Patent Publication Number: US-2007117186-A1

Title: Highly efficient secretory signal peptide and a protein expression system using the peptide thereof

Description:
TECHNICAL FIELD  
      The present invention relates to secretory signal peptides exhibiting higher secretion efficiency than conventional secretory signal peptides, for example.  
     BACKGROUND ART  
      Up to the present, a variety of proteins have been expressed and produced with the use of a variety of hosts, such as  E. coli,  yeast, insect cell, or animal cell. Among such protein production systems, the  E. coli  host is one of the most commonly used hosts. When proteins derived from animals such as humans are produced in  E. coli  hosts, however, these proteins are often insolubilized, and such insolubilized proteins are expressed as denatured proteins or cannot be expressed in some cases.  
      In contrast, protein production systems involving the use of cultured cell hosts, such as insect or animal cell hosts, are capable of producing proteins derived from animals such as humans as properly folded proteins with high probability. These protein production systems utilizing cultured cell hosts, however, suffer from the following drawbacks: (1) high culture cost; (2) slow growth; and (3) difficulty of enlarging the culture scale.  
      When yeast, i.e., a unicellular eukaryote, is used as a host cell of a protein production system, proteins derived from animals such as humans can be expressed as properly folded proteins with relatively high probability, as with cases involving the use of cultured cells. As with cases of microorganisms such as  E. coli,  culture utilizing a yeast host can be carried out in a less expensive manner, in terms of culture equipment, culture duration, and medium cost, than culture utilizing cultured cells. Since budding yeast, Saccharomyces cerevisiae, is used in the production of alcohol beverages such as beer or wine or fermented foods such as bread, the safety of yeast as an organism has been confirmed. Also, sequencing of the yeast genome was completed earlier than that of other eukaryotes, and yeast has an excellent molecular biological background, i.e., well-organized genomic information is available.  
      Thus, yeast had been extensively utilized as a host for production system of endogenous/exogenous proteins, with the utilization of recombinant DNA techniques.  
      As described above, expression and production of a variety of proteins, including human-derived proteins, have been heretofore attempted. Compared with the intracellular expression of soluble proteins, it is generally known that expression of membrane proteins and secretory proteins is difficult. This is because many membrane proteins and secretory proteins are required to mature as functional proteins by various types of modifications in a protein intracellular transportation system through endoplasmic reticulum/Golgi bodies from cytoplasm.  
      In the research for development of new drugs, functional/structural analysis of human membrane proteins and secretory proteins is important. Such analysis requires highly efficient expression systems for membrane proteins and secretory proteins. In the case of the extracellular expression of secretory proteins, purification of expressed proteins can be carried out more easily than in the case of intracellular proteins, and degradation caused by intracellular proteases can be inhibited. Because of such advantages, extracellular expression of secretory proteins is regarded important in the industrial production of proteins. In addition to the aforementioned advantages, yeast advantageously has an intracellular protein transportation system similar to that of animal cells. Thus, expression of membrane proteins and secretory proteins derived from animals is relatively easier than that involving the use of prokaryotic hosts.  
      In general, membrane proteins or secretory proteins comprise at their N termini secretory signal peptides. It is reported that substitution of the secretory signal peptides of the membrane proteins or secretory proteins with secretory signal peptides exhibiting high secretion efficiency, including those derived from hosts, can enhance the expression efficiency and the success rate of expression of such membrane proteins or secretory proteins in a protein expression system. In order to improve the efficiency of membrane and secretory protein expression systems, it is critical to improve the secretion efficiency of secretory signal peptides. Examples of signal peptides of membrane proteins and secretory proteins derived from  Saccharomyces cerevisiae  or yeast viruses that are used in conventional membrane and secretory protein expression systems in yeast include secretory signal peptides derived from α-factor (Non-Patent Document 1), α-factor receptor (Non-Patent Document 2), preprotoxin, SUC2 proteins and PHO5 proteins (Non-Patent Document 3), BGL2 proteins (Non-Patent Document 4), and AGA2 proteins (Non-Patent Document 5). However, only a few secretory signal peptides have been used for the expression of membrane proteins and secretory proteins in the past. Accordingly, it is unknown whether or not such secretory signal peptides used so far exhibit the highest level of efficiency.  
      Computer programs that predict the sequences of secretory signal peptides have been provided. Examples of such computer programs include SOSUI signal Beta (http://sosui.proteome.bio.tuat.ac.jp/˜sosui/protcome/sosuisignal/sosuisignal_submit.html), SignalP (http://www.cbs.dtu.dk/services/SignalP/), PSORT (http://psort.nibb.ac.jp/), and Phobius (http://phobius.cgb.ki.se/). Use of these computer programs enables the prediction of the presence of the sequences of the secretory signal peptides in membrane proteins and secretory proteins based on the genomic information. At present, however, it is difficult to predict the efficiency of each secretory signal peptide with the use of these computer programs. Specifically, it is impossible to predict whether or not a predicted secretory signal peptide can actually be used to express proteins such as membrane proteins and secretory proteins and whether or not such peptide could be used for efficient mass-production of proteins.  
      In the past, the present inventors developed an expression system in yeast at low temperature by analyzing the response mechanism of  Saccharomyces cerevisiae  at low temperature (Patent Documents 1 and 2 and Non-Patent Document 6). The present inventors conducted an experiment concerning the expression of human proteins with the use of this expression system. As a result, they found that this expression system could more effectively inhibit the insolubilization of expressed proteins and the degradation caused by proteases at low temperature and would yield higher expression efficiency with the use of low-temperature-inducible promoters that are induced by low-temperature stimuli, compared with the conventional expression systems involving budding yeast at moderate temperature. In the present expression system, however, the expression efficiency of membrane proteins and secretory proteins were lower than that of the soluble proteins in yeast cells.  
      Patent Document 1: WO 2004/003197;  
      Patent Document 2: JP Patent Publication (Kokai) No. 2005-160357 A;  
      Non-Patent Document 1: M. K. Ramjee, J. R. Petithory, J. McElver, S. C. Weber, and J. F. Kirsch., A novel yeast expression/secretion system for the recombinant plant thiol endoprotease propapain, Protein Engineering, 1996, vol. 9, pp. 1055-1061;  
      Non-Patent Document 2: V. Sarramegna, F. Talmont, P. Demange, and A. Milon, Heterologous expression of G-protein-coupled receptors: comparison of expression systems from the standpoint of large-scale production and purification, Cellular and Molecular Life Sciences, 2003, vol. 60, pp. 1529-1546;  
      Non-Patent Document 3: A. Eiden-Plach, T. Zagorc, T. Heintel, Y. Carius, F. Breinig, and M J. Schmitt, Viral preprotoxin signal sequence allows efficient secretion of green fluorescent protein by  Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae,  and  Schizosaccharomyces pombe.,  Applied and Environmental Microbiology, 2004, vol. 70, pp. 961-966;  
      Non-Patent Document 4: T. Achstetter, M. Nguyen-Juilleret, A. Findeli, M. Merkamm, and Y. Lemoine, A new signal peptide useful for secretion of heterologous proteins from yeast and its application for synthesis of hirudin., Gene, 1992, vol. 110, pp. 25-31;  
      Non-Patent Document 5: D. Huang and E V. Shusta, Secretion and surface display of green fluorescent protein using the yeast  Saccharomyces cerevisiae.,  Biotechnology Progress, 2005, vol. 21, pp. 349-357;  
      Non-Patent Document 6: T. Sahara, T. Goda, and S. Ohgiya, Comprehensive expression analysis of time-dependent genetic response in yeast cells to low temperature., Journal of Biological Chemistry, 2002, vol. 277, pp. 50015-50021  
     DISCLOSURE OF THE INVENTION  
      As described above, the expression of membrane proteins and secretory proteins requires the use of secretory signal peptides exhibiting high secretion efficiency.  
      Accordingly, it is an object of the present invention to identify and provide secretory signal peptides exhibiting higher efficiency of transportation to cell organelles, including cell membranes, endoplasmic reticulum, and Golgi bodies, and higher efficiency of extracellular secretion, than conventional secretory signal peptides, for example.  
      The present inventors have conducted concentrated studies in order to attain the above object. As a result, they succeeded in discovering secretory signal peptides exhibiting a higher ability for transportation to cell organelles, including cell membranes, endoplasmic reticulum, and Golgi bodies, and a higher ability for extracellular secretion under moderate and low temperature conditions, than secretory signal peptides used in conventional membrane and secretory protein expression systems, from membrane proteins and secretory proteins existing in the  Saccharomyces cerevisiae  genomes, with the use of low-temperature-inducible promoters (Patent Document 1 and Non-Patent Document 6) and a reporter assay system involving the use of secretory luciferase as a reporter protein (International Application Number: PCT/JP2006/311597, claiming the priority right of JP Patent Application No. 2005-169768). This has led to the completion of the present invention.  
      The present invention includes the following.  
      (1) DNA encoding the following secretory signal peptide (a) or (b):  
      (a) a secretory signal peptide consisting of the amino acid sequence as shown in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, and 18; or  
      (b) a secretory signal peptide consisting of an amino acid sequence derived from the amino acid sequence of the secretory signal peptide (a) by deletion, substitution, or addition of one or several amino acids and having secretory signal activity at 30° C.  
      (2) DNA according to (1), wherein said DNA is any one of the following DNA (a) to (c) encoding a secretory signal peptide:  
      (a) DNA consisting of the nucleotide sequence as shown in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, and 17;  
      (b) DNA consisting of a nucleotide sequence derived from DNA (a) by deletion, substitution, or addition of one or several nucleotides and encoding a secretory signal peptide having secretory signal activity at 30° C.; and  
      (c) DNA hybridizing under stringent conditions with DNA consisting of a nucleotide sequence complementary to DNA (a) and encoding a secretory signal peptide having secretory signal activity at 30° C.  
      (3) A secretory signal peptide encoded by DNA according to (1) or (2).  
      (4) An expression vector comprising DNA according to (1) or (2) and a foreign gene.  
      (5) A transformant transformed by the expression vector according to (4).  
      (6) The transformant according to (5), wherein the host is yeast.  
      (7) The transformant according to (6), wherein the yeast is  Saccharomyces cerevisiae.    
      (8) A method for producing a protein, wherein the transformant according to any one of (5) to (7) is cultured at 20° C. to 42° C.  
      (9) DNA encoding the following secretory signal peptide (a) or (b):  
      (a) a secretory signal peptide consisting of the amino acid sequence as shown in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, and 102; or  
      (b) a secretory signal peptide consisting of an amino acid sequence derived from the amino acid sequence of the secretory signal peptide (a) by deletion, substitution, or addition of one or several amino acids and having secretory signal activity at 15° C.  
      (10) DNA according to (9), wherein said DNA is any one of the following DNA (a) to (c) encoding a secretory signal peptide:  
      (a) DNA consisting of the nucleotide sequence as shown in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, and 101;  
      (b) DNA consisting of a nucleotide sequence derived from DNA (a) by deletion, substitution, or addition of one or several nucleotides and encoding a secretory signal peptide having secretory signal activity at 15° C.; and  
      (c) DNA hybridizing under stringent conditions with DNA consisting of a nucleotide sequence complementary to DNA (a) and encoding a secretory signal peptide having secretory signal activity at 15° C.  
      (11) A secretory signal peptide encoded by DNA according to (9) or (10).  
      (12) An expression vector comprising DNA according to (9) or (10) and a foreign gene.  
      (13) A transformant transformed by the expression vector according to (12).  
      (14) The transformant according to (13), wherein the host is yeast.  
      (15) The transformant according to (14), wherein the yeast is  Saccharomyces cerevisiae.    
      (16) A method for producing a protein, wherein the transformant according to any one of (13) to (15) is cultured at 0° C. to 20° C.  
      This specification includes part or all of the contents as disclosed in the specification and/or drawings of each of Japanese Patent Applications Nos. 2005-339383 and 2006-297923, which are priority documents of the present application. 
    
    
     PREFERRED EMBODIMENTS OF THE INVENTION  
      Hereafter, the present invention is described in detail.  
      DNA encoding a secretory signal peptide according to the present invention can be identified by identifying a gene encoding a protein containing the secretory signal peptide derived from  Saccharomyces cerevisiae.  At the outset, 1,037 genes included in the categories of the plasma membrane, the integral membrane, the cell periphery, the cell wall, the extracellular, the endoplasmic reticulum (ER), and Golgi from the subcellular localization table of MIPS CYGD (http://mips.gsf.de/genre/proj/yeast/), which is the  Saccharomyces cerevisiae  (budding yeast) genomic database, are selected. Based on the amino acid sequences encoded by the nucleotide sequences of the genes selected from the database, the transmembrane sites and the secretory signal peptides are then predicted using the prediction programs for transmembrane domains and the prediction programs for the secretory signal peptides, TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM/), Phobius (http://phobius.cgb.ki.se/), SOSUI signal Beta (http://sosui.proteome.bio.tuat.ac.jp/˜sosui/proteome/sosuisignal/sosuisignal_submit.html), and SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/). Based on the results of this analysis, the gene regions encoding secretory signal peptides are extracted from the aforementioned database via several prediction programs, concerning the genes, for which the secretory signal peptides have been predicted.  
      Further, a reporter gene containing a DNA fragment in which a secretory luciferase gene lacking the original secretory signal peptide has been ligated downstream of the gene region encoding the predicted secretory signal peptide is prepared, and reporter assay (International Application Number: PCT/JP2006/311597, claiming the priority right of JP Patent Application No. 2005-169768) is carried out using the resulting reporter gene to identify the secretory signal peptide having secretion ability that is more than twice, at moderate temperature (30° C.) and low temperature (15° C.), that of the secretory signal peptide (the α-factor-derived secretory signal peptide (Non-Patent Document 1)) used in conventional membrane and secretory protein expression systems, with the use of extracellular luciferase activity as an indicator.  
      As a result, 9 genes containing novel gene regions encoding secretory signal peptides having high secretion ability at moderate temperature were identified. The identified genes are shown in Table 1.  
                   TABLE 1                          Secretory signal peptides having secretion ability more than twice that of the α-factor-derived           secretory signal peptide at moderate temperature (30° C.)                                                             Secretion                                       efficiency relative                   to α-factor-       SEQ       SEQ           Systematic   Common   derived secretory       ID       ID           gene name   gene name   signal peptide   Amino acid sequence   NO:   Nucleotide sequence   NO:                                                             1   YDR420w   HKR1   5.64   MVSLKIKKLLLVSLLNAIEAYSNDTI    2   ATGGTCTCATTGAAAATAAAAAAAATT   1                           YSTSYNNGIESTPSYSTSAISSTGSS       TTACTCCTGGTGTCATTGTTAAATGCA                       NKENAITSSSETTTMAGDYGESGSTT       ATCGAGGCCTATAGTAACGATACAATA                       IMDEQETGTSSQYISVTTTTQ       TATTCAACTTCATACAATAATGCAATA                               GAAAGCACACCCTCATATTCAACATCC                               GCGATATCCAGTACCGGATCTAGCAAC                               AAAGAGAATGCAATAACATCAAGCTCT                               GAAACCACCACAATGGCTGGCCAATAT                               GGTGAAAGTGGAAGCACAACAATAATG                               GATGAACAAGAAACTGGTACGTCCAGC                               CAGTATATTAGTGTGACGACGACAACG                               CAA               2   YBR187w       3.37   NGGNMAIKKASLIALLPLFTAAAAAA    4   ATGGGAAATATGATAAAGAAGGCATCT   3                       TDAETSNESGSSSHLKS       TTAATAGCCCTCCTACCGCTGTTCACC                               GCCGCAGCCGCTGCAGCTACTGATGCG                               GAGACATCTATGGAATCTGGGAGTTCT                               TCACATTTGAAGTCT               3   YGL126w   SCS3   3.27   NSSKNFNAIHLLVCPLTVLVGYLMNAY    6   ATGTCTAGCAAATGGTTTAATGCTATA   5                       GYGAALQATLNKDGLVNAMLVKKG       CACCTACTGGTGTGCCCGTTGACGGTA                               CTGGTAGGATATCTCATGAACGCGTAT                               GGCTACGGTGCGGCGCTGCAAGCAACC                               CTGAATAAGGATGGTCTGGTAAATGCT                               ATGTTGGTAAAGAAAGGG               4   YHR139c   SPS100   2.69   MKFTSVLAFFLATLTASATFLYKRQN    8   ATGAAATTCACATCAGTGCTAGCATTT   7                       VTSGGGTVPIITGGPAVSGSQSNVTT       TTTCTTGCAACTTTAACAGCTTCTGCA                       TTLFNSTSTLNITQLYQIATDVNDTL       ACACCACTTTACAAGAGGCAGAACGTT                       QSESSS       ACTTCTGGCGGCAGGTACGGTCCCCGT                               GATCATCACGGGTGGACCTGCTGTATC                               TGGTAGCCAGTCAAACGTTACTACCAC                               AACGCTATTCAACTCTACTTCCACCTT                               AAACATCACTCAACTTTACCAAATTGC                               TACTCAAGTTAATCAGACTTTACAAAG                               CGAATCGTCTTCC               5   YGR014w   MSB2   2.66   MQFPFACLLSTLVISGSLARASPFDF   10   ATGCAGTTTCCATTCGCTTGTCTCCTA   9                       IFGNGTQQAQSQSESQGQVSFTNEAS       TCGACCCTTGTAATTAGTGGGTCATTG                       QDSSTTSLVTAYSQGVHSHQSATIVS       GCCCGGGCCAGCCCCTTCGACTTTATA                       ATISSLPSTWYDASSTSQTSVS       TTCGGCAATGGAACGCAACAAGCTCAG                               AGCCAAAGCGAGAGTCAAGGTCAAGTT                               TCTTTCACCAATGAAGCTTCTCAGGAT                               AGTTCCACCACCTCTTTGGTAACAGCC                               TATTCTCAAGGTGTTCATTCGCACCAG                               TCTGCAACAATAGTGAGTGCCACAATC                               TCTTCCCTCCCATCTACTTGGTATGAT                               GCGAGCTCCACTTCCCAGACTTCTGTG                               TCA               6   YBR078w   ECM33   2.48   MQFKNALTATAILSASALAANSTTSI   12   ATGCAATTCAAGAACGCTTGACTGCTA   11                        PSSCSIGTSATATAQADLDKISQCST       CTGCTATTCTAAGTGCCTCCGCTCTAG                       IVGNLTITGDLGSAALASIQEIDGSL       CTGCTAACTCAACTACTTCTATTCCAT                       TIFNSSSLSSFSADIKKI       CTTCATGTAGTATTGGTACTTCTGCCA                               CTGCTACTGCTCAAGCTGATTTGGACA                               AAATCTCCGGTTGTAGTACCATTGTTG                               GTAACTTGACCATCACCGGTGACTTGG                               GTTCCGCTGCTTTGGCTAGTATCCAAG                               AGATTGATGGTTCCTTGACTATCTTCA                               ACTCCAGTTCTTTATCTTCTTTCTCCG                               CTGACTCTATGAAGAAAATC               7   YNL300w   TOS6   2.46   MKFSTLSTVAAIAAFASADSTSDGVT   14   ATGAAATTCTCTACTCTCTCCACCGTT   13                        YVDVTTTPQSTTSMVSTVKTTSTPYT       GCTGCCATTGCCGCATTTGCTTCCGCA                       TSTIATLSTKSISSQANTTTHEIST       GATTCCACCTCTGATGGTGTCACTTAC                               GTAGATGTTACCACCACCCCACAAAGT                               ACTACATCTATGGTCTCCACCGTGAAA                               ACTACTTCCACTCCATACACTACAAGT                               ACCATTGCCACTCTATCCACTAAATCT                               ATCAGTAGCCAAGCTAACACCACCACC                               CATGAGATCAGCACA               8   YLR084c   RAX2   2.27   MFVHRLWTLAFPFLVEISKASQLENI   16   ATGTTTGTTCATCGTCTCTGGACACTC   15                        KSLLDIEDNVLPNLNISQNNSNAVQI       GCATTTCCTTTTCTTGTGGAGATATCG                       LGGVDALSFYEYTGQQNFTKEIGPET       AAGGCATCACAGTTGGAGAATATTAAA                       SSHGLVYYSNNTYIQLEDASDD       TCTCTTCTGGACATCGAAGATAATGTG                               CTACCGAATTTGAATATATCGCAAAAT                               AATAGCAACGCAGTACAAATCCTCGGG                               GGTGTGGACGCCTTATCTTTTTACGAG                               TACACAGGCCAACAAAATTTCACTAAA                               GAAATAGGTCCAGAAACAAGCTCACAT                               GGATTAGTTTATTACTCTAACAACACC                               TATATCCAGTTGGAAGATGCCTCTGAT                               GAT               9   YMR008c   PLB1   2.04   MKLQSLLVSAAVLTSLTENVNAMSPN   18   ATGAAGTTGCAGAGTTTGTTGGTTTCT   17                        NSYVPANVTCDDDINLVREASGLSDN       GCTGCAGTTTTGACTTCTCTAACAGAG                       EYEMLKKRDAYTKE       AACGTTAACGCTTGGTCACCAAATAAC                               AGTTACGTCCCTGCGAACGTAACCTGT                               GATGATGATATTAACTTAGTCAGAGAA                               GCATCTGGTTTGTCAGATAACGAAACA                               GAATGGCTGAAAAAAAGAGATGCATAC                               ACCAAGGAG                  
 
      Table 1 shows the systematic and common names of genes from which the gene regions encoding secretory signal peptides are derived, relative secretion efficiency in relation to α-factor-derived secretory signal peptide, the amino acid sequences of the secretory signal peptides, and the nucleotide sequences of the gene regions encoding secretory signal peptides, concerning secretory signal peptides having secretion ability that is more than twice that of α-factor-derived secretory signal peptide at moderate temperature (30° C.). SEQ ID NOs: of the amino acid sequences and those of the nucleotide sequences are indicated in the rightmost column.  
