNA for raf oneogene 71.350 73.0kDa
M318 A3 H-X03557 interferon-induced protein 56 52.69 50.0kDa
A3 H-X03747 ATPase, Na+/K+ transporting, 33.44 45 beta 1 polypeptide 76
M305 D2 H-X04297 ATPase, Na+/K+ transporting, 112.64 99 alpha subunit
M305 A5 H-X04327 2,3-bisphosphoglycerate mutase 28.6 36
M271 G5 H-X04588 tropomyosin TM30nm, 26.29 40.0kDa cytoskeletal
M305 C8 H-X04741 ubiquitin related protein 23.43 28.0kDa
M236 A5 H-X05231 matrix metalloproteinase 1 51.7 53.0kDa (interstitial collagenase) [MMP1], CLEAVES COLLAGENS
166-53 H-X05246 "phosphoglycerate kinase, testis 50 45.94 specific"
M236 A1 H-X05908 annexin l, REGULATES 38.17 40 PHOSPHOLIPASE A2 ACTIVITY, Binds CALCIUM IONS
M250 A4 H-X06234 SI 00 calcium-binding protein A8 10.34 lO.OkDa (calgranulin A)
M266 B6 H-X06323 ribosomal protein L3, isoform 1 38.39 39
M313 A7 H-X06617 ribosomal protein SI 1 17.49 27
M416 E4 H-X06948 High affinity IgE receptor alpha- 28.38 36.0kDa subunit (FcERI)
M421 H7 H-X07203 Human mRNA for CD20 receptor 32.78 40.0kDa (S7)
217-2 H-X07743 pleckstrin 38 38.57
217-73 H-X07767 "cAMP-dependent protein kinase, 45 38.68 alpha-catalytic subunit"
M305 B3 H-X07898 troponin C, skeletal, fast 17.71 25
M306 El H-X07979 integrin, beta 1 87.89 110
Al l H-X08004 ras-related protein rap IB 20.24 38
M235 A7 H-X12387 Cytochrome P450 IIIA3 55.44 60.0kDa (nifedipine oxidase chain 3)
M315 F1 H-X12496 glycophorin C 14.19 24
M316 D3 H-X12517 small nuclear ribonucleoprotein 17.6 30.0kDa U1, C
M236 E5 H-X12534 guanine nucleotide-binding 20.24 34.0kDa protein rap2, ras-oncogene related
M266 E3 H-X12597 High-mobility group (nonhistone 23.76 37 chromosomal) protein 1, placenta
217-14 H-X12656 human mRNA for protein 40 34.06 phosphatase 2A (beta type)
H4 H-X 12662 H.sapiens arginase gene exon 1 35.53 50 and flanking regions (EC 3.5.3.1) (and joined CDS)
Cl H-X 12953 RAB2, member RAS oneogene 23.43 29 family
F5 H-X13956 Human 12S RNA induced by 9.13 19 poly(rl), poly(rC) and Newcastle disease virus
M297 Al H-X 15005 laminin receptor 1 33.11 48.0kDa
M315 E3 H-X 15088 guanine nucleotide binding 38.61 45 protein (G protein), alpha transducing (transducin) activity polypeptide 1 [GNAT1] 77
G2 H-X15183 Human mRNA for 90-kDa heat- 80.63 80 shock protein
M385 Cl H-X 15422 mannose-binding lectin, soluble 27.39 27.0kDa (opsonic defect) [MBL]
M271 D7 H-X 15606 INTERCELLULAR ADHESION 30.36 37.0kDa MOLECULE-2 PRECURSOR [Homo sapiens].
M298 C5 H-X 15653 uracil-DNA glycosylase 33.55 37
M302 B4 H-X15822 cytochrome-c oxidase, Vila 9.24 20 subunit, liver
M305 A6 H-X 15940 ribosomal protein L31 13.86 18
M236 G5 H-X 15949 interferon regulatory factor 2 , 38.5 54.0kDa BINDS AND REPRESSES REGULATORY REGION OF TYPE I IFN AND IFN- INDUCIBLE MHC CLASS I GENES.
M236 C2 H-X 16064 translationally-controlled tumor 19.03 35 protein
M512 B5 H-X 16323 Hepatocyte growth factor 80.19 lOO.OkDa (hepapoietin A)
M315 C3 H-X 16461 cell division cycle 2, GI to S and 32.78 40 G2 to M [CDC2]
M297 G2 H-X 16832 cathepsin H 36.96 45.0kDa
M271 B1 H-X 16983 integrin, alpha 4 (CD49D, alpha 4 114.29 114 subunit of VLA-4 receptor) [ITGA4], IMPORTANT FOR CELL-CELL ADHESION FUNCTION.