      Also, 51 genes containing novel gene regions encoding secretory signal peptides having high secretion ability at low temperature are shown in Table 2.  
                   TABLE 2                          Secretory signal peptides having secretion ability more than twice that of the α-factor-derived           secretory signal peptide at moderate temperature (30° C.)                                                             Secretion                                       efficiency relative                   to α-factor-       SEQ       SEQ           Systematic   Common   derived secretory       ID       ID           gene name   gene name   signal peptide   Amino acid sequence   NO:   Nucleotide sequence   NO:                                                              1   YBR243c   ALG7   5.76   NLRLFSLALITCLIYYSKNQGPSALV   20   ATGTTGCGACTTTTTTCACTGGCACTT   19                           AAVGFGIA       ATCACATGCTTAATCTACTATTCCAAA                               AATCAGGGGCCCATCTGCTCTTGTTGC                               GGCCGTGGGATTTGGTATAGCA                2   YNL237w   YTP1   5.67   NTAANKNIVFGFSRSISAILLICFFF   22   ATGACAGCAGCTAATAAGAATATTGTT   21                       FKVCGDMEHDHGHDDTSGYTRPEIVQ       CTTCGGATTTTCCAGATCCATTAGCGC                       AGSKS       AATTCTACTAATATGCTTTTTCTTTGA                               AAAAGTCTGCGGTGATATGGAGCATGA                               TATGGGCATGGATGATACTTCGGGATA                               CACGAGGCCAGAAATTGTGCAGGCTGG                               GTCGAAATCT                3   YCL043c   PDI1   5.61   MKFSAGAVLSMSSLLLASSVFAQQEA   24   ATGAAGTTTTCTGCTGGTGCCGTCCTG   23                       VAPEDSAVVAKLATDSFNEYIQSHD       TCATGGTCCTCCCTGCTGCTCGCCTCC                               TCTGTTTTCGCCCAACAAGAGGCTGTG                               GCCCCTGAAGACTCCGCTGTCGTTAAG                               TTGGCCACCGACTCCTTCAATGAGTAC                               ATTCAGTCGCACGAC                4   YKL096w   CWP1   5.55   MKFSTALSVALFALAKMVIADSEEFG   26   ATGAAATTCTCCACTGCTTTGTCTGTC   25                       LVSIRSGSDLGYLSVYSDNGTLKLGS       GCTTTATTCGCCTTGGCTAAGATGGTC                       GSGSFEATITDDGKLKFDDDKYAVVN       ATTGCCGATTCCGAAGAATTCGGCCTG                       EDGSFKEGSESD       GTGAGTATCCGTTCCCGCTCGGATTTA                               CAATACTTGAGTGTTTACAGTGATAAC                               GGCACTTTGAAACTTGGCAGCGGTAGT                               GGCTCATTTGAGGCAACTATTACCGAT                               GACGGTAAACTGAAATTTGACGACGAT                               AAGTATGCTGTTGTCAATGAGGATGGC                               TCATTCAAAGAAGGTTCTGAGAGCGAT                5   YBR078w   ECM33   5.17   MQFKNALTATAILSASALAANSTTSI   12   ATGCAATTCAAGAACGCTTTGACTGCT   11                       PSSCSIGTSATATAQADLDKISGCST       ACTGCTATTCTAAGTGCCTCCGCTCTA                       IVGNLTITGDLGSAALASIQEIDGLS       GCTGCTAACTCAACTACTTCTATTCCA                       TIFNSSSLSSFSADSIKKI       TCTTCATGTAGTATTGGTACTTCTGCC                               ACTGCTACTGCTCAAGCTGATTTGGAC                               AAAATCTCCGGTTGTAGTACCATTGTT                               GGTAACTTGACCATCACCGGTGACTTG                               GGTTCCGCTGCTTTGGCTAGTATCCAA                               GAGATTGATGGTTCCTTGACTATCTTC                               AACTCCAGTTCTTATCTTCTTTCTCCGC                               TGACTCTATCAAGAAAATC                6   YLR250w   SSP120   5.05   HRFLRGFVFSLAFTLYKVTATAEIGS   28   ATGAGGTTTTTGAGGGGATTTGTATTT   27                       EINVENEAPPDGLSWEEKHCIDHEHQ       TCTTTGGFCTTTCACTCTATATAAAGT                       LKDYTPET       AACTGCCACGGCAGAAATAGGATCTGA                               AATTAATGTGGAAAATGAGGCACCACC                               TGATGGTTTATCCTGGGAGGAGTGGCA                               TATGGACCATGAGCATCAGCTAAAAGA                               TTACACTCCAGAGACT                7   YEL001c       4.76   MRFSNLIGFNLLTALSSFCAAISANN   30   ATGAGGTTTTCTATGTTAATCGGGTTT   29                       SDNVEHEQEVAEAVAPPSINIEVKYD       AATTTATTAACTGCCTTGAGCAGCTTC                       VVGKESENHDSFLEFYAEDTATLYNV       TGTGCTGCCATATCAGCTAATAATAGC                       TNHEDTNITIFGVNGTIVTYP       GACAATGTCGAACATGAACAGGAAGTT                               GCGGAGGCGGTAGCGCCACCTTCTATT                               AATATAGAGGTGAAATATGATGTCGTT                               GGGAAGGAATCAGAAAATCATGATTCT                               TTCCTTGAGTTTTACGCGGAGGATACC                               GCTACCTTAGCCTATAATGTTACTAAT                               TGGGAAGATACTAATATCACAATTTTT                               GGTGTCAACGGAACAATTGTTACATAT                               CCA                8   YMR008c   PLB1   4.71   MKLQSLLVSAAVLTSLTENVNAHSPN   18   ATGAAGTTGCAGAGTTTGTTGGTTTCT   17                       NSYVPANVTCDDDINLVREASGLSDN       GCTGCAGTTTTGACTTCTCTAACAGAG                       ETEWLKKRDAYTKE       AACGTTAACGCTTGGTCACCAAATAAC                               AGTTACGTCCCTGCGAACGTAACCTGT                               GATGATGATATTAACTTAGTCAGAGAA                               GCATCTGGTTTGTCAGATAACGAAACA                               GAATGGCTGAAAAAAAGAGATGCATAC                               ACCAAGGAG                9   YNL238w   KEX2   4.70   HKVRKYITLCFVHVAFSTSALVSSQQ   32   ATGAAAGTGAGGAAATATATTACTTTA   31                       IPLKDHTSRQYFAVESNETLSRLEEK       TGCTTTTGGTGGGCCTTTTCAACATCC                       HPWWKYEKHDVRGLPNHVVFSKELLK       GCTCTTGTATCATCACAACAAATTCCA                       LGKRSSLEELQGDNNDHILSVHDL       TTGAAGGACCATACGTCACGACAGTAT                               TTTGCTGTAGAAAGCAATGAAACATTA                               TCCCGCTTGGAGGAAATGCATCCAAAT                               TGGAAATATGAACATGATGTTCGAGGG                               CTACCAAACCATTATGTTTTTTCAAAA                               GAGTTGCTAAAATTGGGCAAAAGATCA                               TCATTAGAAGAGTTACAGGGGGATAAC                               AACGACCACATATTATCTGTCCATGAT                               TTA               10   YLR084c   RAX2   4.61   HFVHRLHTLAFPFLVEISKASQLENI   16   ATGTTTGTTCATCGTCTCTGGACACTC   15                       KSLLDIEDNVLPNKLNISQNNSNAVQ       GCATTTCCTTTTCTTGTGGAGATATCG                       ILQGVDALSFYEYTGQQNFTKEIGPE       AAGGCATCACAGTTGGAGAATATTAAA                       TSSHGLVYYSNNTYIQLEDASDD       TCTCTTCTGGACATCGAAGATAATGTG                               CTACCGAATTTGAATATATCGCAAAAT                               AATAGCAACGCAGTACAAATCCTCGGG                               GGTGTGGACGCCTTATCTTTTTACGAG                               TACACAGGCCAACAAAATTTCACTAAA                               GAAATAGGTCCAGAAACAAGCTCACAT                               GGATTAGTTTATTACTCTAACAACACC                               TATATCCAGTTGGAAGATGCCTCTGAT                               GAT               11   YGR189c   CRH1   4.01   NKVLDLLTVLSASSLLSTFAAAESTA   34   ATGAAAGTGCTTGACCTACTAACGGTA   33                       TADSTTAASSTASCNPLKTTGCTPDT       CTCAGTGCCTCTTCATTATTATCTACA                       ALATSFSEDFSSSSK       TTCGCGGCTGCCGAAAGTACTGCTACT                               GCAGACAGTACAACTTGCAGCTTCTAG                               CACTGCTTCGTGTAACCCGTTAAAAAC                               TACAGGTTGTACGCCGGATACAGCTTT                               GGCAACTAGTTTTAGCGAAGATTTCTC                               ATCTTCATCCAAA               12   YLR286c   CTS1   3.95   MSLLYIILLFTQFLLLPTDAFDRSAN   36   ATGTCACTCCTTTACATCATTCTTCTA   35                       TNIAVYVSQGNASAGTQESLATYCES       TTCACACAATTCTTACTACTGCCAACC                       SDAD       GATGCCTTTGATAGGTCTGCTAACACA                               AATATTGCTGTTTATTGGGGTCAAAAC                               TCAGCAGGAACGCAAGAATCCTTAGCT                               ACTTACTGTGAATCTTCTGATGCTGAT               13   YMR006c   PLB2   3.78   HQLRNILQASSLISGLSLAADSSSTT   38   ATGCAATTACGGAACATATTACAGGCT   37                       GDGYAPSIIPCPSDDTSLVRNASGLS       AGCTCGCTAATTTCTGGACTTTCGCTC                       TEATDRLKKRDAYTKEALHSFLSRAT       ACTGCAGATTCGTCGTCCACTACTGGT                       SNFSDTSLLSTLFSSNSSN       GATGGTTATGCTCCATCAATAATTCCT                               TGTCCCAGTGATGATACCTCTTTAGTT                               AGAAACGCGTCTGGCTTATCTACCGCT                               GAAACTGATTGGTTAAAGAAAAGAGAT                               GCGTACACTAAAGAAGCTTTACATTCC                               TTCTTAAGCAGAGCTACTTCTAACTTC                               AGTGACACTTCTTTGCTATCCACTCTT                               TTCAGTAGTAACTCTTCCAAT               14   YKL096w-a   CWP2   3.70   MQFSTVASVAFVALNFVAAESAAAIS   40   ATGCAATTCTCTACTGTCGCTTCCGTT   39                       QITDQIQATTTATTEATTTAAPSSTV       GCTTTCGTCGCTTTGGCTAACTTTGTT                       ETVSPSSTETISQQTEN       GCCGCTGAATCCGCTGCCGCCATTTCT                               CAAATCACTGACGGTCAAATCCAAGCT                               ACTACCACTGCTACCACCGAAGCTACC                               ACCACTGCTGCCCCATCTTCCACCGTT                               GAAACTGTTTCTCCATCCAGCACCGAA                               ACTATCTCTCAACAAACTGAAAAT               15   YCR028c   FEN2   3.64   MHKESKSITQHEVERESVSSKRAIKK   42   ATGATGAAGGAATCGAAATCTATCACT   41                       RLLLRKIDLFVLSFVCLQYWINYVDR       CAACATGAGGTTGAGAGAGAATCTGTT                       VGFTNAYISQHKEDLKNVGNDLT       TCTTCCAAGCGTGCCATTAAAAAGAGA                               TTACTTCTGTTTAAAATAGACTTGTTT                               GTGCTATCATTTGTTTGCTTGCAATAC                               TGGATTAATTATGTCGACCGTGTCGGT                               TTCACCAATGCATATATATCCGGTATG                               AAGGAAGATCTTAAGATGGTCGGGAAA                               CGATTTGACC               16   YGL126w   SCS3   3.54   LISSSKWFNAIHLLVCPLTVLGYLMN   6   ATGTCTAGCAAATGGTTTAATGCTATA   5                       AYGYGAALQATLNKDGLVNAHLVKKG       CACCTACTGGTGTGCCCGTTGACGGTA                               CTGGTAGGATATCTCATGAACGCGTAT                               GGCTACGGTGCGGGCGCTGCAAGCAAC                               CCTGAATAAGGATGGTCTGGTAAATGC                               TATGTTGGTAAAGAAAGGG               17   YMR200w   ROT1   3.46   NWSKKFTLAKKLILGGYLFAQKVYCE   44   ATGTGGTCGAAAAAGTTTACATTAAAA   43                       DESNSIYGTWSSKASNQVFTGPGFDP       AAGCTAATCTTAGGCGGGTATTTGTTT                       VDELLIEPSLPGLSYSFTEDGHYEEA       GCTCAAAAGGTCTATTGTGAAGACGAA                       TYQVSGNPRNPTCPMASLIYQHG       AGTAACTCTATATACGGTACCTGGTCA                               TCTAAATCAAATCAAGTGTTCACGGGA                               CCGGGGTTTTATGATCCCGTAGATGAA                               CTATTGATAGAACCTTCATTGCCCGGG                               CTTAGCTATTCGTTCACTGAAGATGGT                               TGGTACGAAGAAGCTACTTACCAGGTA                               AGTGGCAATCCTCGTAACCCAACTTGC                               CCCATGGCTTCGTTGATTTATCAGCAT                               GGT               18   YDR304c   CPR5   3.44   MKLQFFSFITLFACLFTTAIFAKEDT   46   ATGAAGCTTCAATTTTTTCCTTTATTA   45                       AEDPETITHKVYFDINHGDKQIGRI       CCTTATTTGCTTGTCTCTTCACAACAG                               CCATTTTTGCGAAAGAGGACACGGCAG                               AAGATCCTGAGATCACACACAAGGTCT                               ACTTTGACATTAATCACGGTGATAAAC                               AAATTGGTAGAATT               19   YLR110c   CCW12   3.33   NQFSTVASIAAVAAVASAAANVTTAT   48   ATGCAATTTTCTACTGTCGCTTCTATC   47                       VSQESTTLVTITSCEDHVCSETVSPA       GCCGCTGTCGCCGCTGTCGCTTCTGCC                       LVSTATVTVDDVITQYTTWCPLTTEA       GCTGCTAACGTTACCACTGCTACTGTC                       PKNGTSTAAPVSTSTEPKNITS       AGCCAAGAATCTACCACTTTGGTCACC                               ATCACTTCTTGTGAAGACCACGTCTGT                               TCTGAAACTGTCTCCCCAGCTTTGGTT                               TCCACCGCTACCGTCACCGTCGATGAC                               GTTATCACTCAATACACCACCTGGTGC                               CCATTGACCACTGAAGCCCCAAAGAAC                               GGTACTTCTACTGCTGCTCCAGTTACC                               TCTACTGAAGCTCCAAAGAACACCACC                               TCT               20   YDR518w   EUG1   3.21   MQVTTRFISAIVSFCLFASFTLAENS   50   ATGCAAGTGACCACAAGATTTTATATC   49                       ARATPGSDLLVLTEKKFKSFIESH       TGCGATAGTCTCGTTTTGCCTGTTTGC                               TTCTTTCACGTTGGCTGAAAACAGCGC                               AAGAGCTACGCCGGGATCAGATTTACT                               CGTTCTAACAGAGAAGAAATTTAAATC                               ATTCATCGAATCTCAT               21   YEL040w   UTR2   3.16   NAIVNSNLICLVSIFSFVVRVEAATF   52   ATGGCAATCGTTAATAGTTGGCTAATT   51                       CNATQACPEDKPCCSQYGECGTGQYC       TGTTTAGTCAGTATTTTTTCCTTCGTG                       LNNCDVRYSFSHDSC       GTACGTGTAGAGGCCGCTACATTTTGC                               AATGCAACTCAAGCATGTCCCGAAGAT                               AAACCATGTTGCTCACAATATGGTGAA                               TGTGGTACTGGTCAATATTGTCTGAAC                               AACTGTGATGTAAGATATTCGTTTAGT                               CATGATTCATGT               22   YLR332w   MID2   3.00   MLSFTTKNSFRLLLLILSCISTIRAQ   54   ATGTTGTCTTTCACAACCAAGAATAGT   53                       FFVQSSSSNSSAVSTARSSVSRVSSS       TTCCGCTTATTACTTTTAATACTGTCA                       SSILSSSHVSSSSADSSSLTSSTSSR       TGCATATCGACGATACGCGCACAATTT                       SLVSHTSSSTSIASISFTSFSF       TTCGTGCAATCATCATCTTCGAATTCT                               TCAGCAGTATCTACTGCACGTTCTTCC                               GTAAGTAGAGTTAGTTCTTCAAGTTCC                               ATTTTGTCATCCAGTATGGTTTCTTCC                               TCAAGTGCTGACTCATCTTCCCTTACT                               TCATCGACATCAAGTAGGTCCCTCGTG                               TCACATACGAGTTCGTCTACCAGCATT                               GCCTCCATATCGTTCACATCATTCAGT                               TTC               23   YDR056c       2.97   MLVRLLRVILLASMVFCADILQLSYS   56   ATGCTTGTGCGGCTGTTGCGTGTGATT   55                       DDAKDAIPLGTFEIDSTSDGNVTVTT       TTATTGGCCAGCATGGTTTTCTGTGCT                       VNIQDVEVSGEYCLNAQIE       GATATTTTACAATTAAGCTATTCAGAT                               GATGCGAAAGACGCTATACCCCTAGGA                               ACATTTGAGATTGATAGTACATCCGAT                               GGGAATGTTACAGTAACAACCGTTAAT                               ATACAGGATGTTGAAGTTTCTGGAGAA                               TACTGTTTGAATGCCCAGATTGAA               24   YJL193w       2.83   NKQQLSASIRHNAHIIFLCISWYFIS   58   ATGTTTCAACAGCTGTCGGCATCCATT   57                       SLASQVTKQVLTVCPLPL       AGGCACAATGCACACATAATTTTTTTA                               TGCATAAGTTGGTATTTTATTTCATCG                               TTGGCATCTCAGGTAACGAAGCAGGTA                               CTAACGGTTTGCCCATTACCACTT               25   YHR139c   SPS100   2.76   MKFTSVLAFFLATLTASATPLYKRQN   8   ATGAAATTCACATCAGTGCTAGCATTT   7                       VTSGGGTVPIITGGPAVSGSQSNVTT       TTTCTTGCAACTTTAACAGCTTCTGCA                       TTLFNSTSTLNITQLYQIATQVNQTL       ACACCACTTTACAAGAGGCAGAACGTT                       QSESSS       ACTTCTGGCGGCGGTACGGTCCCCGTG                               ATCATCACGGGTGGACCTGCTGTATCT                               GGTAGCCAGTCAAACGTTACTACCACA                               ACGCTATTCAACTCTACTTCCACCTTA                               AACATCACTCAACTTTACCAAATTGCT                               ACTCAAGTTAATCAGACTTTACAAAGC                               GAATCGTCTTCC               26   YGR014w   MSB2   2.69   NQFPFACLLSTLVISGSLARASPFDF   10   ATGCAGTTTCCATTCGCTTGTCTCCTA   9                       IFGNGTQQAQSQSESQQQVSFTNEAS       TCGACCCTTGTAATTAGTGGGTCATTG                       QDSSTTSLVTAYSQGVHSHQSATIVS       GCCCGGGCCAGCCCCTTCGACTTTATA                       ATISSLPSTWYDASSTSQTSVS       TTCGGCAATGGAACGCAACAAGCTCAG                               AGCCAAAGCGAGAGTCAAGGTCAAGTT                               TCTTTCACCAATGAAGCTTCTCAGGAT                               AGTT               27   YPL234c   TFP3   2.59   MSTQLASNIYAPLYAPFFGFAGCAAA   60   ATGTCAACGCAACTCGCAAGTAACATA   59                       MVLSCLGAAIGTAKSGIGIAGIGTFK       TATGCTCCATTGTACGCTCCCTTTTTC                       PELIKKSLIP       GGGTTCGCAGGTTGTGCAGCTGCCATG                               GTGCTTTCCTGTTTGGGAGCTGCCATT                               GGTACAGCTAAGTCAGGTATTGGTATC                               GCCGGTATAGGTACTTTCAAGCCGGAA                               TTGATCATGAAGTCTTTGATTCCT               28   YDR057w   YOS9   2.56   HQAKIIYALSAISALIPLGSSLLAPI   62   ATGCAAGCTAAAATTATATATGCTCTG   61                       EDPIVSNKYISYIDEDDWSDRILQNQ       AGCGCAATTTCTGCGTTGATTCCGTTA                       SVMNSGY       GGATCATCACTATTAGCACCTATAGAA                               GACCCCATAGTATCGAATAAGTACCTC                               ATATCTTACATCGATGAGGACGACTGG                               AGTGATAGGATATTACAAAATCAGTCT                               GTCATGAACTCGGGATAT               29   YDR055w   PST1   2.54   MQLHSLIASTALLITSALAATSSSSS   64   ATGCAATTACATTCACTTATCGCTTCA   63                       IPSSCTISSHATATAQSDLDKVSRCD       ACTGCGCTCTTAATAACGTCAGCTTTG                       T       GCTGCTACTTCCTCTTCTTCCAGCATA                               CCCTCTTCCTGTACCATAAGCTCACAT                               GCCACGGCCACAGCTCAGAGTGACTTA                               GATAAATATAGCCGCTGTGATACG               30   YHR110w   ERP5   2.50   MKYNIVHGICLLFAITQAVGAVHFYA   66   ATGAAATATAATATAGTGCATGGAATT   65                       KSGETKCFYEHLSRGNLLIGDLDLYV       TGCCTATTATTTGCTATTACCCAAGCT                       EKDGLFEEDPESSLTITVDETFDNDH       GTAGGGGCTGTCCATTTTTATGCGAAG                       RVLNQKNSHTGDVTFTALDTGE       TCCGGGGAAACCAAATGCTTCTATGAA                               CACTTATFCCCGGGGAAACCTACTGAT                               TGGGGATTTAGACCTATATGTAGAAAA                               GGATGGTCTGTTTGAAGAGGACCCTGA                               ATCCAGTCTGACAATAACTGTCGATGA                               AACATTCGATAACGACCATCGTGTCCT                               AAATCAAAAAAACTCACACACAGGTGA                               TGTTACTTTTACAGCTTTAGACACAGG                               TGAA               31   YOL011w   PLB3   2.48   MIRPLCSKIIISYIFAISQFLLAANA   68   ATGATACGTCCATTATGTTCAAAAATT   67                       WSPTDSYVPGTVSCPDDINLVREATS       ATTATCAGTTACATATTCGCAATTTCT                       ISQNESAWLEKRNKVTSVALKDFLTR       CAGTTTCTACTGGCCGCTAATGCGTGG                       ATANFSDSSEVLSKLFNDGNSE       TCGCCCACAGATAGTTATGTTCCTGGC                               ACCGTGTCGTGTCCCGATGACATAAAT                               CTGGTAAGAGAGGCTACGTCTATATCT                               CAGAATGAGAGCGCATGGTTGGAAAAG                               AGGAATAAAGTCACTAGTGTAGCTTTA                               AAAGATTTCTTGACTAGGGCTACTGCA                               AATTTTTCAGATAGCTCAGAAGTTTTG                               TCGAAGCTATTTAATGATGGCAACAGC                               GAA               32   YDR134c       2.47   MQFSTVASIAAIAAVASAASNITTAT   70   ATGCAATTCTCTACCGTCGCTTCTATC   69                       VTEESTTLVTITSCEDHVCSETVS       GCTGCTATTGCCGCTGTTGCCTCCGCC                               GCTTCTAACATTACCACTGCTACTGTC                               ACAGAAGAATCTACCACTTTGGTCACT                               ATCACTTCTTGTGAGGACCACGTTTGT                               TCTGAAACAGTTTCC               33   YBR296c   PHO89   2.42   BALHQFDYIFAIAHLAFAFLDAFNIG   72   ATGGCTTTACATCAATTTGACTATATT   71                       ANDVANSFASSISSRS       TTTGCCATTGCCATGTTATTTGCATTT                               TTGGATGCCTTTAACATCGGGGCAAAC                               GACGTGGCGAACTCATTCGCGTCGTCG                               ATCTCTTCTAGATCT               34   YDR144c   MKC7   2.37   HKLSVLTFVVDALLVCSSIVDAGVTD   74   ATGAAATTATCTGTCCTCACATTTGTC   73                       FPSLPSNEVYVKNNFQKKYGSSFENA       GTAGATGCATTACTTGTCTGCAGCTCA                       LDDTKGRTRLHTRDDYELVELTNQNS       ATAGTAGATGCCGGTGTTACAGATTTT                       FYS       CCATCCTTACCAAGTAATGAAGTCTAT                               GTCAAAATGAATTTTCAGAAGAAATAC                               GGCAGTTCATTTGAAAACGCTTTGGAT                               GATACAAAAGGTAGAACGCGTTTGATG                               ACAAGAGATGATGATTACGAGCTGGTG                               GAACTGACTAATCAAAAACAGTTTTTA                               TTCG               35   YDR077w   SED1   2.37   HKLSTVLLSAGLASTTLAQFSNSTSA   76   ATGAAATTATCAACTGTCCTATTATCT   75                       SSTDVTSSSSISTSSGSVITSSEAPE       GCCGGTTTAGCCTCGACTACTTTGGCC                       SDNGTSTAAPTETSTE       CAATTTTCCAACAGTACATCTGCTTCT                               TCCACCGATGTCACTTCCTCCTCTTCC                               ATCTCCACTTCCTCTGGCTCAGTAACT                               ATCACATCTTCTGAAGCTCCAGAATCC                               GACAACGGTACCAGCACAGCTGCACCA                               ACTGAAACCTCAACAGAG               36   YDR276c   PMP3   2.35   MDSAKIINIILSLFLPPVAVFLARGN   78   ATGGATTCTGCCAAGATCATTAACATT   77                       GTDCIVDI       ATATTATCCCTTTTCTTACCACCAGTC                               GCCGTTTTTCTAGCCCGTGGGTGGGGT                               ACTGACTGTATAGTGGATATC                37   YNL291c   MID1   2.35   MIVWQALFVVYCLFTTSIHGLFQDFM   80   ATGATAGTGTGGCAAGCACTATTCGTG   79                       PFANKNISLKFPSLNRWEKNVMATGQ       GTTTACTGCCTATTTACCACTTCTATT                       QTIINSDSIYE       CACGGTTTATTCCAAGACTTCAATCCT                               TTCGCAAATAAGAATATTTCCTTAAAG                               TTTCCCAGCCTAAATAGATGGGAGAAA                               AACGTTATGGCTACTGGTCAACAAACA                               ATCATCAATTCGGATAGCATTTATGAA               38   YAL058w   CNE1   2.32   MKFSAYLWWLFLNLALVKGTSLLSNV   82   ATGAAATTTTCTGCGTATTTATGGTGG   81                       LAEDSFWEHFQAYTNTKHLNQEWITS       CTGTTTTTGAATCTAGCGTTGGTGAAA                       EAVNNEGSKIYGACWRLSQGRLQGSA       GGCACTTCATTGCTATCCAACGTTACA                       WDKGIAVRTGNAAAMIGHLLE       TTAGCGGAAGATTCTTTCTGGGAGCAT                               TTTCAGGCTTACACTAATACAAAGCAT                               TTAAACCAAGAGTGGATCACAAGTGAA                               GCCGTCAACAATGAAGGCTCTAAAATA                               TATGGTGCACAATGGCGACTATCACAG                               GGTCGATTGCAAGGATCCGCATGGGAT                               AAAGGAATCGCAGTTCGAACAGGCAAT                               GCCGCAGCTATGATAGGACATCTCTTG                               GAG               39   YLR300w   EXG1   2.31   MLSLKTLLCTLLTVSSVLATPVPARD   84   ATGCTTTCGCTTAAAACGTTACTGTGT   83                       PSSIQFVHEENKKRYYDYDHGSLGE       ACGTTGTTGACTGTGTCATCAGTACTC                               GCTACCCCAGTCCCTGCAAGAGACCCT                               TCTTCCATTCAATTTGTTCATGAGGAG                               AACAAGAAAAGATACTACGATTATGAC                               CACGGTTCCCTCGGAGAA               40   YIL140w   AXL2   2.30   MTQLQISLLLTATISLLHLVVATPYE   86   ATGACACAGCTTCAGATTTCATTATTG   85                       AYPIGKQYPPVARVNESFTFQISNDT       CTGACAGCTACTATATCACTACTCCAT                       YKSSVDXTAQITYNCFDLPSWLSFDS       CTAGTAGTGGCCACGCCCTATGAGGCA                       SSRTFSGEPSSDLLSDANTTLY       TATCCTATCGGAAAACAATACCCCCCA                               GTGGCAAGAGTCAATGAATCGTTTACA                               TTTCAAATTTCCAATGATACCTATAAA                               TCGTCTGTAGACAAGACAGCTCAAATA                               ACATACAATTGCTTCGACTTACCGAGC                               TGGCTTTCGTTTGACTCTAGTTCTAGA                               ACGTTCTCAGGTGAACCTTCTTCTGAC                               TTACTATCTGATGCGAACACCACGTTG                               TAT               41   YBR187w       2.26   MGNHIKKASLIALLPLFTAAAAAATD   4   ATGGGAAATATGATAAAGAAGGCATCT   3                       AETSMESGSSSHLKS       TTAATAGCCCTCCTACCGCTGTTCACC                               GCCGCAGCCGCTGCAGCTACTGATGCG                               GAGACATCTATGGAATCTGGGAGTTCT                               TCACATTTGAAGTCT               42   YBR070c   SAT2   2.19   MKTAYLASLVLIVSTAYVIRLIAILP   88   ATGAAAACGGCCTACTTGGCGTCATTG   87                       FFHTQAGTEKDTKDGVNLLKIRKSSK       GTGCTCATCGTACGACAGCATATGTTA                       K       TTAGGTTGATAGCGATTCTGCCTTTTT                               TCCACACTCAAGCAGGTACAGAAAAGG                               ATACGAAAGATGGAGTTAACCTACTGA                               AAATACGAAAATCGTCAAAGAAA               43   YJR118c   ILM1   2.16   MAQALNSTNIAFFRVAFLFTIAFFCL   90   ATGGCTCAAGCCTTGAACTCCACCAAT   89                       KNVNSILQNTYFIVLTQAMNLPQLTL       ATTGCTTTTTTCAGAGTAGCATTTTTA                       SR       TTCACGATCGCCTTCTTTTGTTTAAAG                               AACGTTAATTCTATTTTGCAAAATACA                               TATTTCATAGTCTTAACGCAAGCGATG                               AATTTACCGCAGTTAACACTGTCACGT               44   YOR190w   SPR1   2.14   MVSFRGLTTLTLLFTKLVNCNPVSTK   92   ATGGTTTCGTTCAGAGGGCTGACTACA   91                       NRDSIQFIYKEKDSIYSAINNQAINE       CTAACACTACTTTTTACCAAATTAGTA                       K       AACTGTAATCCTGTTTCCACAAAAAAT                               AGGGACTCTATACAGTTTATTTATAAA                               GAAAAGGATAGTATATACTCTGCCATC                               AACAATCAAGCCATCAATGAAAAA               45   YDR519w   FPR2   2.13   MHFNIYLFVTFFSTILAGSLSDLEGI   94   ATGATGTTTAATATTTACCTTTTCGTC   93                       IKRIPVEDCLIKANPGDKVKVHYTGS       ACTTTTTTTTCCACCATTCTTGCAGGT                       LLESGTVFDSSYSR       TCCCTGTCAGATTTGGAAATCGGTATT                               ATCAAGAGAATACCGGTAGAAGATTGC                               TTAATTAAGGCAATGCCAGGTGATAAA                               GTTAAGGTTCATTATACAGGATCTTTA                               TTAGAATCGGGAACTGTATTTGACTCA                               AGTTATTCAAGA               46   YIL090w       2.13   MTSLSKSFMQSGRICAACFYLLFTLL   96   ATGACCAGTTTGTCCAAAAGCTTCATG   95                       SIPISFKVGGLECG       CAGAGTGGACGAATCTGCGCAGCATGT                               TTCTATCTGTTATTCACACTACTTTCA                               ATTCCAATCTCGTTTAAAGTTGGTGGT                               TTGGAATGCGGG               47   YER113c       2.06   MRVRPKRSVITLMAIVVVKLILRNQF   98   ATGAGAGTAAGACCAAAGAGGTCGGTC   97                       YSSRTRGHGQPEVISSSQKNLYDGWI       ATAACACTCATGGCGATAGTAGTCGTG                       TPNFYRKQDPLELIVNKVESDLTQLP       ATGCTCATCCTCAGAAACCAGTTCTAC                       YAYYDLPFTCPPTHHKKPLHLS       TCATCACGAACGCGAGGGCATGGGCAG                               GAACCAGTCATCTCCTCCAGCCAAAAG                               AATCTTTATGACGGGTGGATAACACCC                               AATTTCTATAGAAAGGGTGATCCCTTG                               GAATTGATTGTGAATAAGGTAGAATCT                               GACTTAACGCAATTGCCATACGCATAT                               TATGACTTGCCCTTCACTTGTCCTCCT                               ACTATGCATAAAAAACGGCTTCATTTA                               TCT               48   YDR261c   EXG2   2.05   MPLKSFFFSAFLVLCLSKFTQGVGTT   100   ATGCCTTTGAAGTCGTTTTTTTTTTCA   99                       EKEESLSPLENILQNKFASYYANDTI       GCATTTCTAGTTTTATGCCTGTCTAAA                       TVKGITIGGWLVTEPYITPSLYRNAT       TTCACGCAAGGCGTTGGCACCACAGAG                       SLAKQQNSSSNISIVDEFTLC       AAGGAAGAATCGTTATCGCCTTTGGAA                               CTAAATATTTTACAAAACAAATTCGCC                               TCCTACTATGCAAACGACACTATCACC                               GTGAAAGGTATTACTATTGGCGGCTGG                               CTAGTAACAGAACCTTATATCACGCCA                               TCATTATATCGTAATGCTACGTCACTG                               GCAAAACAGCAAAACTCTTCCAGCAAT                               ATCTCCATTGTCGACGAATTTACTCTT                               TGT               49   YDR420w   HKR1   2.04   MVSLKIKKILLLVSLLNAIEAYSNDT   2   ATGGTCTCATTGAAAATAAAAAAAATT   1                       IYSTSYNNGIESTPSYSTSAISSTGS       TTACTCCTGGTGTCATTGTTAAATGCA                       SNKENAITSSSETTTMAGQYGESGST       ATCGAGGCCTATAGTAACGTACAATAT                       TIMDEQETGTSSQYISVTTTTQ       ATTCAACTTCATACAATAATGGAATAG                               AAAGCACACCCTCATATTCAACATCCG                               CGATATCCAGTACCGGATCTAGCAACA                               AAGAGAATGCAATAACATCAAGCTCTG                               AAACCACCACAATGGCTGGCCAATATG                               GTGAAAGTGGAAGCACAACAATAATGG                               ATGAACAAGAAACTGGTACGTCCAGCC                               AGTATATTAGTGTGACGACGACAACGC                               AA               50   YFL051c       2.03   MSIPHSVFSALLFVALATTTLASTEA   102   ATGTCTATACCCCATTCCGTATTTTCG   101                       CLPTNKREDGNNINFYEYTIGDQTTY       GCACTCTTGGTCTTCGTGGCGCTAGCT                       LEPEYQGYEYSNTKK       ACTACAACCTTAGCCAGTACAGAAGCT                               TGCTTACCAACAAACAAAAGGGAAGAT                               GGTATGAATATTAATTTTTATGAGTAC                               ACAATAGGCGACCAAACCACATACTTG                               GAGCCTGAATATATGGGCTATGAATAC                               TCCAATACAAAGAAG               51   YNL300w   TOS6   2.02   MKFSTLSTVAAIAAFASADSTSDGVT   14   ATGAAATTCTCTACTCTCTCCACCGTT   13                       YVDVTTTPQSTTSMVSTVKTTSTPYT       GCTGCCATTGCCGCATTTGCTTCCGCA                       TSTIATLSTKSISSQANTTTHEIST       GATTCCACCTCTGATGGTGTCACTTAC                               GTAGATGTTACCACCACCCCACAAAGT                               ACTACATCTATGGTCTCCACCGTGAAA                               ACTACTTCCACTCCATACACTACAAGT                               ACCATTGCCACTCTATCCACTAAATCT                               ATCAGTAGCCAAGCTAACACCACCACC                               CATGAGATCAGCACA                  
 
      Table 2 shows the systematic and common names of genes from which the gene regions encoding secretory signal peptides are derived, relative secretion efficiency in relation to α-factor-derived secretory signal peptides, the amino acid sequences of the secretory signal peptides, and the nucleotide sequences of the gene regions encoding secretory signal peptides, concerning secretory signal peptides having secretion ability that is more than twice that of α-factor-derived secretory signal peptide at low temperature (15° C.). SEQ ID NOs: of the amino acid sequences and the nucleotide sequences are indicated in the rightmost column. The systematic and common names of genes (or gene regions encoding secretory signal peptides) identical to genes (or gene regions encoding secretory signal peptides) shown in Table 1 are shaded.  