M270 A7 H-X 17025 plasminogen activator-inducible 25.19 34 c54, Human homolog of yeast IPP isomerase
M302 C3 H-X 17042 proteoglycan 1, secretory granule 17.49 26
Bl H-X 17206 ribosomal protein S2 24.42 45
B4 H-X 17254 Transcription factor Eryf 1 45.54 53
M311 H2 H-X17610 beta-1-glycoprotein, pregnancy- 46.97 48.0kDa specific (GB:X 17610)
M315 D1 H-X 17644 GI to S phase transition protein 55 55 (GST1)
M340 GI H-X51415 lipase, hormone-sensitive [LIPE] 84.59 98.0kDa
M464 A7 H-X51688 Cyelin A 47.63 47.0kDa
M313 G1 H-X51745 major histocompatibility complex, 40.26 50 class I, A
M297 A2 H-X51804 putative receptor protein PMI 21.23 30
D4 H-X51952 Human UCP gene for uncoupling 33.88 37 protein exons 1 and 2
M300 B1 H-X52011 muscle determining factor 26.73 39
M419 GI H-X52479 "protein kinase c, alpha type" 82.28 85.0kDa
A2 H-X52486 Uracil-DNA glycosylase 35.97 36
E3 H-X52520 Tyrosine aminotransferase 50.05 58
Bl H-X52638 6-phosphofructo-2- 51.92 47 kinase/fructose-2,6- bisphosphatase 78
M509 C4 H-X52730 Human gene for 31.13 35.0kDa phenylethanolamine N-methylase (PNMT) (EC 2.1.1.28)
M235 C5 H-X52839 ribosomal protein L17 15.51 18
M426 C2 H-X52943 Human mRNA for ATF-a 53.24 64.0kDa transcription factor
M266 G5 H-X53777 ribosomal protein L23 20.35 31
B4 H-X53961 Lactotransferrin 78.32 78
M462 C6 H-X54150 Fc fragment of IgA, receptor for 31.68 37.0kDa
M302 A6 H-X54304 myosin, light polypeptide 2, 18.92 32.0kDa regulatory
M311 G2 H-X54802 cytochrome-c oxidase, IV subunit 18.7 23.0kDa
M270 H3 H-X54871 guanine nucleotide-binding 23.76 33.0kDa protein Rab5B, ras-oncogene related [RAB5B], PROTEIN TRANSPORT. PROBABLY INVOLVED IN VESICULAR TRAFFIC (BYSIMILARITY).
M313 B6 H-X54936 placenta growth factor [PLGF*] 16.5 22.0kDa
M496 B2 H-X55079 Human lysosomal alpha- 104.83 98.0kDa glucosidase gene exon 1
Dl H-X55330 Aspartylglucosaminidase 38.17 36
El H-X55448 H.sapiens G6PD gene for 25.41 30 glucose-6-phosphate dehydrogenase
M421 G6 H-X56253 Human MPR46 gene for 46kd 30.58 52.0kDa mannose 6-phosphate receptor
169-89 H-X56468 14-3-3 protein tau 34 27.02
M300 B4 H-X56549 fatty-acid-binding protein, muscle 14.74 17
M298 D2 H-X56740 guanine nucleotide-binding 23.87 31.0kDa protein rab 11 [RAB11*]
M266 E5 H-X56932 highly basic protein, 23 kDa 22.44 30.0kDa
M318 G1 H-X57025 insulin-like growth factor I 16.94 18
M305 F5 H-X57348 protein kinase C inhibitor 27.39 35.0kDa
M236 D6 H-X57351 interferon-induced protein 1-8D 14.63 24
H3 H-X57352 interferon-induced protein 1-8U 14.74 38
M305 B6 H-X58079 S-100 protem, alpha chain 10.45 11
E6 H-X59131 H.sapiens D13S106 mRNA for a 34.76 50 highly charged amino acid sequene
M248 H5 H-X59268 transcription factor IIB [TCF2B*] 34.87 49
E2 H-X59357 Epstein-Barr virus small RNA- 14.19 36 associated protein
M236 D4 H-X59417 macropain, iota subunit , THE 27.17 36 INTERACTION OF CALPONIN WITH ACTIN INHIBITS ACTOMYOSIN MG-ATPASE ACTIVITY
M271 H4 H-X59618 ribonucleotide reductase, small 42.9 46 subunit 79
M250 G3 H-X59710 CAAT-box DNA-binding protein, 22.66 34 subunit B, CCAAT-BINDING TRANSCRIPTION FACTOR SUBUNIT A [Homo sapiens]
M423 E2 H-X59711 Nuclear transcription factor Y, 38.28 48.0kDa alpha
M271 C7 H-X59798 Cyelin Dl (PRAD1; parathyroid 32.56 40.0kDa adenomatosis 1). ESSENTIAL FOR THE CONTROL OF THE CELL CYCLE AT THE Gl/S (START) TRANSITION.