      Hereafter, a secretory signal peptide (or a gene region encoding a secretory signal peptide) shown in Table 1 is referred to as a “secretory signal peptide at moderate temperature (or a gene region encoding a secretory signal peptide at moderate temperature),” and a secretory signal peptide (or a gene region encoding a secretory signal peptide) shown in Table 2 is referred to as a “secretory signal peptide at low temperature (or a gene region encoding a secretory signal peptide at low temperature).” Also, a “secretory signal peptide at moderate temperature (or a gene region encoding a secretory signal peptide at moderate temperature)” and a secretory signal peptide at low temperature (or a gene region encoding a secretory signal peptide at low temperature) are collectively referred to as “secretory signal peptides (or gene regions encoding secretory signal peptides).” 
      DNA according to the present invention encodes a secretory signal peptide consisting of the amino acid sequence of a secretory signal peptide shown in Table 1 or 2. DNA according to the present invention can be easily obtained by preparing a primer based on the amino acid sequence of a secretory signal peptide shown in Table 1 or 2 and conducting PCR using the genomic DNA extracted from  Saccharomyces cerevisiae  as a template.  
      DNA according to the present invention may be DNA encoding a secretory signal peptide consisting of an amino acid sequence derived from the amino acid sequence of a secretory signal peptide shown in Table 1 or 2 by deletion, substitution, or addition of one or several amino acids (e.g., 1 to 10 or 1 to 5 amino acids), and having secretory signal activity at 30° C. regarding the amino acid sequence derived from the amino acid sequence of the secretory signal peptide at moderate temperature shown in Table 1, or having secretory signal activity at 15° C. regarding the amino acid sequence derived from the amino acid sequence of the secretory signal peptide at low temperature shown in Table 2.  
      The term “secretory signal activity” used herein refers to the ability for extracellular protein secretion of secretory signal peptides expressed as fusion proteins with proteins, such as membrane proteins and secretory proteins, or the ability for transporting proteins to intracellular organelles, including cell walls, cell membranes, endoplasmic reticulums, and Golgi bodies. Secretory signal activity of the secretory signal peptide encoded by DNA according to the present invention can be evaluated by, for example, utilizing a reporter gene, such as the luciferase gene. For example, an expression vector, wherein a reporter gene is ligated, as a fusion protein, to a site downstream of DNA according to the present invention, is constructed. Subsequently, an adequate host (e.g., yeast) is transformed using the resulting expression vector. The resulting transformant is cultured at 30° C. or 15° C., the amount of the reporter protein secreted extracellularly or the amount thereof transported to intracellular organelles, including cell walls, cell membranes, endoplasmic reticulums, and Golgi bodies, is quantified to result in the activity level of the reporter protein or to result in the protein level via an immunological method (e.g., Western blotting, ELISA, or flow cytometry). Thus, secretory signal activity of the secretory signal peptide encoded by DNA according to the present invention can be evaluated. Alternatively, the ability for transportation to the intracellular organelles as secretory signal activity can be evaluated by inspecting the localization of the fusion protein encoded by a foreign gene ligated downstream of DNA according to the present invention in intracellular organelles via immunostaining using an antibody or other means.  
      Further, DNA according to the present invention may be DNA consisting of a nucleotide sequence of a gene region encoding a secretory signal peptide shown in Table 1 or 2, or DNA cosisting of a nucleotide sequence derived from the nucleotide sequence of a gene region encoding a secretory signal peptide shown in Table 1 or 2 by deletion, substitution, or addition of 1 or several (e.g., 1 to 10 or 1 to 5) nucleotides and encoding a secretory signal peptide having secretory signal activity at 30° C. regarding the nucleotide sequence derived from the nucleotide sequence of the gene region encoding a secretory signal peptide at moderate temperature shown in Table 1, or encoding a secretory signal peptide having secretory signal activity at 15° C. regarding the nucleotide sequence derived from the nucleotide sequence of the gene region encoding a secretory signal peptide at low temperature shown in Table 2. Accordingly, DNA according to the present invention may be the full-length DNA consisting of the nucleotide sequence of the gene region encoding a secretory signal peptide shown in Table 1 or 2. As long as the amino acid sequence encoded by DNA according to the present invention exhibits secretion signal activity at 30° C., such DNA may be part of the DNA consisting of the nucleotide sequence of the gene region encoding a secretory signal peptide at moderate temperature shown in Table 1. Alternatively, such DNA may be part of the DNA consisting of the nucleotide sequence of the gene region encoding a secretory signal peptide at low temperature shown in Table 2, provided that the amino acid sequence encoded by such DNA exhibits secretory signal activity at 15° C.  
      DNA according to the present invention includes a DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to DNA consisting of a nucleotide sequence of a gene region encoding a secretory signal peptide shown in Table 1 or 2 and that encodes a secretory signal peptide exhibiting secretory signal activity at 30° C. regarding a gene region encoding a secretory signal peptide at moderate temperature shown in Table 1 or at 15° C. regarding a gene region encoding a secretory signal peptide at low temperature shown in Table 2.  
      Under stringent conditions, for example, hybridization with phosphorus-32-labeled probe DNA is carried out in a hybridization solution comprising 5×SSC (0.75 M NaCl, 0.75 M sodium citrate), 5× Denhart&#39;s reagent (0.1% ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin), and 0.1% sodium dodecyl sulfate (SDS) at 45° C. to 65° C., and preferably at 55° C. to 65° C. The step of washing is carried out in a washing solution comprising 2×SSC and 0.1% SDS at 45° C. to 55° C., and preferably in a washing solution comprising 0.1×SSC and 0.1% SDS at 45° C. to 55° C. When probe DNA labeled with an enzyme using the AlkPhos direct labeling module kit (Amersham Bioscience) is used, hybridization is carried out in a hybridization solution (containing 0.5 M NaCl and a 4% blocking reagent) having the composition described in the manufacturer&#39;s instructions of the kit at 55° C. to 75° C. The step of washing is carried out in a primary washing solution (containing 2M urea) described in the manufacturer&#39;s instructions of the kit at 55° C. to 75° C. and in a secondary washing solution at room temperature. Another detection method may be employed. In such a case, such detection method may be carried out under its standard conditions.  
      Once the nucleotide sequence of DNA according to the present invention has been determined, DNA of the present invention can be obtained by chemical synthesis, by PCR using the cloned probe as a template, or by hybridization using a DNA fragment having the aforementioned nucleotide sequence as a probe. Further, DNA that is a mutant of the DNA of the present invention and has functions equivalent to those before mutation can be synthesized via, for example, site-directed mutagenesis.  
      Mutation can be introduced into DNA of the present invention by conventional techniques, such as the Kunkel method, the Gapped duplex method, or a method in accordance therewith. For example, a mutagenesis kit (e.g., Mutant-K that utilizes site-directed mutagenesis, TaKaRa) or the LA PCR in vitro Mutagenesis Series kit (TaKaRa) may be used to introduce mutation. Other mutagenesis techniques may also be employed.  
      The secretory signal peptide of the present invention is encoded by DNA of the present invention. The secretory signal peptide of the present invention can be obtained as a fusion protein with a protein to be expressed (e.g., a membrane or secretory protein).  
      An expression vector comprising DNA of the present invention and a foreign gene (hereafter referred to as the “expression vector of the present invention”) can be obtained by inserting DNA of the present invention and a foreign gene into an adequate vector. The foreign gene may be located downstream or upstream of DNA of the present invention in the expression vector of the present invention. Alternatively, DNA of the present invention may be present in the foreign gene. Preferably, the foreign gene is located adjacent to a site downstream of DNA of the present invention. DNA of the present invention is inserted into the expression vector of the present invention in-frame with the foreign gene.  
      Any vector may be used without particular limitation, provided that the vector can be replicated in a host. Examples thereof include a plasmid, a shuttle vector, and a helper plasmid. When the vector is incapable of replication, a DNA fragment that becomes replicable upon insertion thereof into a host chromosome may be used.  
      Examples of plasmid DNAs include  E. coli -derived plasmid DNAs (e.g., pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, and pBluescript),  Bacillus subtilis -derived plasmid DNAs (e.g., pUB110 and pTP5), and yeast-derived plasmid DNAs (e.g., a YEp plasmid such as YEp13 and a YCp plasmid such as YCp50). Examples of phage DNAs include λ phages, such as Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, and λZAP. Animal virus vectors, such as retrovirus or vaccinia virus vectors, and insect virus vectors, such as baculovirus vectors, can also be used.  
      DNA of the present invention may be inserted into a vector by first cleaving the purified DNA with an adequate restriction enzyme, inserting the cleavage product into a restriction site or multicloning site of an adequate vector DNA, and ligating the product to a vector. With the provision of a homologous region at a part of the vector and at a part of DNA of the present invention, the vector may be ligated to DNA of the present invention via, for example, the in vitro method involving PCR or the in vivo method using yeast.  
      A foreign gene may be inserted into a vector at a site downstream or upstream of DNA of the present invention, or a foreign gene may be inserted into a vector so as to locate DNA of the present invention within the foreign gene in the same manner as with the insertion of DNA of the present invention into the vector.  
      In the expression vector according to the present invention, a foreign gene located downstream or upstream of DNA of the present invention or a foreign gene containing DNA of the present invention therein may be any protein or peptide. Examples thereof include a membrane protein and a secretory protein.  
      Further, a transformant can be obtained by introducing the expression vector of the present invention into a host. Any host can be used without particular limitation, provided that DNA of the present invention and a foreign gene can be expressed therein. An example thereof is yeast. Examples of yeast include  Saccharomyces cerevisiae,  experimental yeast, fermentation yeast, edible yeast, and industrial yeast.  
      The expression vector of the present invention may be introduced into yeast via any method without particular limitation, provided that DNA is introduced into yeast by such method. Examples thereof include electroporation, spheroplast, and lithium acetate methods. Also, yeast transformation involving substitution and/or insertion with a chromosome may be carried out using vectors such as a YIp vector or a DNA sequence homologous to an arbitrary region in the chromosome. Further, the expression vector of the present invention may be introduced into yeast by any method described in general experiment guidebooks or academic articles.  
      A transformant can also be obtained not only by introducing the expression vector of the present invention into the aforementioned yeast but also by introducing the expression vector into bacteria of  Escherichia  such as  Escherichia coli, Bacillus  such as  Bacillus subtilis,  or  Pseudomonas  such as  Pseudomonas putida,  animal cells such as COS cells, insect cells such as Sf9 cells, or plants belonging to genera such as  Brassicaceae.  When a bacterial host is used, it is preferable that the expression vector of the present invention may be capable of autonomous replication therein and that the vector may be composed of a promoter, a ribosome binding site, DNA of the present invention, a foreign gene, and a transcription termination sequence.  
      The expression vector of the present invention may be introduced into bacteria by any method without particular limitation, provided that DNA is to be introduced into bacteria by such method. Examples thereof include a method involving the use of calcium ions and electroporation.  
      When an animal cell host is used, for example, monkey cells (COS-7 cells or Vero cells), Chinese hamster ovarian cells (CHO cells), or mouse L cells may be used. The expression vector of the present invention may be introduced into animal cells by, for example, electroporation, the calcium phosphate method, or lipofection.  
      When an insect cell host is used, for example, Sf9 cells or the like may be used. The expression vector of the present invention may be introduced into insect cells by, for example, the calcium phosphate method, lipofection, or electroporation.  
      When a plant host is used, for example, the entire plant, a plant organ (e.g., a leaf, petal, stem, root, or seed), plant tissue (e.g., epidermis, phloem, parenchyma, xylem, or fibrovascular bundle), or cultured plant cells may be used. The expression vector of the present invention may be introduced into a plant host by, for example, electroporation, the agrobacterium method, the particle gun method, or PEG.  
      Whether or not DNA of the present invention and a foreign gene were incorporated into a host can be examined by, for example, PCR, Southern hybridization, or Northern hybridization. For example, DNA is prepared from a transformant, a DNA-specific primer is designed, and PCR is then carried out. Thereafter, the amplification product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, or capillary electrophoresis, the resultant is stained with ethidium bromide or an SYBR Green solution, and the amplification product is detected as a band to confirm the transformation. Also, PCR may be carried out using a primer labeled in advance with a fluorescent dye or the like and the amplification product may be detected. Further, the amplification product may be bound to a solid phase support, such as a microplate, to confirm the amplification product via, for example, fluorescence or enzyme reactions.  
      In the method for producing proteins according to the present invention, the aforementioned transformant obtained with the use of the expression vector of the present invention (hereafter referred to as the “transformant of the present invention”) is cultured, and a fusion protein of the secretory signal peptide encoded by DNA according to the present invention and a protein encoded by a foreign gene is secreted and produced extracellularly or transported to and expressed in the intracellular organelles, including cell walls, cell membranes, endoplasmic reticulums, and Golgi bodies. When DNA of the present invention introduced into the transformant of the present invention is related to the gene region encoding a secretory signal peptide at moderate temperature, the transformant of the present invention is cultured at 20° C. to 42° C., and preferably at 25° C. to 37° C. When DNA of the present invention introduced into the transformant of the present invention is related to the gene region encoding a secretory signal peptide at low temperature, the transformant of the present invention is cultured at 0° C. to 20° C., and preferably at 4° C. to 15° C. Other culture conditions (e.g., the composition of the medium) for the transformant of the present invention can be adequately selected in accordance with the type of host to be used.  
      After the transformant of the present invention has been cultured, a protein encoded by a foreign gene can be isolated, extracted, and/or purified from the culture supernatant or culture product, as a fusion protein of the secretory signal peptide of the present invention with a protein encoded by a foreign gene. Alternatively, the secretory signal peptide of the present invention may be cleaved by, for example, signal peptidase in the transformant, and a protein encoded by a foreign gene can be selectively isolated, extracted, and/or purified from the culture supernatant or culture product. Proteins can be isolated, extracted, and purified in accordance with relevant general techniques.  
      According to the present invention, membrane proteins and secretory proteins can be expressed with higher efficiency than is possible with conventional membrane and secretory protein expression systems in yeast or the like with the use of secretory signal peptides. The expression system according to the present invention is superior to conventional membrane and secretory protein expression systems in terms of secretion efficiency. For example, the amount of protein production can be further increased in conventional expression of membrane proteins and secretory proteins in yeast.  
     EXAMPLES  
      Hereafter, the present invention is described in greater detail with reference to the examples, although the technical scope of the present invention is not limited to these examples.  
      Concerning secretory signal peptides that are present in many of the genes encoding membrane proteins and secretory proteins in the  Saccharomyces cerevisiae  genome, the secretion abilities thereof were evaluated via the reporter assay system (see International Application Number: PCT/JP2006/311597, claiming the priority right of JP Patent Application No. 2005-169768) involving the use of secreted  Cypridina noctiluca  luciferase (hereafter referred to as “CLuc;” nucleotide sequence: SEQ ID NO: 103; amino acid sequence: SEQ ID NO: 104) as a reporter protein. CLuc is used as a mature protein lacking a naturally occurring secretory signal peptide (hereafter referred to as “mature CLuc”).  
      The secretory signal peptides exhibiting higher secretion efficiency than the secretory signal peptides used in conventional expression systems in yeast (see Non-Patent Documents 1 to 5) were identified in the following manner.  
     Example 1  
     Production of Reporter Vector pCLuRA-s  
      The reporter vector, pCLuRA-s, was produced utilizing a gene encoding mature CLuc as a reporter gene in the following manner.  
      The pUG35-MET25-EGFP3+MCS plasmid (see International Application Number: PCT/JP2006/311597, claiming the priority right of JP Patent Application No. 2005-169768) produced from pUG35 (http://mips.gsf.de/proj/yeast/info/tools/hegemann/gfp.html) was cleaved with HindIII and XbaI, the DNA fragment was fractionated via agarose gel electrophoresis, and a vector fragment of approximately 5.1 kbp was obtained. This vector fragment is hereafter referred to as “DNA fragment A.” 
      The pCLuRA plasmid comprises a gene having, at the 5′ end of DNA encoding mature CLue, DNA encoding the α-factor-derived secretory signal peptide ligated thereto (see International Application Number: PCT/JP2006/311597, claiming the priority right of JP Patent Application No. 2005-169768). A gene encoding mature CLuc (i.e., a protein consisting of an amino acid sequence derived from the amino acid sequence of CLuc as shown in SEQ ID NO: 104 by deletion of a secretory signal peptide sequence consisting of amino acids 1 to 18) (hereafter referred to as the “mature CLuc gene”) was amplified from the pCLuRA plasmid via PCR.  
      The following primers were used. cLuc ORF−Sig F+HindIII comprises at its 5′ end the HindIII cleavage site, and downstream thereof, a sequence complementary to a 21-bp region encompassing the 5′ end of the mature CLuc gene. cLuc ORF−Sig R+XbaI comprises at its 5′ end the XbaI cleavage site, and downstream thereof, a sequence complementary to a 24-bp-sequence including a termination codon of the mature CLuc gene.  
      cLuc ORF−Sig F+HindIII: GCGC-AAGCTT-CAGGACTGTCCTTACGAACCT (SEQ ID NO: 105)  
      cLuc ORF−Sig R+XbaI: GCGC-TCTAGA-CTATTTGCATTCATCTGGTACTTC (SEQ ID NO: 106)  
      PCR was carried out using 50 μl of a reaction solution comprising 300 nM of each primer, 1 mM MgSO 4 , 200 μM dNTP (a mixed solution of 4 types of deoxynucleotide triphosphates), 1 ng of the pCLuRA plasmid, and 1× buffer and KOD-Plus-1U included in the KOD-Plus-(TOYOBO). The PCR cycle comprised: the first step at 94° C. for 2 minutes; 35 cycles of the second step at 94° C. for 15 seconds (denaturation), at 50° C. for 30 seconds (annealing), and at 68° C. for 2 minutes (elongation); and the third step at 68° C. for 5 minutes.  
      The amplified DNA fragment was cleaved at the restriction enzyme cleavage sites at both of its ends with HindIII and XbaI, a DNA fragment was fractionated via agarose gel electrophoresis, and a DNA fragment containing approximately 1.6 kbp of the mature CLuc gene was obtained. This DNA fragment is hereafter referred to as “DNA fragment B.” 
      Subsequently, DNA fragments A were ligated to DNA fragments B using the DNA Ligation Kit ver. 2.1 (TaKaRa), and the ligation products were then introduced into  E. coli  DH5α. The resulting transformants were cultured overnight, plasmids were extracted using the GenElute Plasmid Miniprep Kit (Sigma-Aldrich), and transformants having a plasmid of interest were identified based on the restriction enzyme cleavage patterns and via nucleotide sequence analysis. A plasmid was prepared from the transformant, the plasmid was cleaved with SpeI and BamHI, a DNA fragment was fractionated via agarose gel electrophoresis, and a DNA fragment of approximately 6.7 kbp was obtained. This DNA fragment is hereafter referred to as “DNA fragment C.” 