M270 H5 H-X59834 calmodulin 41.14 53.0kDa
M416 D5 H-X59871 Transcription factor 7 (T-cell 29.59 36.0kDa specific)
M485 D6 H-X60036 Phosphate carrier, mitochondrial 39.82 37.0kDa
M250 D4 H-X60489 translation elongation factor 1, 24.86 33.0kDa beta
F5 H-X60592 Human CDw40 mRNA for nerve 30.58 46 growth factor receptor-related B- lymphocyte activation molecule
M312 F3 H-X61587 ras-related rhoG 21.12 21.0kDa
F9 H-X61622 cyclin-dependent kinase 2 32.89 56 [CDK2]
M313 E3 H-X61970 macropain, zeta subunit 26.62 35.0kDa
M428 Dl H-X62055 tyrosine phosphatase, non- 65.78 66.0kDa receptor type 6
M248 C4 H-X62534 high mobility group protein 2, 23.1 37 BINDS PREFERENTIALLY SINGLE-STRANDED DNA AND UNWINDS DOUBLE STRANDED DNA.
M305 F3 H-X62753 folate-binding protein 28.38 36
M476 G2 H-X63468 H.sapiens mRNA for transcription 48.4 53.0kDa factor TFIIE alpha
G6 H-X63469 General transcription factor TFIIE 32.12 56 beta subunit, 34 kD
G4 H-X63522 H.sapiens mRNA DAUDI6 for 58.74 54 retinoic acid X receptor b
M316 G2 H-X63526 translation elongation factor 1, 48.18 52.0kDa gamma
M305 C5 H-X63527 ribosomal protein L19 21.67 33
E2 H-X63629 Cadherin 3 (P-cadherin) 91.3 110
D4 H-X64037-2 General transcription factor IIF, 56.98 64 polypeptide 1 (74kD subunit)
M302 C6 H-X64559 tetranectin 22.33 32.0kDa
M271 H1 H-X64728 choroideremia-like [CHML], 72.27 98 H.sapiens CHML mRNA
M270 El H-X64810 proprotein convertase 82.94 90 subtilisin/kexin type 1 [PCSK1], INVOLVED IN PROCESSING OF HORMONE AND OTHER PROTEIN PRECURSORS
M311 F4 H-X64877 complement factor H-related 29.81 36.0kDa protein 80
M388 D1 H-X65293 protein kinase C, epsilon 81.18 96.0kDa [PRKCE]
B5 H-X65873 kinesin, heavy polypeptide 106.04 34
F4 H-X66079 Spi-B transcription factor (Spi- 28.93 54 1/PU.l related)
F3 H-X66114 2-oxoglutarate carrier protein 0 37 [OGMT*]
M305 C6 H-X66141 myosin, light polypeptide 2, 18.37 31 regulatory, ventricular
M419 H1 H-X66357 cell division protein kinase 3 33.620 44.0kDa
166-13 H-X66358 serine/threonine-protein kinase 45 39.45 KKIALRE
166-25 H-X66360 serine/threonine-protein kinase 60 57.60 PCTAIRE-2
M419 A2 H-X66363 serine/threonine-protein kinase 54.600 64.0kDa PCTAIRE-1
166-37 H-X66364 H.sapiens mRNA PSSALRE for 38 32.19 serine/threonine protein kinase
M419 B2 H-X66365 cell division protein kinase 6 35.900 46.0kDa
H3 H-X66839 H.sapiens MaTu MN mRNA for 50.6 54 p54/58N protein
M266 G3 H-X67325 interferon, alpha-inducible gene 13.53 13 p27
M462 H7 H-X67594 Melanocortin 1 receptor (alpha 34.98 44.0kDa melanocyte stimulating hormone receptor)
M236 C5 H-X67951 Proliferation-associated gene A 22 34 (natural killer-enhancing factor A), PAGA
H3 H-X68486 Adenosine receptor A2 45.43 45
M429 E3 H-X68561 Sp4 transcription factor 86.35 86.0kDa
M430 F2 H-X69151 ATP synthase, H+ transporting, 42.13 58.0kDa subunit C, vacuolar
M236 C3 H-X69392 ribosomal protein L26 16.06 29
B3 H-X69532 H.sapiens gene for inter-alpha- 100.32 98 trypsin inhibitor heavy chain HI, exons 1-3
M236 F5 H-X69654 ribosomal protein S26 12.76 18