      A low-temperature-inducible promoter, i.e., an HSP12 gene promoter (hereafter referred to as “HSP12 promoter;” SEQ ID NO: 107), was amplified via PCR from the pLTex321s vector (see Patent Document 2).  
      The following primers were used. −610-HSP12 IGR F+SpeI comprises at its 5′ end a SpeI cleavage site, and downstream thereof, a sequence complementary to a 19-bp region encompassing the 5′ end of the HSP12 promoter region. −610-HSP12 IGR R+BamHI comprises at its 5′ end a BamHI cleavage site, and downstream thereof, a sequence complementary to a 28-bp region encompassing the 3′ end of the HSP12 promoter region.  
      −610-HSP12 IGR F+SpeI: GG-ACTAGT-GATCCCACTAACGGCCCAG (SEQ ID NO: 108)  
      −610-HSP12 IGR R+BamHI: CG-GGATCC-TGTTGTATTTAGTTTTTTTTGTTTTGAG (SEQ ID NO: 109)  
      PCR was carried out using 50 μl of a reaction solution comprising 300 nM of each primer, 1 mM MgSO 4 , 200 μM dNTP, 1 ng of the pLTex321s vector, and 1× buffer and KOD-Plus-1U included in the KOD-Plus-. The PCR cycle comprised: the first step at 94° C. for 2 minutes; 35 cycles of the second step at 94° C. for 15 seconds (denaturation), at 50° C. for 30 seconds (annealing), and at 68° C. for 1 minute (elongation); and the third step at 68° C. for 5 minutes.  
      The amplified DNA fragment was cleaved at restriction enzyme cleavage sites at both ends thereof with SpeI and BamHI, a DNA fragment was fractionated via agarose gel electrophoresis, and a fragment of approximately 600 bp of the HSP12 promoter region was obtained. This fragment of the HSP12 promoter region is hereafter referred to as “DNA fragment D.” 
      DNA fragments C were ligated to DNA fragments D using the DNA Ligation Kit ver. 2.1, and the ligation products were introduced into  E. coli  DH5α. The resulting transformants were cultured overnight, plasmids were extracted using the GenElute Plasmid Miniprep Kit, and a transformant having a plasmid of interest was identified based on the restriction enzyme cleavage pattern and via nucleotide sequence analysis. The pCLuRA-s plasmid was prepared from the transformant. In the pCLuRA-s plasmid, the mature CLuc gene is inserted downstream of the HSP12 promoter.  
     Example 2  
     Isolation of  Saccharomyces cerevisiae -derived Secretory Signal Peptide  
      The secretory signal peptides existing in membrane proteins and secretory proteins derived from the budding yeast  Saccharomyces cerevisiae  were extracted in the following manner.  
      Thousand and thirty seven genes included in the categories of the plasma membrane, the integral membrane, the cell periphery, the cell wall, the extracellular, the endoplasmic reticulum (ER), and Golgi from the subcellular localization table of MIPS CYGD (http://mips.gsf.de/genre/proj/yeast/), which is the  Saccharomyces cerevisiae  genomic database, were selected.  
      Based on the amino acid sequences encoded by the nucleotide sequences of the genes selected from the database, the transmembrane sites and the secretory signal peptides were predicted using the prediction programs for transmembrane sites and the prediction programs for the secretory signal peptides, such as TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM/), Phobius (http://phobius.cgb.ki.se/), SOSUI signal Beta (http://sosui.proteome.bio.tuat.ac.jp/˜sosui/proteome/sosuisignal/sosuisignal_submit.html), and SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/). Based on the results of this analysis, the gene regions encoding the predicted secretory signal peptides were extracted from the aforementioned database via several prediction programs, concerning genes encoding 440 types of proteins, for which the secretory signal peptides had been predicted (Table 3).  
      Table 3 below shows: the systematic and common names of the genes encoding 440 types of proteins relating to the secretory signal peptides extracted from the budding yeast genome information; the nucleotide sequences and the amino acid sequences of the gene regions encoding the predicted secretory signal peptide of the protein-encoding genes (all SEQ ID NOs of these nucleotide sequences and the amino acid sequences are indicated in the rightmost column in Table 3); and the primers used for amplifying the gene regions encoding secretory signal peptides (nucleotide sequences of the synthetic primers used for the amplifying the 1st PCR products; all SEQ ID NOs of such primers are indicated in the rightmost column in Table 3).  
                   TABLE 3                          Amino acid sequences of secretory signal peptides extracted from the budding yeast genome information,           nucleotide sequences encoding the same, and synthetic primers                                                                 Systematic   Common       SEQ       SEQ   Synthetic primer   SEQ   Synthetic primer   SEQ               gene name   gene name   Amino acid sequence   ID NO:   Nucleotide sequence   ID NO:   (Forward)   ID NO:   (Reverse)   ID NO:                                                                         1   YAL022c   FUN26   KSTSADTDTIKKPILAVPEPALAD   111   ATGAGTACTAGTGCGGACACTGATACCATCAAGAAGCCAAT   110   aaatacaacaATGAGT   888   gacagtcctgATTTTT   889                       THSEEISRSGEENHESENKEHSDE       CCTTGCGGTGCCAGAGCCTGCACTGGCCCATACGCATTCAG       ACTAGTGCGGACA       CAACTTTTTTCTTA                   EGDHYSEREQSVSTEPLDTLPLKK       AGGAGATATCACGCTCTGGAGAAGAACATGAATCAGAGAAG                   KLKR       AACGAGCACTCAGATGAAGAAGGCGATAATTATTCTGAAAG                           AGAGCAATCTGTGTCAACCGAACCACTGGATACATTGCCGT                           TAAGAAAAAAGTTGAAAAAT               2   YBL042c   FUI1   KPVSDSGFDNSSKTMKDDTIPTED   113   ATGCCGGTATCTGATTCTGGATTCGATAATTCTTCAAAAAC   112   aaatacaacaATGCCG   890   gacagtcctgAAATGT   891                   YEEITKESEXGDAATKITSKIDAN       AATGAAGGACGACACTATACCGACGGAGGATTACGAAGAAA       GTATCTGATTCTG       CCTAATTGGATTTT                   VIEKKDTDSENNITIAQDDEKVSV       TCACGAAAGAGTCCGAGATGGGTGATGCGACTAAAATTACT                   LQRVVEFFEVKWDSTQLADHXPER       TCTAAGATTGATGCTAATGTCATTGAAAAAAAAGATACCGA                   PIRTF       TTCAGAAAACAATATAACCATTGCTCAGGATGACGAAAAGG                           TGTCTTGGCTGCAAAGAGTTGTCGAGTTTTTTGAGGTTAAG                           AATGACTCTACCGATTTGGCCGATCATAAGCCCGAAAATCC                           AATTAGGACATTT               3   YBR021w   FUR4   MPDWLSLHLSGSSKRLHSRQLMES   115   ATGCCAGACAATCTATCATTACATTTAAGCGGCTCTTCAAA   114   aaatacaacaATGCCA   892   gaccgtcctgTTTCTT   893                   SNETFAPNQVDLEKEYKSSQSNIT       AAGATTGAACTCTCGCCAACTTATGGAATCTTCCAATGAGA       GACAATCTATCATT       CCAGAATGAGCTGT                   TEVYEASSFEEKVSSEKPQYSSFH       CCTTTGCGCCAAATAATGTGGATTTGGAAAAAGAGTATAAG                   KK       TCATCTCAGAGTAATATAACTACCGAAGTTTATGAGGCATC                           GAGCTTTGAAGAAAAAGTAAGCTCAGAAAAACCTCAATACA                           GCTCATTCTGGAAGAAA               4   YBR023c   CHS3   MTGLKGDDPDDYYLAHLNQDEESL   117   ATGACCGGCTTGAATGGAGATGATCCTGATGACTACTATCT   116   aaatacaacaATGACC   894   gacagtcctgAAATTT   895                   LRSRHSVGSGAPHRQGSLVRPERS       GAACCTTAATCAAGATGAAGAGTCTCTACTTAGGTCAAGAC       GGCTTGAATGGA       GCTTCTCACTGAGC                   RLNHPDNPHFYYAQKTQEQMHHLD       ACAGTGTCGGCTCAGGAGCACCTCATAGACAAGGCTCTTTA                   VLPSSTGGVKPHATRRSGSLRSKG       GTGCGGCCCGAAAGAAGCCGACTGAACAATCCTGATAATCC                   SVRSKF       ACATTTTTATTATGCGCAGAAAACGCAGGAGCAGATGAATC                           ACCTGGATGTTTTACCATCAAGTACCGGTGTAAACCCAAAT                           GCAACTCGTCGGAGTGGCTCCCTGCGGTCCAAAGGCTCAGT                           GAGAAGCAAATTT               5   YBR038w   CHS2   MTRNPFMVEPSNGSPHRRGASNLS   119   ATGACGAGAAACCCGTTTATGGTGGAACCTTCGAATGGCTC   118   aatacaacaATGACGA   896   gacagtcctgAGACTC   897                   KFYARANSNSRVANPSEESLEDSY       TCCTAATAGACGTGGTGCTTCAAACCTCTCCAAATTTTACG       GAAACCCGTTT       TTGTAGATTAGCAG                   DGSNVFQGLPASPSRAALRVSPDR       CAAACGCTAACAGCAACTCTCGGTGGGCTAATCCCAGTGAG                   HHRTQFYRDSAHNSPVAPNRYAAN       GAGAGTTTGAGGAGGATAGCTATGACCAATCTAACGTTTTC                   LQES       CAAGGCCTTCGGGCATCTCCTTCGAGAGCTGCACTAAGATA                           CTCCCCAGACCGTCGCCATAGAACTCAATTTTACCGCGATA                           GTGCCCATAACTCTCCAGTTGCTCCGAACAGGTATGCTGCT                           AATCTACAAGAGTCT               6   YBR041w   FAT1   MSPIQVVFALSRIFLLLFRLIKLI   121   ATGTCTCCCATACAGGTTGTTGTCTTTGCCTTGTCAAGGAT   120   acatacaacaATGTCT   898   gacagtcctgATCCTC   899                   ITPIQKSLFYLFGNTFDELDRKYR       TTTCCTGCTATTATTCAGACTTATCAAGCTAATTATAACCC       CCCATACAGGTTGT       CTTGTATCTATATT                   YKED       CTATCCAGAAATCACTGGGTTATCTATTTGGTAATTATTTT                           GATGAATTAGACCGTAAATATAGATACAAGGAGGAT               7   YBR068c   BAP2   MLSSEDFGSSGKKETSPDSISIRS   123   ATGCTATCTTCAGAAGATTTTGGATCTTCTGGGAAAAAGGA   122   aaatacaacaATGCTA   900   gccagtcctgCGACTTC   901                   FSAGNKFQSSSSEKTYSKQXSGSD       AACTTCTCCTGATTCGATATCGATACGTTCCTTTAGGGCCG       TCTTCAGAAGATTT       ATGGACTTTTTCA                   KLIHRFADSFKRAESGSTTRTKQI       GGAATAATTTCCAATCATCATCAAGTGAGAAAACTTATTCT                   NENTSDLEDGVESITSDSKLKKSH       AAGCAAAAATCCGGGAGTGACAAACTTATACATAGATTTGC                   KS       GGATTCATTCAAAAGAGCCGAGGGTAGCAGTACAAGAACTA                           AGCAAATAAATGAAATACGTCTGATTTAGAGGATGGCGTTG                           AGTCTATCACGTCGGATTCCAAGTTGAAAAAGTCCATGAAG                           TCG               8   YBR069c   TAT1   MDDSVSFIAKEASPADYSHSLHER   125   ATGGACGATAGTGTCAGTTTCATTGCCAAAGAGGCCAGTCC   124   aaatacaacaATGGAC   902   gcccgtcctgAGATTT   903                   THSEKQXRDFTITEKQDEVSGQTA       AGCACAATATTCGCACAGTTTGCATGAAAGAACACACAGTG       GATAGTGTCAGTTT       GATCGACTTTGTCA                   EPRRTDSKSILQRKCKEFFDSFKR       AAAAACAAAAAGAGAGACTTTACAATAACAGAAAAACAAGA                   QLPPDRNSELESQEKKCHLTKSIK       TGAGGTATCTGGACAAACAGCGGAGCCTCGAAGGACGGACA                   S       GCAAATCCATATTACAGAGGAAATGCAAAGAATTCTTCGAC                           TCTTTTAAAAGGCAGCTGCCACCAGACCGTAATTCCGAACT                           AGAGTCCCAAGAAAAAACAACCTGACAAAGTCGATCAAATC                           T               9   YBR086c   IST2   MSQTITSLDPNCVIVFNKTSSANE   127   ATGTCGCAGACAATTACATCTCTAGATCCGAATTGTGTTAT   126   acctacaacaATGTCG   904   gacagtcctgCAGTAT   905                   KSLNVEFKRLNIHSIIEPGHDLQT       TGTATTCAATAAAACTTCGAGTGCAAACGAGAAGAGTTTGA       CAGACAATTACATC       TTTTGATTTGCTGA                   SYAFIRIHQDMAKPLFSFQKLDFI       ATGTCGAATTCAAACGTTTGAATATACATTCTATTATCGAA                   ESIIPYHDTELSDDLHXLISISKS       CCTGGCCATGATCTGCAAACAAGCTATGCGTTTATTAGAAT                   KIL       CCATCAGGATAATGCGAAACCGCTTTTTTCATTTTTGCAGA                           ATCTGGACTTCATTGAATCCATCATACCATATCATGATACT                           GAATTGTCCGATGATTTGCATAAACTGATTTCTATCAGCAA                           ATCAAAAATACTG               10   YBR140c   IRA1   KRQSFPQDKKHFPCEYSLTKHLFF   129   ATGAATCAAAGCGATCCGCAAGACAAAAAAAATTTCCCAAT   128   acctacaacaATGAAT   906   gacagtcctgAACCAA                   DRLLLVLPIESNLKTYADVEADSV       GGAATACTCTTTGACCAAGCATCTTTTTTTTGATAGGCTTC       CAAAGCGATCCGCA       TATAGAATGGGCAA                   FRSCRSIILNIAITKDLNPIIENT       TACTTGTTCTTCCCATAGAATCTAATTTGAAAACATATGCT                   LGLIDLIVQDEEITSDNITDDIAN       GATGTGGAGGCAGATTCAGTTTTCAATTCGTGTCGGTCCAT                   SILV       CATTTTGAATATAGCCATCAGTAAGGACTTGAACCCGATTA                           TCGAAAACACATTAGGTTTAATTGACTTGATTGTGCAAGAT                           GAAGAAATTACGTCTGAGAATATTACAGATGATATTGCCCA                           TTCTATATTGGTT               11   YBR294w   SUL1   MSRKSSTEYVHMQEDADIEVFESE   131   ATGTCACGTAAGAGCTCGACTGAATATGTGCATAATCAGGA   130   aaatacaacaATGTCA   908   gacagtcctgTAGATT   909                   YRTYRESEAAEMRDGLHMGDEENG       GGATGCTGATATCGAAGTATTTGAATCAGAATACCGCACAT       CGTAAGAGCTCGA       GTTTTTGATAGAAT                   KVNSSKQKFGVTKNELSDVLYDSI       ATAGGGAATCTGAGGCGGCAGAAAACAGAGACGGACTTCAC                   PAYEESTVTLKEYYDHSIKNXL       AATGGTGATGAGAAAATTGGAAGGTTAATAGTAGTAAGCAG                           AAATTTGGGGTAACGAAAAATGAGCTATCAGATGTCCTGTA                           CGATTCCATTCCAGCGTATGAAGAGAGCACAGTCACTTTGA                           AGGAGTACTATGATCATTCTATCAAAAACAATCTA               12   YBR298c   MAL31   KXGLSSLIMRKKDFKDSHLDEIEN   133   ATGAAGGGATTATCCTCATTAATAAACAGAAAAAAAGACAG   132   aaatacaacaATGAAG   910   gacagtcctgGAGTGG   911                   GVNATEFHSIEKEEQGKKSDFDLS       GAACGACTCACACTTAGATGAGATCGAGAATGGCGTGAACG       GGATTATCCTCATT       CATTCCCCTCT                   HLEYGPGSLIPKDWNEEVPDLLDE       CTACCGAATTCAACTCGATAGAGATGGAGGAGCAAGGTAAG                   ANQDAKEADESERGMPL       AAAAGTGATTTTGATCTTTCCCATCTTGAGTACGGTCCAGG                           TTCACTAATACCAAACGATAATAATGAAGAAGTCCCCGACC                           TTCTCGATGAAGCTATGCAGGACGCCAAAGAGGCAGATGAA                           AGTGAGAGGGGAATGCCACTC               13   YCL025c   AGP1   MSSSKSLYELKDLKNSSTEIKATG   135   ATGTCGTCGTCGAAGTCTCTATAGGAACTGAAAGACTTGAA   134   acatccaacaATGTCG   912   gacagtcctgTTCTAG   913                   QDNEIEVFETGSHDRPSSQPHLGY       AAATAGCTCCACAGAAATACATGCCACGGGGCAGGATAATG       TCGTCGAAGTCTCT       TTCTTGAGCCTGTC                   EQHNTSAVRRFFDSFKRADQGPQD       AAATTGAATATTTCGAAACAGGCTCCAATGACCGTCCATCC                   EVEATQHNDLTSAISPSSRGAQEL       TCACAACCTCATTTAGGTTACGAACAGCATAACACTTCTGC                   E       CGTGCGTAGGTTTTTCGACTCCTTTAAAAGAGCGGATCAGG                           GTCCACAGGATGAAGTAGAAGCAACACAAATGAACGATCTT                           ACGTCGGCTATCTCACCTTCTTCTAGACAGGCTCAAGAACT                           AGAA               14   YCR011c   ADP1   MGSHRRYLYYSILSFLLLSCSVVL   137   ATGGGAAGTCATCGACGTTATCTCTACTATAGTATATTATC   138   aaatacaacaATGGGA   916   gacagtcctgCGGTGA   917                   AKQDKTPFFEGTSSKNSWLTAQDX       ATTTCTATTATTATCCTGCTCAGTGGTACTTGCAAAACAAG       AGTCATCGACGTTA       TAGACCGCCA                   GHDTCPPCFNCHLPIFECKQFSEC       ATAAGACCCCATTCTTTGAAGGTACTTCTTCGAAAAATTCG                   HSYTGRECCIEGFAGDCGSLPLCG       CGTCTAACTGCACAAGATAAGGGCAATGATACATGCCCGCC                   GLSP       ATGTTTTAATTGTATGCTACCTATTTTTGAATGCAAACAGT                           TTTCTGAATGCAATTCGTACACTGGTAGATGTGAGTGTATA                           GAAGGGTTTGCAGGTGATGATTGCTCTCTGCCCCTCTGTGG                           CGGTCTATCACCA               19   YCR034w   FEN1   MNSLVTQYAAPLFERYPQLHDYLP   145   ATGAATTCACTCGTTACTCAATATGCTGCTCCGTTGTTCGA   144   aaatacaacaATGAATT   924   gacagtcctgTTCACCT   925                   LERPFFNISLNEHFDDVVTRVTNG       GCGTTATCCCCAACTTCATGACTATTTACCAACTTGGAGCG       CACTCGTTACTGA       GCAATGAATTGG                   RFVPSEFQFIAGE       ACCATTTTTTAATATTTCGTTGTGGGAACATTTCGATGATG                           TCGTCACTCGTGTAACTAACGGTAGATTTGTTCCAAGCGAA                           TTCCAATTCATTGCAGGTGAA               20   YCR098c   GIT1   MEDKEITSVHEKEVWENTMPRIIA   147   ATGGAAGACAAAGATATCACATCGGTAAATGAGAAGGAAGT   146   aaatccaacaATGGAAG   926   gccagtcctgCACTTTT   927                   KYDAERRATQTERSKKDKVKNIVT       GAACGAGAACACTAATCCTAGAATAATAAAATATGATGCCG       ACAAGATATCAC       GAGCTATAGTTTT                   IIASGFALISDGYVWGSHSMLNKV       AGAGGCGTGCAACCCGTACTGAAACCTCAAAGAAAGATAAA                   FVWEYGKKNYSSKV       TGGAAAAATAGTTACAATCATTGCGTCCGGTTTTGCTCTGA                           TAAGTGATGGTTACGTAAATGGTTCAATGAGTATGCTAAAC                           AAGGTTTTTGTTATGGAGTACGGTAAGAAAAACTATAGCTC                           AAAAGTG               21   YDL035c   GPR1   MITEGFFPPMLNALKGSSLLEKRV   149   ATGATAACTGAGGGATTTCCCCCGAATTTAAACGCGTTGAA   148   aaatacaacaATGATAA   928   gacagtcctgTAACTGCA   929                   DSLRQLNTTTVHQLLGLPGMTTST       AGGGTCATCCTTACTAGAAAAGAGAGTTGATTCTCTCCGAC       CTGAGGGATTTCC       ACAGTGCGG                   FTAPQLLQL       AGCTTAACACTACCACGGTTAACCAGCTGCTGGGGTTGCCG                           GGGATGACCTCTACATTCACGGCTCCGCAACTGTTGCGTTA               22   YDL138w   RGT2   KNDSQNCLRQREENSHLNPGNDFG   151   ATGAACGATAGCCAAAACTGCCTACGACAGAGGGAAGAAAA   150   aatacaacaATGAACGA   930   gacagtcctgTAGTAGAA   931                   HHQGAECTNHKNMPHRMAYTESTN       TAGTCATCTGAATCCTGGAAATGACTTCGGCCACCACCAGG       TAGCCAAAACTG       TGGACGTGGTTT                   DTEACSIVCCDDPNAYQISYYNNE       GTGCAGAATGTACGATAAATCATAACAACATGCCACACCGC                   PAGDGAIETTSILL       AATGCATACACAGAATCTACGAATGACACGGAAGCAAAGTC                           CATAGTGATGTGCGACAGATCCTAACGCATACCAAATTTCC                           TACACAAATAATGAGCCGGCGGGGAGATGGAGCTATAGAAA                           CCACGGTCCATTCTACTA               23   YDL194w   SNF3   KDPHSNSSSTLRQEKQGFLDKALQ   153   ATGGATCCTAATAGTAACAGTTCTAGCGAAACATTACGCCA   152   aaatacaaaccATGGAT   932   gacagtcctgAGATTGTT   933                   RVKGIALRRANNSKXDHTTDDTTG       AGAGAAACAGGGTTTCCTAGACAAAGCTCTTCAGAGGGGTG       CCTAATAGTAACAG       TCTGAGGAGGCT                   SIRTPTSLQRQNSDRQSNQTSVFT       AAGGGCATAGCACTGCGACGAAACAATAGTAACAAAGATCA                   DDISTIDDNSILFSEPPQKQS       TACAACAGATGATACGACAGGTAGCATACGAACCCCTACGA                           GCTTGCAGCGGCAAAATTCTGACAGGCAATCTAATATGACA                           TCCGTGTTTACGGATGACATTTCTACGATAGACGACAACTC                           AATTTTATTTCAGAGCCTCCTCAGAAACAATCT               24   YDL210w   UGA4   MSKISSKKENKISVEQRISTDIGQ   155   ATGAGTATGTCAAGCAAAAACGAGAATAAGATATCAGTAGA   154   aaatacaacaATGAGTA   934   gacagtcctgTGAAAATT   935                   AYQLQGLGSNLRSIRSKTGAGEVN       ACAAAGAATATCCACTGATATCGGTCAGGCTTACCAGTTAC       TGTCAAGCAAAAA       GTCTTTTTAATT                   YIDAAKSVNDKQLLAEIGYKQELK       AAGGCCTTGGGTCTAACCTAAGGTCGATTCGCTCCAAGACT                   RQFS       GGTGCCGGTGAAGTGAACTATATCGATGCTGCTAAATCTGT                           AAATGATAACCAACTGCTTGCAGAGATCGGTTATAAACAAG                           AATTAAAAAGACAATTTTCA               25   YDL245c   HXT15   DASEQSSPEIRADWLNSSAADVHV   157   ATGGCAAGCGAACAGTCCTCACCAGAAATTAATGCAGATAA   156   aaatacaacaATGCAAG   936   gacagtcctgTCTCTTCG   937                   QPPGEKEHSDGFYDXEVINGNTPD       TCTAAACAGTAGTGCAGCTGACGTTCATGTACAGCCACCCG       CGAACAGT       GTGCGTCTG                   APKR       GAGAGAAAGAATGGTCAGACGGGTTTTATGACAAAGAAGTC                           ATTAATGGAAATACGCCAGACGCACCGAAGAGA               26   YDL247w   MPH2   KXMLSFLIKRRKERTSDSHVYPGK   159   ATGAAAAACTTATCTTTTCTCATAAACAGAAGAAAGGAAAA   158   aaatacaacaATGAAAA   938   gccagtcctgCATTCCTC   939                   AKSHEPSHIENDDQTKKDGLDIVH       TACAAGTGACTCGAATGTATACCCAGGAAGGCTAAGTCGCA       ACTTATCTTTTCT       TTTCACTTTCG                   VEFSPDTRAPSDSKKVITEIFDAT       TGAACCCAGCTGGATAGAAATGGATGATCAAACTAAGAAGG                   EDAKEADESERGH       ACGGCTTAGATATTGTTCATGTTGAGTTCAGTCCGGATACA                           AGAGCGCCAAGCGATAGCAATAAAGTAATAACAGAGATATT                           TGACGCTACTGAGGATGCCAAGGAGGCAGACGAAAGTGAAA                           GAGGAATG               27   YDR011w   SNQ2   KSHIKSTQDSSHNAVARSSSASFA   161   ATGAGCAATATCAAAAGCACGCAAGATAGCTCTCATAATGC   160   aaatacaccaATGAGCA   940   gacagtcctgATCTGAAT   941                   ASEESFTGITNDXDEQSDTPADXL       TGTCGCTAGAAGCTCAAGCGCTTCTTTTGCAGCTTCAGAAG       ATATCAAAAGCAC       CCATATTAAAGG                   TKMLTGPARDTASQISATYSEMAP       AATCATTTACGGGCATAACCCATGACAAAGATGAGCAGAGC                   DVVSKVESFADALSRHTTRSGAFK       GATACCCCGGCGGATAAACTAACAAAAATGCTGACAGGACC                   NDSD       TGCAAGAGACACTGCGAGCCAGATTAGTGCCACTGTGTCTG                           AAATGGCGCCAGATGTCGTATCTAAAGTGGAGTCATTTGCA                           GATGCACTATCCCGTCATACAACGAGAAGCGGTGCCTTTAA                           TATGGATTCAGAT               28   YDR033w   MRH1   MSTFETLIKRGGNEAIKINPPTGA   163   ATGTCTACCTTTGAAACTTTAATTAAAAGAGGTGGTAACGA   162   aaatacaacaATGTCTA   942   gacagtcctgATCGGAAC   943                   DFHITSRGSD       AGCCATCAAAATTAACCCTCCAACCGGTGCGGATTTCCACA       CCTTTGAAACTTT       CACGACTAGTGA                           TCACTAGTCGTGGTTCCGAT               29   YDR046c   BAP3   HSDPIVTSSKHEKSAEFEVTDSAL   165   ATGTCAGATCCTATAGTAACGTCTTCCAAAATGGAAAAAAG   164   aaatacaacaATGTCAG   943   gacagtcctgTCTAGATT   945                   YNHFNTSTTASLTPEIKEHSEESR       TGCAGAGTTTGAAGTAACAGACTCTGCTTTATATAATAACT       ATCCTATAGTAAC       TCATTGACTTTT                   NGLVHRFVDSFRRAEQSRLEENDL       TCAATACATCAACAACAGCTTCACTAACTCCGGAGATTAAG                   EDDGTKSHKSMKHLKKSNXSR       GAACATTCTGAGGAATCTCGCAATGGGTTAGTTCACAGATT                           CGTCGACTCATTCAGAAAGAGCCAAAGCCAACGTTTAGAAG                           AAGACAATGACTTGGAGGATGGTACCAATCGATGAAATCTA                           ATAACCACTTAAAAAAGTCAATGAAATCTAGA               30   YDR093w   DNF2   NSSPSKPTSPFVDDIEHESGSASW   167   ATGTCAAGTCCCTCCAAACCCACTTCTCCCTTCGTGGATGA   166   aaatacaacaATGTCAA   946   gacagtcctgTTTTTTTA   947                   GLSSHSPFDDSFQFEKPSSAHGHI       TATTGAGCATGAATCGGGATCAGCATCTAATGGTCTGTCGT       GTCCCTCCAAA       CCATTGAGTTCAT                   IEVAKTGGSVLKRQSKPEXDISTP       CCATGTCACCATTTGACGATAGTTTTCAATTTGAAAAACCC                   DLSKVTFDGIDDYSKDVDIHDDDE       AGTAGTGCGCATGGAAATATTGAAGTAGCGAAAACCGGCGG                   LNGKK       TTCTGTTTTGAAGCGACAATCTAAGCCAATGAAAGATATTA                           GCACACCCGATCTCTCAAAAGTTACTTTTGATGGAATCGAT                           GATTATAGTAACGTAATGATATTAATGATGATGATGAACTC                           AATGGTAAAAAA               31   YDR276c   PMP3   MDSAKIINIILSLFLPPVAVFLAR   78   ATGGATTCTGCCAAGATCATTAACATTATATTATCCCTTTC   77   aaatacaacaATGGATT   948   gacagtcctgGATATCCA   949                   GWGTDCIVDI       TTACCACCAGTCGCCGTTTTTCTAGCCCGTGGGTGGGGTAC       CTGCCAAGATCA       CTATACAGTCAG                           TGACTGTATAGTGGATATC               32   YDR342c   HXT7   KSQDAAIAEQTPVEHLSAVDSASH   169   ATGTCACAAGACGCTGCTATTGCAGAGCAAACTCCTGTGGA   168   aaatacaacaATGTCAC   950   gacagtcctgTCTCTTTG   951                   SVLSTPSKXAERDEIKAYGEGEEW       GCATCTCTCTGCTGTTGACTCAGCCTCCCACTCGGTTTTAT       AAGACGCTGCTAT       GAATTTCAACGA                   EPVVEIPKR       CTACACCATCAAACAAGGCTGAAAGAGATGAAATAAAAGCT                           TATGGTGAAGGTGAAGAGCACGAACCTGTCGTTGAAATTCC                           AAAGAGA               33   YDR343c   HXT6   HSQDAAIAEQTVEHLSAVDSASHS   171   ATGTCACAAGACGCTGCTATTGCAGAGCAAACTCCTGTGGA   170   aaatacaacaATGTCAC   952   gacagtcctgTCTCTTTG   953                   VLSTPSKXAERDEIKAYGEGEEHE       GCATCTCTCTGCTGTTGACTCAGCCTCCCACTCGGTTTTAT       AAGACGCTGCTAT       GAATTTCAACGA                   PVVEIPRK       CTACACCATCAAACAAGGCTGAAAGAGATGAAATAAAAGCT                           TATGGTGAAGGTGAAGAGCACGAACCTGTCGTTGAAATTCC                           AAAGAGA               34   YDR345c   HXT3   HNSTPDLISPQXSSENSNADLPSN   173   ATGAATTCAACTCCAGATTTAATATCTCCACAAAAGTCAAG   172   aaatacaacaATGAATT   954   gacagtcctgTTTACCTGTATT   955                   SSQVCXMPEEKGVQDDFQSEDQVL       TGAGAATTCGAATGCTGACCTGCCTTCGAATAGCTCTCAGG       CAACTCCAGATTT       TGGGTTGG                   TNPWTGK       TAATGAACATGCCTGAAGAAAAAAGGTGTTCAAGATGATTT                           CCAAGCTGAGGCCGACCAAGTACTTACCAACCCAAATACAG                           GTAAA               35   YDR384c   ATO3   MTSSASSPQDLEKGENTLENIETL   175   ATGACATCGTCTGCTTCTTCTCCACAGGATTTGGAAAAGGG   174   aactacaacaATGACAT   956   gacagtcctgGAACTGGTGCGG   957                   PQGSIAGVSQGFPNIQEIYSDRDF       TGTGAACACTCTAGAAAATATTGAGACGCTCCCCCAGCAGG       CGTCTGCTTCTTC       AGTATA                   ITLGSSTYRRRDLLNLDRGDEEGN       GTTCGATTGCAGGCGTTTCGCAGGGCTTCCCTAATATTCAA                   CAKYTPWQF       GAGATATACTCCGACAGAGACTTCATTACTCTAGGATCCTC                           CACCTACAGGCGCAGAGATTTGCTCAATGCACTAGACAGAG                           GGGATGGGGAGGAAGGAAACTGTGCAAAGTATACTCCGCAC                           CAGTTC               36   YDR420w   HKR1   HVSLKIKKILLLVSLLHAIEAYSN   2   ATGGTCTCATTGAAAATAAAAAAAATTTTACTCCTGGTGTC   1   aactcccccaATGGTCT   958   gcccgtcctgTTGCGTTGTCGT   959                   DTIYSTSYHKSGIESTPSYSTSAI       ATTGTTAAATGCAATCGAGGCCTATAGTAACGATACAATAT       CATTGAAAATAAA       CGTCA                   SSTGSSNKEQAITSSSETTTMAGQ       ATTCAACTTGATACAATAATGGAATAGAAAGCACACCCTCA                   YGESGSTTICDEQETGTSSQYISV       TATTCAACATCCGCGATATCCAGTACCGGATCTAGCAACAA                   TTTTQ       AGAGAATGCAATAACATCAAGCTCTGAAACCACCACAATGG                           CTGGCCAATATGGTGAAAGTGGAAGCACAACAATAATGGAT                           GAACAAGAAACTGGTACGTCCAGCCGAGTATATTAGTGTGA                           CGACGACAACGCAA               37   YDR497c   ITR1   KQIHIPYLTSKTSQSHVGDAVGNA   177   ATGGGAATACACATACCATATCTCACGTCAAAGACATGGCA   176   aaatacaacaATGGGAA   960   GACAGTCCTGgttaaaagtgat   961                   DSVEFNSEHDSPSKRGKITLESHE       ATCAAATGTTGGTGATGCCGTTGGCAACGGTGATAGTGTAG       TACACATACCATA       catgaccg                   IGRAPASQDEDRIQIKPVHDEDQT       AGTTCAACAGTGAGCATGACTCACCTTCAAAGAGAGGTAAA                   SVDITFN       ATTACACTTGAGTCACATGAAATACAGAGGGCTCCCGCTAG                           CGATGATGAAGATAGGATTCAAATTAAACCCGTGAACGACG                           AGGATGACACGTCGGTCATGATCACTTTTAAC               40   YEL083c   CAN1   MTRSKEDADIEEKHKYNEVTTLFH   183   ATGACAAATTCAAAAGAAGACGCCGACATAGAGGAGAAGCA   182   aaatacaacaATGACA   966   gacagtcctgTCTTGC   967                   DVEASQTHHRRGSIPLKDEKSKEL       TATGTACAATGAGCCGGTCACAACCCTCTTTCACGACGTTG       AATTCAAAAGAAGA       TTAAGCTCTCTC                   YPLRSFPTRVNGEDTFSKEDGIGE       AAGCTTCACAAACACACCACAGACGTGGGTCAATACCATTG                   DEGEVQNAEVKRELKQR       AAAGATGAGAAAAAGTAAAGAATTGTATCCATTGCGCTCTT                           TCCCGACGAGAGTAAATGGCGAGGATACGTTCTCTATGGAG                           GATGGCATAGGTGATGAAGATGAAGGAGAAGTACAGAACGC                           TGAAGTGAAGAGAGAGCTTAAGCAAAGA               41   YEL065w   SIT1   KDPGIANHTLPEEFEEVVPEHLEK   185   ATGGACCCTGGTATTGCTAATCATACCCTCCCCGAGGAATT   184   aactccaacaATGGAC   968   gacagtcctgGTTGTA   969                   VGAKVDVKPTLTTSGPAPSYIELD       TGAAGAGGTTGTCGTTCCCGAGATGCTGGAAAGGAAGTAGG       CCTGGTATTGCTAA       CATTTCTGCGTAAA                   PGVHKIIEIYAEEYN       AGCCAAGGTCGATGTCAAGCCAACACTAACCACATCTTCTC                           CAGCACCTTCTTACATTGAATTGATAGATCCAGGTGTGCAT                           AACATCGAGATTTACGCAGAAATGTACAAC               42   YEL069c   HXT13   KSSAQSSIDSDGDVRDADIHVAPP   187   ATGTCTAGTGCGCAATCCTCTATTGATAGCGATGGAGATGT   186   aaatacaaacaATGTC   970   gacagtccgTCTTTTT   971                   VEKRWSDGFDDNEVIRGDNVEPPX       TCGAGATGCTGATATTCATGTCGCACCACCCGTGAAAAAGA       TAGTGCGCAATCCT       GGTGGCTGAAC                   R       GTGGTCAGATGGATTTGATGACAACGAAGTCATAAACGGGG                           ATAACGTTGAGCCACCAAAAAGA               43   YER008c   SEC3   KRSSKSPFKRKSHSRETSHDENTS   189   ATGAGGTCCTCGAAGTCTCCTTTTAAAAGGAAGTCTCACAG   188   aaatacaacaATGAGG   972   GACAGTCCTGcttatg   973                   FFHKRTISGSSAHHSRNVSQGAVP       