M421 C8 H-X70218 Protein phosphatase 4 (formerly 33.88 X), catalytic subunit
M266 H5 H-X70848 protein phosphatase 1, alpha 36.41 37 catalytic subunit
El H-X70940 Eukaryotic translation elongation 51.04 60 factor 1 alpha 2
M270 FI H-X72215 [PIT1], POU domain, class 1, 32.12 40.0kDa transcription factor 1 (Pitl, growth hormone factor 1)
M271 A7 H-X72760 Laminin, beta 2 (laminin S), S- 67.87 75.0kDa
LAMΓNΓN IS A LAMΓNIN-LIKE ADHESIVE PROTEIN CONCENTRATED IN THE SYNAPTIC CLEFT OF THE NEUROMUSCULAR JUNCTION. 81
M235 Bl H-X72841 Human retinoblastoma-binding 46.86 52.0kDa protein (RbAp46) mRNA, complete eds, IEF 7442 (GB:X72841)
217-25 H-X73428 DNA-binding protein inhibitor 20 17.08 ID-3
M305 B5 H-X73459 signal recognition particle, 15.07 20 subunit 14
M250 D6 H-X73460 ribosomal protein L3, isoform 2, 44.44 50.0kDa COMPONENT OF THE LARGE SUBUNIT OF CYTOPLASMIC RIBOSOMES
M462 D8 H-X74008 Protein phosphatase 1, catalytic 35.64 46.0kDa subunit, gamma isoform
M266 G2 H-X74104 Signal sequence receptor, beta; 20.24 27 translocon-associated protein, beta subunit
M266 E7 H-X74262 retinoblastoma binding protein 46.86 50.0kDa RbAp48
HI H-X74330 DNA primase polypeptide 1 46.31 51 (49kD)
M313 F3 H-X74570 gal beta (1-3/1-4) GlcNAc alpha- 36.3 46.0kDa 2,3 sialyltransferase (GB:X74570)
M429 H3 H-X74764 H.sapiens mRNA for receptor 94.120 98.0kDa protein tyrosine kinase
M271 E6 H-X75042 V-rel avian reticuloendotheliosis 68.2 88 viral oneogene homolog
M305 G2 H-X75252 phosphatidylethanolamine- 20.68 30 binding protein
M302 G1 H-X75593 guanine nucleotide-binding 22.44 32.0kDa protein rab 13
166-49 H-X75958 H.sapiens trkB mRNA for 55 52.54 protein-tyrosine kinase
C4 H-X76013 H.sapiens QRSHs mRNA for 85.36 85 glutaminyl-tRNA synthetase
A2 H-X76029 H.sapiens mRNA for neuromedin 19.25 20
U
M305 D5 H-X76228 ATP synthase, H+ transporting, 24.97 36 subunit E, vacuolar
M298 F6 H-X76648 glutaredoxin 11.77 l l.OkDa
M311 A4 H-X76717 metallothionein 11 6.82 14
C4 H-X77533 H.sapiens mRNA for activin type 56.43 61 II receptor
H2 H-X77548 H. sapiens cDNA for RFG 67.65 67
169-41 H-X77743 H.sapiens CDK activating kinase 45 38.1 3 mRNA
A4 H-X77909 H.sapiens IKBL mRNA 42.02 52
M305 Cl H-X78136 heterogeneous nuclear 40.26 40.0kDa ribonucleoprotein E2
M306 G2 H-X78416 casein, alpha [CSN1] 20.46 33
M271 C2 H-X78678 ketohexokinase (fructokinase) 32.89 39 [KHK], H.sapiens KHK mRNA for ketohexokinase, clone pHKTOGa 82
M305 D4 H-X79193 cyclin-dependent kinase 7 38.17 35 (homolog of Xenopus MO 15 cdk- activating kinase) [CDK7]
M431 F2 H-X79389 glutathione S-transferase Tl 26.51 34.0kDa
M298 C6 H-X79537 glycogenin 30.8 34.0kDa
M440 Cl H-X79865 H.sapiens Mrpl7 mRNA 21.89 31.0kDa
M298 F5 H-X80229 protein kinase PKN 52.8 64.0kDa
167-39 H-X80230 H.sapiens mRNA (clone C-2k) 42 40.99 mRNA for serine/threonine protein kinase
217-49 H-X80343 H.sapiens p35 mRNA for 40 33.84 regulatory subunit of cdk5 kinase
M270 D7 H-X80695 cytochrome oxidase-assembly 47.96 50 protein, OXAl, H.sapiens OXAlHs mRNA