TCGTGAGACATCACACGATGAAAACACATCGTTTTTCCACA       TCCTCGAAGTCT       gtcgggtctggaaa                   SSAPPYVSGGMYSHXRNVSRASRS       AGAGAACAAATATCCGGTAGCAGTGCTCACCATTCTAGAAA                   SQTSNFLAEQY       CGTCAGTCAAGGTGCAGTACCCTCTTCCGCACCACCTGTTT                           CTGGTGGAAATTATTCGCATAAGAGAAACGTGTCGAGAGCT                           TCAAATTCCTCTCAAACTTGAATTTTTTAGCCGAACAATAT                           GAAAGGGATAGGAAAGCCATAATTAATTGCTGCTTTTCCAG                           ACCCGACCATAAG               44   YER056c   FCY2   KLEEGNVYEIQQLEKRSPYIGSSL   191   ATGTTGGAAGAGGAAATAATGTTTACGAAATCCAAGACTTG   190   aaatacaaaca   974   gacagtcctgATTCAG   975                   ENEKKVAASETFTATSEDDQQYIV       GAGAAGAGATCTCCTGTAATAGGCTCAAGCTTGAAAACGAA       ATGTTGGAAGAGGAAAT       TATGGAATCGTCCG                   ESSEATKSLHRFKFFASNAETKGV       AAGAAGGTAGCCGCTTCTGAAACTTTCACAGCAACTTCCGA                   EPVTEDEKTSSILN       AGTGACCAACAGTATATCGTTGAATCATCAGAGGCCACAAA                           ATTATCGTGGTTCCATAAGTTCTTTGCCAGTTTGAATGCAG                           AAACAAAGGGTGTTGAACCAGTTAGAGAGGATGAAAAAAAC                           GGACGATTCCATACTGAAT               45   YER072w   VTC1   DSSAPLLQRTPGKKAILPTRVEPK   193   ATGTCTTCAGCACCATTATTACAAAGAACACCTGGGAAAAA   192   aaatacaacaATGTCT   976   gacagtcctgCAAAAA   977                   VFFANERTFL       GATCGCTTTGCCCACACACGAGTTGAGCCAAAAGTGTTCTT       TCAGCACCATTATT       GGTACGCTCATTGG                           TGCCAATGAGCGTACCTTTTTG               46   YER118c   SHO1   MSISSKIRPTPRKPSRCIATDHSF   195   ATGTCAATATCATCAAAGATAAGACCAACTCCTCGTAAACC   914   aaatacaacaATGTCAATATCATC   978   gacagtcctgAAAACG   979                   KDKKFYADPFAISSISLAIVSVVI       TTCACGTATGGCTACCGACCATTCTTTTAAAATGAAAAATT       AAAGAT       TGGGAAGGATTC                   ATGGSISSASSTNESFPRF       TTATGCCGATCCTTTCGCTATATCATCAATTTCTTGGCAAT                           TGTATCGTGGGTCATCGCCATCGGGGGCTCCATCTCATCTG                           CATCCACCAATGAATCCTTCCCACGTTTT               47   YER145c   FTR1   HPNKVFNVAVFFVVFRECLEAVIS   197   ATGCCTAACAAAGTGTTTAACGTGGCCGTTTTCTTCGTTGT   196   aaatacaaacaATGCC   980   gacagtcctgCTGAAT                   ISVLLSFLKQAIGEHDRALYRKRI       GTCAGAGAGTGCTTGGAAGCAGTGATTGTTATTTCCGTGCT       TAACAAAGTGTTTAA       CCTTAATTTACGGT                   Q       GCTATCGTTTTTTGAAACAGGCAATCGGGAACATGACCGGG                           CGCTGTACCGTAAATTAAATTCAG               48   YER166w   DNF1   WSGTFKGDGHAPNSPFEDTFQFED   199   ATGTCTGGAACTTTTCATGGCGATGGGCATGCTCCCATGTC   198   aaatacaacaATGTCT   982   gacagtcctgTAATTC   983                   NSSNEDIHIAPTHFDDGATSKKYS       GCCCTTTGAAGATACATTTCAATTTGAAGATAATAGCAGTA       GGAACTTTTCATGG       CACATCATCAAACG                   RPQVSFNDETPKKKREDAEEFTFN       ATGAAGATACACATATTGCACCTACCCATTTTGATGATGGT                   KKTEYDNHSFQPTPKLNNGSGTFD       GCAACAAGCAACAAATACAGCCGGCCACAGGTCAGCTTCAA                   DVEL       TGATGAAACACCCAAAAATAAACGTGAAGATGCAGAAGAAT                           TCACATTTAACGATGACACAGAATATGACAATCATTCCTTT                           CAGCCAACACCGAAACTTAATAATGGATCTGGGACGTTTGA                           TGATGTGGAATTA               49   YFL011w   HXT10   HVSSSVSILGTSAKASTSLRKDEI   201   ATGGTTAGTTCAAGTGTTTCCATTTTGGGGACTAGCGCCAA   200   acatacaacaTGGTTA   984   gacagtcctgGGGTTT   985                   TKLTPETREASLDIPYKP       GGCATCCACTTCTCTAAGTAGAAAGGATGAAATTAAACTAA       GTTCAAGTGTTTC       GTATGGAATGTCCA                           CCCCTGAAACAAGGGAAGCTAGCTTGGACATTCCATACAAA                           CCC               50   YFL026w   STE2   HSDAAPSLSNLFYDPTYNPGQSTI   203   ATGTCTGATGCGGCTCCTTCATTGAGCAACTATTTTATGAT   202   aaatacaacaATGTCT   986   gacagtcctgaACCTT   987                   NYTSIYGNGSTITFDELDQ       CCAACGTATAATCCTGGTCAAAGCACCATTAACTACACTTC       GATGCGGCTCCTT       GCAACTCATCGAA                           CATATATGGAATGGATCTACCATCACTTTCGATGAGTTGCA                           AGGT               51   YFL041w   FET5   MLFYSFVHSVLAASVALAKTHXLN   205   ATGTTCTTCTACTCGTTCGTGTGGTCTGTACTGGCCGCTAG   204   aaatacaacaATGTTG   988   gacagtcctgCATAGA   989                   YTASWVTANPDGLHEXRWIGHNGE       TGTTGCTTTGGCAAAGACACATAAGTTAAACTATACCGCTT       TTCTACTCGTTCGT       AGGACCATCCATT                   HPLPDIHVEKGDRVELYLTNGFQD       CTTGGGTAACTGCCAATCCTGATGGATTGCATGAAAAAAGG                   NTATSLHFHGLFQNTSLQNQLQND       ATGATTGGTTTTAATGGCGAATGGCCACTTCCAGATATCCA                   GPSSI       TGTTGAAAAAGGAGATCGTGTTGAGCTTTATTTGACTAACG                           GCTTTCAAGACAATACTGCTACTTCTCTACATTTCCATGGT                           CTTTTCCAGAATACGAGTTTGGGGAACCAGCTTCAAATGGA                           TGGTCCTTCTATG               52   YFL050c   ALR2   MSSLSTSFDSSSDLPRSKSVDNTA   207   ATGTCGTCCTTATCCACTTCATTTGATTCATCGTCAGATTT   206   aaatacaacaATGTCG   9909   gacagtcctgTTCTGC   991                   ASSKTGKYPKLENYRGYSDAQPIR       ACCAAGGTCAAAACCGTTGACAATACGGCGGCTTCCATGAA       TCCTTATCCACTTC       TTCACCTCAAA                   HEALALKVDETKDSRHXFSSSNGE       GACAGGCAAGTACCCAAAATTAGAGAACTATAGGCAGTATT                   NSGVENGGYVEKTNISTSGRNDFE       CTGATGCACAACCAATACGTCACGAAGCGCTTGCATTGAAA                   GEAE       GTGGACGAAACGAAAGATTCTAGACACAAATTTAGTTCCTC                           TAACGGGGAGAATAGTGGAGTGGAAAATGGAGGCTATGTGG                           AGAAAACGAATATATCCACAAGTGGCCGCATGGATTTTGAA                           GGTGAAGCAGAA               53   YGL008c   PMA1   MTDTSSSSSSSSASSVSAHQPTQE   209   ATGACTGATACATCATCCTCTTCATCATCCTCTTCAGCATC   208   acatacaagaATGACT   992   gacagtcctgGTACTT   993                   KPAKTYDDAASESSDDDDIDALIE       TTCTGTTTCAGCTCATCAGCCAACTCAAGAAAAGCCTGCTA       GATACATCATCCTC       CTTTCTTCTTTTCA                   ELQSNHGVDDEDSDNDGPVAAGEA       AGACTTACGATGACGCTGCATCTGAATCTTCTGACGATGAC                   RPVPEEYLQTDPSYGLTSDEVLKR       GATATCGATGCTTTAATCGAAGAACTACAATCTAATCACGG                   RKKY       TGTCGACGACGAAGACAGTGATAACGATGGTCCAGTTGCCG                           CCGGTGAAGCTAGACCAGTTCCAGAAGAATATTTAGAAACT                           GACCCATCTTACGGTTTAACTTCCGATGAAGTTTTGAAAAG                           AAGAAAGAAGTAC               54   YGL077c   HNM1   HSIRNDWASGGYHQPDQSSNASHH   211   ATGAGTATTCGGAATGATAATGCTTCCGGTGGCTATATGCA   210   aaatacaacaATGAGT   994   gacagtcctgTGACTT   995                   KRDRVEEEIKPLDDNHSKGAVAAD       GCCGGATCAATCTTCGAAGGCTTCTATGCACAAAAGAGACT       ATTCGGAATGATAA       TCTTAGATGAACTT                   GEVHLRKS       TAAGAGTTGAGGAGGAAATAAAGCCATTGGATGATATGGAT                           AGCAAGGGTGCTGTAGCAGCAGATGGTGAAGTTCATCTAAG                           AAAGTCA               55   YGR014w   MSB2   MQFPFACLLSTLVISSSLARASPF   10   ARTGCAGTTTCCATTCGCTTGTCTCCTATCGACCCTTGTAA   9   aaatacaacaATGCAG   996   gacagtcctgTGACAC   997                   DFIFGRGTQQAWSQSESQGVSFTN       TTAGTGGGTCATTGGCCCGGGCCAGCCCCTTCGACTTTATA       TTTCCATTCGCT       AGAAGTCTGGGAA                   EASQDSSTTSLVTAYSQGVHSHQS       TTCGGCAATGGAACGCAACAAGCTCAGAGCCAAAGCGAGAG                   ATIVSATISSLPSTQVDASSTSQT       TCAAGGTCAAGTTTCTTTCACCAATGAAGCTTCTCAGGATA                   SVS       GTTCCACCACCTCTTTGGTAACAGCCTATTCTCAAGGTGTT                           CATTCGCACCAGTCTGCAACAATAGTGAGTGCCACAATCTC                           TTCCCTCCCATCTACTTGGTASTGATGCGAGCTCCACTTCC                           CAGACTTCTGTGTCA               56   YGR032w   GSC2   HSYNDPNLNGQYYSHGDGTGDGNY   213   ATGTCCTACAACGATCCAAACTTGAATGGACAGTATTACAG   212   aaatacaacaATGTCC   998   gacagtcctgTCCATC   999                   PTQVQDQSAYDEYGQPIYQNQLDD       TAACGGTGATGGGACTGGTGACGTAATTACCCTACGTACCA       TACAACGATCCAA       TTGAGAAGACGG                   GYYDPNEQYVDGTQFPQGQDPSQD       AGTGACACAGGATCAAAGTGCGTACGATGAGTACGGTCAGC                   QGPYNNDASYYNQPPKHNPSSQDS       CAATCTATACACAAAACCAACTGGATGATGGTTATTATGAT                           CCAAACGAACAATACGTTGACGGTACACAATTCCTCAGGGA                           CAAGATCCTTCACAAGACCAAGGTCCTTATAATAACGATGC                           TAGTTACTATAACCAACCCCCCAATATGATGAACCGTCTTC                           TCAAGATGGA               57   YGR055w   MUP1   MSEGRTFLSQLNVFNKEHYQFSSS   215   ATGTCGGAAGGAAGAACGTTTCTGTCACAGTTGAATGTCTT   214   aaatacaacaATGTCG   1000   gacagtcctgCTGCTT   1001                   TTKKEVSHSTVDADHGASDFEAQQ       CAACAAGGAGAACTATCAATTTTCTTCTTCTACTACAAAAA       GAAGGAAGAACGT       TTCACCTTGGTC                   FATELDQGEKQ       AAGAAGTAAGTAACTCGACAGTGGATGCTGACAACGGTGCC                           TCCGATTTTGAGGCAGGCCAGCAATTTGCTACAGAATTGGA                           CCAAGGTGAAAAGCAG               58   YGR121c   MEP1   HESRTTGPLITETYDGPVAFHILG   217   ATGGAGAGTCGAACTACAGGGCCTTTAACGACTGAAACCTA   216   aaactcccacaATGGA   1002   gacagtcctgTGCAGA   1003                   AALVFFHVPGLGFLYSGLARRKSA       CATGGCCCCACTGTGGCCTTGATGATATTAGGTGCCGCCCT       GAGTCGAACTACAGG       CTTCCTTCTTGC                           AGTATTTTTTTTATGGTGCCCGGATTGGGATTCTTGTACTC                           CGATTGGCAAGAAGGAAGTCTGCA               59   YGR191w   HIP1   HPRHPLKKEYRADVVDGFKPATSP   219   ATGCCTAGAAACCCATTGAAAAAGGAATATTGGGCAGATGT   218   aaatacaaacaATGCC   1004   gacagtcctgGGATAG   1005                   AFEHEKESTTFVTELTSKTDSAFP       AGTTGACGGATTCAAGCCGGCTACTTCTCCAGCCTTCAGAG       TAGAAACCCATTG       ATCTTTACTCAGGT                   LSSKDSPGINQTTNDITSSDRFRR       AATGAAAAAGAATCTACTACATTTGTTACCGAACTAACTTC                   NEDYEQEDINNTKLSKDLS       CAAAACCGATTCTGCATTTCCATTAAGTAGCAAGGATTCAC                           CTGGCATAAACCAAACCACAAACGATATTACCTCTTCAGAT                           CGCTTCCGTCGTAATGAAGACACAGAGCAGGAAGACATCAA                           CAAGACCAACCTGAGTAAAGATCTATCC               60   YGR281w   YOR1   MTITVSDAVSETELENKSQVVVLS   221   ATGACGATTACCGTGGGGGATGCAGTTTCCGAGACGGAGCT   220   aaatacaacaATGACG   1006   gacagtcctgTTGTGG   1007                   PKASASSDISTDVKDTSSSWDDKS       GGAAAACAAAAGTCAAAACGTGGTACTATCTCCCAAGGCAT       ATTACCGTGGG       TACTTCTGGAATTT                   LLPTGEYIVDRNKPQTYLNSDDIE       CTGCTTCTTCAGACATAAGCACAGATGTTGATAAGGACACA                   XVTESDIFPQXRLFSFLHSKKIPE       TCGTCTTCTTGGGATGACAAATCTTTGCTGCCTACAGGTGA                   VPQ       ATATATTGTGGACAGAAATAAGCCCCAAACCTACTTGAATA                           GCGATGATATCGAAAAAGTGACAGAATCTGATATTTTCCCT                           CAGAAACGTCTGTTTTCATTCTTGCACTCTAAGAAAATTCC                           AGAAGTACCACAA               61   YHL016c   DUR3   MGEFKPPLPQGAGYAIVLGLGAVF   223   ATGGGAGAATTTAAACCTCCGCTACCTCAAGGCGCTGGGTA   222   acatacaacaATTGGA   1008   gacagtcctgTACAGA   1009                   AGHHVLTTYLLKRYQKEIITAEEF       CGCTATTGTATTGGGCCTATTTGCAGGAATGATGGTTTTGA       GAATTTAAACCTCC       TCTACCGGCGGT                   TTAGRSV       CCACTTATTTACTGAAACGTTATCAAAAGGAAATCATCACA                           GCAGAAGAATTCACCACCGCCGGTAGTCTGTA               62   YHL036w   MUP3   MEPLLFNSGKAHPSQDVFIDVEVD   225   ATGGAACCGCTGCTTTTTAATAGTGGGAAAGCAAATCCCTC   224   aaatacaacaATGGAA   1010   gacagtcctgTCTTCC   1011                   GITTKYGSTNTGSFSSHDTVEAQI       TCAAGATGTTTTCATGAGTGTGGAAGTTGGTGATATTACCA       CCGCTGCTTTT       CTGTGGAAACTTC                   KAETARFHEVPQGR       CAAAATATGGTTCCACAAATACTGGATCATTCAGTTCGATG                           GATACTGTGGAAGCGCAGGCGATAAAGGCAGAGACGGCAAG                           ATTCATGGAAGTTCCACAGGGAAGA               63   YHR092c   HXT4   HSEEAAYQEDTAVQNTPADALSPV   227   ATGTCTGAAGAAGCTGCCTATCAAGAGGATACAGCAGTCCA   226   aaatccaacaATGTCT   1012   gacagtcctgGGGCTT   1013                   ESDSNSALSTPSKKAERDDKHDFD       AAATACTCCAGCTGATGCTTTGTCGCCAGTTGAATCCGATT       GAAGAAGCTGCCTA       CTTTGGAATTTC                   ENHEESKRYVEIPKKP       CTAATTCCGCTTTGTCTACTCCATCCAACAAAGCTGAAAGA                           GATGACATGAAAGATTTCGACGAGAATCACGAAGAATCTAA                           TAACTACGTTGAAATTCCAAAGAAGCCC               64   YHR094c   HXT1   HNSTPDLISQXSNSSNYELESGRS   229   ATGAATTCAACTCCCGATCTAATATCTCCTCAGAAATCCAA   228   acatacaacaATGAAT   1014   gacagtcctgTCCGGT   1015                   KAKNTPEGKNESFHDMLSESQVQP       TTCATCCAACTCATATGAATTGGAATCTGGTCGTTCAAAGG       TCAACTCCCGATCT       GTTTGGAGGGG                   AVAPPNTG       CCATGAATACTCCAGAAGGTAAAAATGAAAGTTTTCACGAC                           AACTTAAGTGAAAGTCAAGTGCAACCCGCCGTTGCCCCTCC                           AAACACCGGA               65   YHR096c   HXT5   HSELENAHQGPLEGSATVSTNSNS   231   ATGTCGGAACTTGAAAAGCGCTCATCAAGGCCCCTTGGAAG   230   aaatacaacaATGTCG   1016   gacagtcctgCGATTT   1017                   YNEKSGNSTAPGTAGYRDKLAGAK       GGTCTGCTACTGTGAGCAAATTCTAACTCATACAACGAGAA       GAACTTGAAAACG       CTTCTCTAGTTTGTT                   VSSYISHEGPPKDELEELQKEVDK       GTCAGGAAACTCGACTGCTCCTGGTACCGCCGGTTACAACG                   QLEKKS       ATAATTGGCACAAGCTAAACCCGTCTCAAGTTACATTTCCC                           ATGAAGGCCCTCCCAAAGACAACTGGAAGAGCTTCAGAAGG                           AGGTTGACAAACAACTAGAGAAGAAATCG               66   YIL013c   PDR11   HSLSKVFNPIPDASVTFDGATVQL   233   ATGTCTCTTTCCAAATATTTTAATCCAATTCCTGACGCTTC   232   aaatacaacaATGTCT   1018   gacagtcctgCTGTTT   1019                   EESLGAVNDEESASEFKNVGHLEI       AGTCACCTTTGATGGGGCTACCGTTCAATTGGAAGAATCCC       CTTTCCAAATATTT       GTACTCATTGTCTT                   SDITFRANEGEVVLVLGNPTSALF       TCGGTGCTGTTCAGAACGATGAAGAGTCCGCATCGGAATTC                   KGLGHGHKHLKYSPEGSIRFKDNE       AAAAACGTAGGCCATTTAGAAATTAGTGATATCACTTTTCG                   YKQ       TGCTAATGAAGGTGAAGTCGTCTTAGTACTGGAAACCCAAC                           ATCAGCGCTCTTCAAAGGTCTATTCCATGGTCACAAGCATC                           