M266 B5 H-X80909 nascent polypeptide-associate 23.76 37.0kDa complex, alpha
M416 D9 H-X80910 Protein phosphatase 1, catalytic 36.08 45.0kDa subunit, beta isoform
E2 H-X81198 Archain 52.03 63
169-6 H-X81817 H.sapiens BAP31 mRNA 32 27.1 3
E4 H-X82018 H.sapiens mRNA for ZID protein 46.75 57
M313 D7 H-X82456 MLN50 28.82 33
A2 H-X82629 H.sapiens mRNA for Mox-2 33.44 42
M236 D1 H-X83006 lipocalin, neutrophil gelatinase 21.89 34.0kDa associated
166-40 H-X83107 H.sapiens Bmx mRNA for 75 74.32 cytoplasmic tyrosine kinase
E3 H-X83425 H.sapiens LU gene for Lutheran 69.19 59 blood group glycoprotein
C6 H-X83703 H.sapiens mRNA for cytokine 35.2 54 inducible nuclear protein
M416 H2 H-X83928 H.sapiens mRNA for transcription 23.32 33.0kDa factor TFIID subunit TAFII28
166-17 H-X85106 H.sapiens mRNA for ribosomal 90 80.70 S6 kinase
166-39 H-X85337 H.sapiens mRNA for myosin light 110 109.0 chain kinase
D2 H-X85750 H.sapiens mRNA for transcript 26.29 30 associated with monocyte to macrophage differentiation
M266 E6 H-X87176 17-beta-hydroxysteroid 81.07 65 dehydrogenase, type 4
M297 F2 H-X87689 CLCP 23.21 33.0kDa
M300 A2 H-X87843 cyelin H assembly factor 34.1 47
M271 E3 H-X89750 homeotic protein, TGIF, 30.03 32.0kDa H.sapiens mRNA for TGIF protein
M235 G1 H-X90529 guanine nucleotide-binding 34.54 40 protein ragA [RAGA]
M302 E6 H-X90583 translocon-associated protein, 19.14 28.0kDa delta M306 G1 H-X90872 gp2512 23.65 33 83
M416 D2 H-X91504 Transcription factor COUP 2 22.22 32.0kDa (a.k.a. ARP1)
M250 B3 H-X92098 transmembrane protein rnp24 22.22 30
M271 G7 H-X92106 bleomycin hydrolase. 50.16 55.0kDa PROTECTING NORMAL AND MALIGNANT CELLS FROM BLM TOXICITY.
F3 H-X92715 Zinc finger protein 74 (Cos52) 63.03 47
M270 H6 H-X92720 H.sapiens mRNA for 70.51 71 phosphoenolpyruvate carboxykinase
H5 H-X92762 H.sapiens mRNA for tafazzins 32.23 37 protein
M298 D3 H-X93036 MAT-8 9.68 16.0kDa
M476 A5 H-X93595 H.sapiens mRNA for NK receptor 50.16 56.0kDa (clone 17.1C)
M417 D2 H-X93920 protein tyrosine phosphatase 41.980 48.0kDa foreskin
A5 H-X95592 H.sapiens mRNA for CID protein 15.62 28
M298 B4 H-X95648 translation initiation factor 2B, 33.66 34.0kDa alpha subunit
F3 H-X95735 H.sapiens mRNA for zyxin 2 63.03 72
M386 B1 H-X96752 L-3-hydroxyacyl-CoA 34.65 45.0kDa dehydrogenase, SCHAD gene
M422 B6 H-X97229 H.sapiens mRNA for NK 41.58 48.0kDa receptor, clone library 15.212
B3 H-X98173 H.sapiens mRNA for MACH- 51.15 51 alpha-2 protein
166-14 H-X99325 H.sapiens mRNA for Ste20-like 55 46.9 3 kinase
C4 H-X99459 H.sapiens mRNA for sigma 3B 21.34 30 protein
M424 C4 H-Y00291 Human hap mRNA encoding a 49.39 59.0kDa DNA-binding hormone receptor
M386 HI H-Y00345 polyadenylate-binding protein 69.74 70.0kDa
M469 A2 H-Y00630 Plasminogen activator inhibitor, 45.76 46.0kDa type II (arginine-serpin)
M305 El H-Y00711 lactate dehydrogenase B 36.85 38.0kDa
H2 H-Y00764 ubiquinol cytochrome c reductase 10.12 33 hinge protein
F5 H-Y07848 H.sapiens EWS, gar22, rrp22 and 36.3 50 bam22 genes
M305 G6 H-Z 11559 iron-responsive element binding 97.9 98 protein 1 [IREBl]
M250 F3 H-Z 11566 Pr22 protein , STATΗMIN 16.5 22.0kDa [Homo sapiens], SERVES AS RELAY (VIA
PHOSPHORYLATION) FOR DIVERSE SECOND MESSENGER PATHWAYS
169-73 H-Z 11695 H.sapiens 40 kDa protein kinase 50 38.35 related to rat ERK2
M475 C8 H-Z11737 Flavin-containing 61.49 70.0kDa monooxygenase 4 84
Cl H-Z 11898 Octamer binding protein 3 39.71 50
M266 H4 H-Z 12830 SSR, alpha subunit 31.57 42.0kDa
A3 H-Z 14000 Ring finger protein 1 41.58 50
M300 El H-Z 14978 actin-related protein 41.47 49
GI H-Z 19002 H.sapiens of PLZF gene encoding 74.14 84 kruppel-like zinc finger protein
HI H-Z21966 POU homeobox protein 33.22 43
M248 G3 H-Z23139 CLASS II 29.04 34
HISTOCOMPATIBILITY ANTIGEN, M BETA CHAIN PRECURSOR [Homo sapiens]
D3 H-Z26876 ribosomal protein L38 7.81 35
F2 H-Z28339 H.sapiens mRNA for delta 4-3- 35.97 43 oxosteroid 5 beta-reductase
M298 B3 H-Z28407 ribosomal protein L8 28.38 39.0kDa
M313 C3 H-Z29330 ubiquitin-conjugating enzyme 20.24 34 UbcH2, 23 kDa
M271 F3 H-Z29677 guanine nucleotide-binding 20.35 28.0kDa protein, ras-related
M465 C2 H-Z30425 H.sapiens mRNA for orphan 38.39 34.0kDa nuclear hormone receptor
M302 F5 H-Z31357 cysteine dioxygenase 22.11 31.0kDa
M340 C1 H-Z31695 inositol polyphosphate 5- 40.04 49.0kDa phosphatase, 43 kDa
E3 H-Z32564-2 H.sapiens FRGAMMA mRNA 26.84 36 (819bp) for folate receptor
M236 H1 H-Z35227 small G protein, TTF, RAS- 21.12 30.0kDa RELATED PROTEIN RACl
A10 H-Z35491 H.sapiens mRNA for novel 30.25 60 glucocortieoid receptor-associated protein
M440 G5 H-Z37986 H.sapiens mRNA for 25.41 28.0kDa phenylalkylamine binding protein
M297 E2 H-Z47087 cyelin A/cyclin-dependent kinase 18.04 30.0kDa 2-associated pl9
FI H-Z48051 H.sapiens gene for myelin 27.28 31 oligodendrocyte glycoprotein (MOG)
A2 H-Z48475 Glucokinase regulator 68.86 70
M302 E4 H-Z48570 sperm zona pellucida-binding 16.72 24 protein
M266 A2 H-Z68907 Human clone ID 193225 NAD 43.34 45.0kDa (H)-specific isocitrate dehydrogenase gamma subunit mRNA, alternatively spliced, partial eds
GI H-Z83850 Human DNA sequence from PAC 45.76 60 82 Jl 1 and cosmid U134E6 on chromosome Xq22. Contains NIK like and Thyroxin-binding globulin precursor (T4-binding globulin, TBG) genes, ESTs and STSs 85
H4 H-Z97171 Homo sapiens GLC1A (trabecular 55.55 55 meshwork induced glucocortcoid response) gene, exon I, joined CDS
M421 D5 H-Z97632 Human DNA sequence from PAC 28.49 38.0kDa 196E23 on chromosome Xq26.1- 27.2. Contains the TAT-SF1 (HIV-1 transcriptional elongation factor TAT cofactor TAT-SF1) gene, the BRS3 (Bombesin Receptor subtype-3 (Uterine Bombesin Receptor, BRS-3) gene, an unknown gene coding for two isoforms, a predicted CpG island, ESTs and STSs
Example 3 - Construction of Expression Plasmids
The following example illustrates the construction of the expression vectors used in the Examples above. Similar modifications can be made in other vectors for use in creating libraries of expressible gene sequences.
The vector pcDNA3.1/V5-His was obtained from Invitrogen (cat #V810-20) and modified slightly so that it carried an gene sequence for Zeocin™ resistance and lacked the multiple cloning site. A lOOμg aliquot was suspended in 200 μl medical irrigation (MI) water. A 5μl aliquot was saved for gel analysis. The remainder was transferred to a 1.7 ml Eppendorf tube. The vector was digested with Hindlll (400 U) using Promega Buffer E (final volume = 400 μl). The reaction ran 3 hours at 37° C. An aliquot was checked for completeness of digestion by running on an 0.8% agarose gel in IX TAE, and visualizing with ethidium bromide.
The digested vector was treated with 200 μl phenol/chloroform (pH7.5) according to standard procedures, and the DNA precipitated from the aqueous phase using 1/10 volume 3M NaOAc and 2 volumes 100% EtOH at room temperature, followed by washing with 80% EtOH. The pellet was resuspended in 100 μl MI water. 86
Two oligonucleotides were added to the resuspended DNA (Topo -H (40 μg) 5'-(P)AGCTCGCCCTTATTCCGATAGTG (SEQ ID NO:3), Topo-4 (12 μg) 5'- (P)AGGGCG (SEQ ID NO:4)), plus 17 μl 10X Promega T4 Ligase buffer. The tube was placed on ice and the volume increased to 170 μl with MI water. The oligos were ligated to the vector using 20U Promega T4 DNA ligase, incubated at 12° C overnight.
The vector was treated with 100 μl phenol/chloroform and the aqueous phase precipitated as described above. The pelleted DNA was resuspended in 150 μl of sterile water the redigested with Hindlll (17 μl Promega Buffer E, 200 U Hindlll - 37° C, 1 hour). The redigested DNA was re-extracted with phenol/chloroform and precipitated with 1/10 volume 3M NaOAc and 7/10 volume isopropanol, then washed with 80% EtOH.
The pelleted DNA was resuspended in 82 μl TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA, pH 8.0). A 2 μl aliquot was used to check the foregoing procedure using agarose gel electrophoresis as described above. The remaining 80 μl was transferred to a Falcon tube and mixed with 16 μg Topo-5 oligonucleotide (5'- (P)CAACACTATCGGAATA (SEQ ID NO:5). To this mixture was added 190 μl NEB Restriction Buffer #1 (room temperature). The total reaction mixture was adjusted to 1.9 mis with MI water. Vaccinia Topoisomerase I enzyme was added (80 μg) and the reaction tube placed in a 37° C water bath for 15 minutes.
After 15 minutes, 200 μl of room temperature Topo- 1 OX stop buffer was added (100 mM Tris 7.4, 110 mM EDTA, bromophenol blue). The entire volume was loaded onto an agarose gel (1.2 gr agarose/ 130 mis IX TAE) and run at 70 volts until the bromophenol blue dye had run down about 1/2 in (volume in the loading well was kept constant by the addition of IX TE). The voltage was reversed for 90 seconds. The contents of the loading well were transferred to a 15 ml Falcon tube and placed on ice. 2 mis of cold Topo-2X Wash Buffer (60 mM Tris 7.4, 1 mM EDTA, 4 mM dithiothreitol (DTT), 200 μg/ml bovine serum albumin (BSA)) was added and the 87
volume then adjusted to 4 mis with cold Topo- IX Enzyme Dilution Buffer (50% glycerol, 50 mM Tris 7.4, 1 mM EDTA, 2 mM DTT, 0.1% Triton X-100, 100 μg/ml BSA) plus 4 mis Topo-Glycerol mix (90% glycerol, 10% 50 mM TE pH 7.4, 0.1% Triton X-100) and stored until needed.
A similar procedure was used to make Topo-adapted pYES2 (Invitrogen cat #
V825-20).
While the foregoing has been presented with reference to particular embodiments of the invention, it will be appreciated by those skilled in the art that changes in these embodiments may be made without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims.

Claims

88That which is claimed is:
1. A method for producing a library of expressible coding regions comprising the steps of: (a) amplifying a plurality of coding regions using at least one coding region specific primer,
(b) inserting each coding region into an expression vector, and
(c) verifying the size and orientation of the inserted coding region.
2. The method according to claim 1 further comprising transforming cells with the vector containing the verified coding region.
3. The method according to claim 1 further comprising purifying the amplified coding region prior to insertion into an expression vector.
4. The method according to claim 1 wherein the coding regions encode full- length proteins.
5. The method according to claim 4 wherein the 5' primer used for amplification starts with the nucleotides C ACC ATG and the 3' primer causes the amplification product to end at the third position of the codon immediately preceding the stop codon of the coding region being amplified plus a single adenine residue.
6. The method according to claim 3 wherein the purification is performed using agarose gel electrophoresis.
7. The method according to claim 6 wherein the agarose is low melt agarose.
8. The method according to claim 1 wherein insertion of the amplified coding region into an expression vector is performed using an enzyme that both cleaves and ligates DNA. 89
9. The method according to claim 3 wherein the purification is performed using low melt agarose gel electrophoresis and insertion of the amplified coding region into an expression vector is performed using an enzyme that both cleaves and ligates DNA.
10. The method according to claim 8 wherein said enzyme is a type I topoisomerase or a site-specific recombinase.
11. The method according to claim 10 wherein said enzyme is vaccinia DNA topoisomerase, lambda integrase, FLP recombinase or Pl-Cre protein.
12. A method according to claim 11 wherein said enzyme is vaccinia DNA topoisomerase.
13. The method of claim 1 wherein the expression vector is a eukaryotic expression vector.
14. The method of claim 13 wherein said eukaryotic expression vector is pYES2/GS or pcDNA3.1/GS.
15. The method of claim 1 wherein the expression vector is a prokaryotic expression vector.
16. The method of claim 15 wherein said prokaryotic expression vector is pBAD.
17. The method according to claim 1 wherein the expression vector comprises one or more elements selected from: a promoter-enhancer sequence, a selection marker sequence, an origin of replication, an affinity purification tag sequence, an inducible element sequence and an epitope-tag sequence.
18. The method of claim 1 wherein size and orientation of the insert is verified using a polymerase chain reaction protocol. 90
19. The method of claim 18 wherein said verification is performed using whole cell lysates.
20. The method of claim 1 wherein the coding regions to be amplified are open reading frame sequences in prokaryotic DNA or eukaryotic DNA.
21. The method according to claim 20 wherein the eukaryotic DNA is obtained from yeast or mammalian cells.
22. The method according to claim 1 wherein the coding regions being amplified encode members of a family of proteins.
23. The method according to claim 22 wherein the proteins are human proteins.
24. The method according to claim 23 wherein the family of proteins are kinases, phosphatases, transcription factors, oncogenes, or tumor suppressors.
25. The method according to claim 1 wherein steps (a) and (b) are performed in a multiwell microtiter plate.
26. The method according to claim 1 wherein coding regions of the correct size and in the correct orientation are roboticly selected for transformation into cells for expression.
27. The method according to claim 2 comprising the additional step of verifying that the transformed cells express the coding region.
28. The method according to claim 2 wherein the transformed cells are eukaryotic cells or prokaryotic cells.
29. A method according to claim 28 wherein the eukaryotic cells are CHO cells or S. cerevisiea cells. 91
30. An expression library of coding regions produced according to the method of claim 1.
31. The library according to claim 30 wherein the coding regions encode yeast proteins.
32. The library according to claim 31 wherein the coding regions encode mammalian proteins.
33. The library according to claim 32 wherein the mammalian proteins are human proteins.
34. The library according to claim 33 wherein the human proteins are kinases, phosphatases, transcription factors, oncogenes, or tumor suppressors.
35. An expression library obtainable from the method of claim 1.
36. An expression vector pYES2/GS.
37. An expression vector pCDN A3.1 /GS .
92
38. A method for producing a library of expressible coding regions comprising the steps of:
(a) amplifying a plurality of coding regions using PCR, wherein the 5' primer comprises the sequence CACCATG and the 3' primer causes the amplification product to end just prior to any stop codon,
(b) purifying the amplified coding regions using low melt agarose electrophoresis,
(c) inserting each of the purified coding regions into an expression vector using vaccinia DNA topoisomerase, wherein said expression vector comprises a promoter-enhancer sequence, a selection marker sequence, an origin of replication, an affinity purification sequence, and an epitope-tag sequence,
(d) transforming bacterial cells with the insert containing expression vector,
(e) growing the transformed cells and verifying the size and orientation of the inserted coding region,
(f) selecting expression vectors containing inserted coding regions in the correct orientation for expression of the gene product, and
(g) transforming cells for expression with said expression vectors.
EP99917335A 1998-04-03 1999-04-02 Methods for producing libraries of expressible gene sequences Withdrawn EP1066404A4 (en)

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WO1996034112A1 (en) * 1995-04-24 1996-10-31 Chromaxome Corp. Methods for generating and screening novel metabolic pathways
WO1996039186A1 (en) * 1995-06-06 1996-12-12 Cedars-Sinai Medical Center Anti-neutrophil cytoplasmic antibody material associated with ulcerative colitis and related methods and kits
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