TGAAATACTCGCCTGAAGGGTCTATTAGATTCAAAGACAAT                           GAGTACAAACAG               67   YIL047c   SYG1   KXFADHLTESAIPWRDKIDYKVGK   235   ATGAAGTTTGCTGACCATCTAACCGAGTCTGCCATCCCGGA   234   aaatacaacaATGAAG   1020   gacagtcctgCAACCA   1021                   KKLRRKEKLDAEEEQSSSYRSWHP       ATGGAGGGACAAATATATTGATTATAAGGTCGGCAAGAAGA       TTTGCTGACCATC       GTCTTCAATGAAGT                   SVSVQTAFQQREPGKSRSDGDYRS       AGCTTCGCCGCTACAAGGAGAAGCTGGATGCCGAAGAAGAG                   GPAFKKDYSALQREFVADFIEDWL       CAATCCAGCTCCTACCGGAGCTGGATGCCCTCTGTGTCGGT                           ATACCAGACTGCATTTCAGCAGAGAGCCCGGCAAAAGTCGC                           AGCGACGGGACTATCGCTCCGGACTTGCGTTCAAGAAAGGA                           CTATTCTGCTTTGCAGAGGGAGTTCGTTGCTGACTTCATTG                           AAGACTGGTTG               68   YIL140w   AXL2   NTQLQISLLLTATISLLHLVVATP   86   ATGACACAGCTTCAGATTTCATTATTGCTGACAGCTACTAT   85   aatacaacaATGACAC   1022   gacagtcctgATACAA   1023                   YEAYPIGKQYPPARVNESFTFQIS       ATCACTACTCCATCTAGTAGTGGCCACGCCCTATGAGGCAT       AGCTTCAGATTTC       CGTGGTGTTCGCAT                   WDTYKSSVDKTAQITYNCFDLPSW       ATCCTATCGGAAAACAATACCCCCCAGTGGCAAGAGTCAAT                   LSFDSSSRTFSGEPSSDLLSDANT       GAATCGTTTACATTTCAAATTTCCAATGATACCTATAAATC                   TLY       GTCTGTAGACAAGACAGCTCAAATAACATACAATTGCTTGA                           CTTACCGAGCTGGCTTTCGTTTGACTCTAGTTCTAGAACGT                           TCTCAGGTGAACCTTCTTCTGACTTACTATCTGATGCGAAC                           ACCACGTTGTAT               69   YIL147c   SLN1   MRFGLPSKLELTPPFRIGIRTQLT   237   ATGCGATTTGGCCTGCCATCAAATTGGAACTCACTCCTCCG   236   aaatacaacaATGCGA   1024   gacagtcctgGTTACC   1025                   ALVSIVALGSLIILAVTTGVYFTS       TTTAGGATAGGCATCCGAACTCAACTAACGGCACTAGTTAG       TTTGGCCTGCC       TGCAACGTAACTTG                   NYKHLRSDRLYIAAGLKSSQIDQT       TATAGTGGCTTTGGCTCACTGATTATTCTGGCTGTAACGAC                   LNYLYQAYYLASRDALQSSLTSYV       AGGGGTCTATTTTACCTCGAACTATAAAAATTTAAGGTCCG                   AGN       ATAGACTGTACATTGCCGCTCAGTTAAAGTCATCACAGATT                           GACCAAACTCTAAACTACTTATATTACCAGGCGTACTATTT                           GGCATCAAGAGACGCCCTGCAAAGCTCACTAACAAGTTACG                           TTGCAGGTAAC               70   YIL170w   HXT12   MGLIVSIFNIGCAIGGIVLSKVGD   239   ATGGGTTTGATTGTCTCAATATTCAACATTGGCTGCGCCAT   238   aaatacaacaATGGGT   1026   gacagtcctgTGTAAT   1027                   IYGRRIGLIY       AGGCGGAATTGTCTTGTCAAAAGTCGGTGATATATATGGTC       TTGATTGTCTCA       CAATCCAATACGAC                           GTCGTATTGGATTGATTACA                                                                                                                                                                                                                                                                                                                                                                                       331   YJR118c   ILM1   HAQALNSTNIAFFVAFLFTIAFFC   90   AATGGCTCAAGCCTTGAACTCCACCAATATTGCTTTTTTTC   89   aaatacaacaATGGCT   1548   gacagtcctgACGTGA   1549                   LKNVNSILQRTVFIVLTQANKLPQ       AGAGTAGCATTTTTATTCACGATCGCCTTCTTTTGTTTAAA       CAAGCCTTGAACT       CAGTGTTAACTGCG                   LTLSR       GAACGTTAATTCTATTTTGCAAAATACATATTTCATAGTCT                           TAACGCAAGCGATGAATTTACCGCAGTTAACACTGTCACGT               332   YKL065a   YET1   MSLYFTTLFLLLVEVVKLFIFVLP   731   ATGAGTTTATACTTTAGACATTATTTTTATTGCTCACTGTT   730   aaatacaacaATGAGT   1550   gacgtcctgCTTCGCT   1551                   LPFRIRRGIFSTYMQLTAK       GAGGTGGTAATGCTCTTCATCTTCGTTTTGCCTTTGCCATT       TTATACTTTACGAC       GTCAATTGGTTAT                           CCGGATCCGTAGGGGGTATTTTTTAGCACCTATAACCAATT                           GACAGCGAAG               333   YLR034c   SMF3   YRSYMQILQXFAKFIGPGILVSVA   733   ATGCGATCTTATATGCAGATTCTTCAAAAATTTGCCAAATT   732   aaatacaacaATGCGA   1552   gacagtcctgATATTG   1553                   YMDPGNYATSVSGGAQYKY       TATTGGGCCAGGGATATTAGTCAGTGTGGCTTATATGGACC       TCTTATATGCAGAT       TATTGAGCACCAC                           CAGGAAATTATGCCACTAGTGTTTCCGTGGTGCTCAATACA                           AATAT               334   YLR050c       KKLGHREQQFYLVVFIVHIPITIF   735   ATGAAGCTAGGACATCGTGAGCAACAATTCTACTTGTGGTA   734   aaatacaacaATGAAG   1554   gacagtcctgTTCTGG   1555                   IDSSVVIPAKWQLGIAQKVVSDHI       CTTGATCGTTCACATTCCCATCACCATATTCATCGACTCAT       CTAGGACATCGTGA       TTTCTCTGATAGCA                   AKQHDFLLSEKPE       CAGTGGTTATTCCCGCTAAATGGCAACTAGGGATTGCGCAA                           AAGGTTGTTAGTGATCACATCGCAAAGCAACACGATTTCCT                           GCTATCAGAGAAACCAGAA               335   YLR207w   HRD3   HITLLLYLCVICHAIVLIRADSIA   737   ATGATAACACTCTTATTATACCTGTGCTAATATGTAACGCA   736   gcatacaacaATGATA   1556   gacagtcctgGAATTG   1557                   DPWPEARHLLNTIAKSRDPKXEAA       ATAGTGTTAATAAGGGCTGATTCGATAGCGGACCCTTGGCC       ACACTCTTATTATA       TTCACTTGATTGTA                   NEPHADEFVGFYVPHDYSPRNEEK       TGAAGCGCGACATCTACTAAATACCATAGCTAAGTCCAGAG                   NYQSIGNNEITDSQRHIYELLVQS       ACCCAATGAAAGAAGCTGCTATGGAACCCAATGCAGATGAA                   SEQF       TTTGTTGGATTCTATGTACCGATGGATTATTCCCCACGTAA                           TGAGGAAAAAAAACTACCAGAGCATTGGCAAACGAAATCAC                           AGATTCTCAACGTCATATTTATGAATTACTTGTACAATCAA                           GTGAACAATTC               336   YLR220w   CCC1   HSIVALKNAVVTLIQKAKGSGGTS   739   ATGTCCATTGTAGCACTAAAGAACGCAGTGGTGACCCTTAT   738   aaatacaacaATGTCC   1558   gacagtcctgTACGCG   1559                   ELGQSESTPLLRGSHSHSSRHDKL       ACAGAAAGCGAAAGGTAGTGGTGGAACCTCAGAGTTGGGGG       ATTGTAGCACTAAA       AGGATCTACTGATT                   SSSSSDIIYGRNSAQDLENSPHSV       GGTCTGAATCAACTCCTTGTTGAGGGGTAGTAATAGCAATA                   GHDKRNGDHDSDKEKANLGFFQSV       GTTCAAGGCATGATAACTTATCCTCATGTAGCTCGGATATT                   DPRV       ATCTATGGTAGAAATTCAGCGCAGGATCTAGAAAACTCACC                           GATGTCAGTAGGGAAAGATAATAGGAATGGCGATAACGGTT                           CGGATAACGAAAAGGCGAACCTAGGGTTCTTCCAATCAGTA                           GATCCTCGCGTA               337   YML012w   ERV25   HQVLQLDLTTLISLVVAVDGLHFD   741   ATGCAGGTGTTACAGTTATGGTTGACAACTTGATCTCTTGG   740   aaatacacaATGCAGG   1560   gacagtcctgAAAACA   1561                   IAASTDPEQVCIRDFVTEGQLVVA       TGGTGGCAGTGCAGGGATTACATTTCGACATTGCAGCATCT       TGTTACAGTTATG       AACGTCGAATGCC                   DIHSDGSVGDGQKLFVRDSVGNEY       ACAGATCCAGAACAGGTTTGTATTCGTGATTTTGTCACTGA                   RRKRDFAGDVRVAFTAPSSTAFDV       AGGTCAATTGGTTGTCGCGGATATTCACTCAGATGGTTCTG                   CF       TTGGTCATGGACAGAAACTAAACCTCTTCGTGCGTGATTCA                           GTTGGAAACGAGTATCGTAGAAAGAGGGACTTTGCAGGCGA                           CGTTCGTGTTGCGTTTACTGCTCCATCCTCCACGGCATTCG                           ACGTTTGTTTT               338   YMR149w   SWP1   MQFFKTLAALVSCISFVLAYVADD   743   ATGCAATTCTTCAAAACACTTGCGGCCTTGGTGTCGTGCAT   742   aactacaacaATGCAA   1562   gacagtcctgAATTCG   1563                   VHVSFPSTAGKSRVWIGKVERPRI       ATCGTTCGTCCTCGCTTACGTGGCACAAGATGTTCATGTAT       TTCTTCAAAACACT       GGTTCAAAAGCCA                   GIDETVPTTITVEDPKEVIQVNHA       CATTCCCCTCCACCGCAGGAAAGTCTAGGGTAATGATCGGG                   IESTKXPFWSTLLIGLPKKLENAF       TAAAGTTGAACCCAGAATAGGAATCGATGAAACTGTTCCGA                   EPEI       CTACAATCACAGTTGAAGACCCTAACGAGGTGATCAAGTAA                           ATTTCGCCATTGAGTCTACCAACAAACCCTTCCAGAACACC                           TTATTGATAGGCTTACCTAATAAGAACCTAGAAATGGCTTT                           TGAACCCGAAATT               339   YMR171c       MSFKFLIESLLLGSISSQIRCGRS   745   ATGTCATTCAAGTTTCTGATAGAATCGCTGCTTCTCGGTTC   744   aaatacaacaATGTCA   1564   gccagtcctgATCCAT   1565                   SVIPRGDVSYGGDDTDELNND       GATAAGCGGACAAATACGGTGTGGTAGATCTTCGGTGATCC       TTCAAGTTTCTGAT       GTTAAGTTCGTCAG                           CCCGTGGCGATGTATCTTATGGGGGAGACGATACTGACGAA                           CTTAACATGGAT               340   YMR200w   ROT1   MZKSKKFTLKKLILGGYLFAQXVY   44   ATGTGGTCGAAAAAGTTTACATTAAAAAAGCTAATCTTAGG   43   acctacaacaATGTGG   1566   gacagtcctgACCATG   1567                   CEDESHSIYGTWSSKSHQVFTGPG       CGGGTATTTGTTGCTCAAAAGGTCTATTGTGAAGACGAAAG       TCGAAAAAGTTTAC       CTGATAAATCAACG                   FYDPVDELLIEPSLPGLSYSFTED       TAACTCTATATACGGTACCTGGTCATCTAAATCAAATCAAG                   GWYEEATQVSGMPRNPTGPHASLI       TGTTCACGGGACCGGGGTTTTATGATCCCGTAGATGAACTA                   YQHG       TTGATAGAACCTTCATTGCCCGGGCTTAGCTATTCGTTCAC                           TGAAGATGGTTGGTACGAAGAAGCTACTTACCAGGTAAGTG                           GAAATCCTCGTAACCCAACTTGCCCCATGGCTTCGTTGATT                           TATCAGCATGGT               341   YMR238w   DFG5   HIVHISAKHILSICFTFLSFFKAT   747   ATGATCGTCAATATTAGTGCGAAGATGATCTTATCGATATG   746   aaatacacaATGATCG   1568   gacagtcctgTAGTGC   1659                   HAKDLDTTSKTSICDATALIQGGW       CTTTACGTTTCTGTCATTTTTTAAAGCCACTCATGCCATGG       TCAATATTAGTGC       ATCGTATAGTAATT                   LDYYEGTRYGGTVGNFQSPYWWHA       ATTTGGATACTACTAGCAAAAACGTCAATTTGTGATGCGAC                   GEAFGGHLEKKFLCENDTYQELLY       AGCGTTAATTCAAGGTGGTATGCTGGATTACTATGAGGGTA                   DAL       CTAGATACGGTGGTACCGTTGGGATGTTTCAGTCACCATAC                           TATTGGTGGCATGCAGGGGAAGCATTTGGTGGCATGTTGGA                           AAAATTGGTTTCTTTGTGAGAATGATACATATCAAGAATTA                           CTATACGATGCACTA               342   YMR274c   RCE1   CLQFSTFLVLLYSISIYVLPLYAT   749   ATGCTACAAATTCTCAACATTTCTAGTGCTCCTATACATCT   748   aaatacaacaATGCTA   1570   gacagtcctgGCGAGA   1571                   SQPEGSKKRDWPRTIKSR       CCATATCCTATGTGCTACCGCTATATGCAACTTCACAACCA       CAATTCTCAACATT       TTTAATCGTTCGGAG                           GAAGGGTCTAAACGAGATAATCCTCGAACGATTAAATCTCG                           C               343   YNR021w       HSSSIQPLTGFLERVNSLHAPYQA   751   ATGTCTAGTTCAATATTGGCCCACTTACGGGTTTTTTGGAG   750   aaatacaacaATGTCT   1572   gaccgtcctgTTTGAC   1573                   LSYDEQKANTIKKRVK       CGTGTCAATTCACTCAATGCGCCCTACCAAGCATTATCATA       AGTTCAATATTTGG       TCTTTGCCAAATAG                           TGACGAACAAAAGGCCATGACTATTTGGCAAAGAGTCAAA               344   YNR030w   ECM39   HKQSVLDTVLLTVISFHLIQAPFT   753   ATGCGTTGGTCTGTCCTTGATACAGTCTATTGACCGTGATT   752   aaatacaacaATGCGT   1574   gccagtcctgTGTTCT   1575                   KVEESFNIQAIDHILTYSVFDISQ       TCCTTTCATCTAATCCAAGCTCCATTCACCAAGGTGGAAGA       TGGTCTGTCCTTGG       AGGGACTACTCCAG                   YDHLKFPGVVPRT       GAGTTTTAATATTCAAGCCATTCATGATATTTTAACCTACA                           GCGTATTTGATATCTCCAATATGACCACTTGAAATTTCCTG                           GAGTAGTCCCTAGAACA               345   YOL013c   HRD1   WVPENKKRKQLAIFVVVTVLLTFY   755   ATGGTGCCAGAAAATAGAAGGAAACAGTTGGCAATTTTTGT   754   aaatacacaATGGTGC   1576   gccagtcctgGCCTTC   1577                   CYYSATKSYSVSFLQVTLKLWEG       AGTTGTCACATATTGCTCACATTTATGCGTGTATTCAGCCA       CAGAAAAATAGAAG       ATTTAGCTTCAGTG                           CCAAGACAAGCGTTTCCTTTTTGCAAGTAACACTGAAGCTA                           AATGAAGGC               346   YOR016c   ERP4   KRYFTLIAILFSSSLLTHAFSSNY   757   ATGCGCGTTTTTACTTTGATTGCGATTTTGTTTAGTTCATC   756   aactacaacaATGCGC   1578   gaccgtcctgACAGAA   1579                   APVGISLPAFIKEGLYYDLSSDXD       TTTGTTAACACATGCATTCTCCTCTAATTATGCTGCTGTAG       GTTTTTACTTTGAT       GTGTACTTACCTA                   VLVVSYQVLTGGFFEDIFITAPDG       GCATATCATTACCTGCTTACCAAAGAATGTCTTTACTATGA                   SVIVERQKKHSDFLLKSFSIGXYT       TTTATCCTCTGATAAAGATGTCCTTGTGGTCAGTTACCAAG                   FC       TTTGACAGGTGGGAATTTCGAGATAGACTTCGATATTACCG                           CCCCTGATGGCTCTGTTATCGTCACTGAAAGACAAAAGAAG                           CATTCTGATTTTCTACTGAAGTCGTTTGGTATAGGTAAGTA                           CACTTTCTGT               347   YOR085w   OST3   KKLFLVSLVFFCGVSTHPALAHSS   759   ATGAATTGGCTGTTTTGGTCTCGCTGGTTTTCTTCTGCGGC   758   aaatacaacaATGAAT   1580   gacagtcctgGGACGA   1581                   SDRLLKLANKSPKKIIPLKDSSFE       GTGTCAACCCATCCTGCCCTGGCAATGTCCAGCAACAGACT       TGGCTGTTTTTGGT       TTTTGCATCCG                   NILAPPHENAYIVALFTATAPEIG       ACTAAAGCTGGCTAATAAATCTCCCAAGAAAATTATACCTC                   CSLCLELESEYDTIVASRHDNNPD       TGAAGGACTGAAGTTTTGAAAACATCTTGGCACCACCTCAG                   AKSS       GAAAATGCCTATATAGTTGCTCTGTTTACTGCCCACAGCGC                           CCGAAATTGGCTGTTCTCTGTGTCTCGAGCTAGGATCCGAA                           TACGACACCATAGTGGCCTCCTGGTTTGATGATGATCCGGA                           TGCAAAATCGTCC                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       422   YCL043c   PDI1   MKFSAGAYLSWSSLLLASSVFAQQ   24   ATGAAGTTTCTGCTGGTGCCGTCCTGTCATGGTCCTCCCTG   23   aaatacaacaATGAAG   1730   gacagtcctgGTCGTG   1731                   EAVAPEDSAVVKLATDSFNEYIQS       CTGCTCGCCTCCTCTGTTTCGCCCAACAAGAGGCTGTGGCC       TTTTCTGCTGGTGC       CGACTGAATGTACT                   HD       CCTGAAGACTCCGCTGTCGTTAAGTTGGCCACCGACTCCTC                           AATGAGTACATTCAGTCGCACGAC               423   YCR069w   CPR4   MVLKSLLLVLYSLVLCQVHAAPSS   863   ATGTGGTTGAAATCCTTGCTGCTCTGCCTGTACTCCTTAGT   862   aaatacacaATGTGGT   1732   gacagtcctgAGTACC   1733                   SKQITSKDVDLQKKYEPSPPATHR       ACTCTGCCAAGTCCACGCTGCACCTTCATCAGGGAAAGCAG       TGAAATCCTTGCT       GTACAACTCAAAAG                   GIITIEYFDPVSKSHKEADLTFEL       ATTACCTCCAAGGATGTTAGATCTTCAGAAAAAATATGAGC                   YGT       CCAGTCCCCCGCCACACATCGTGGAATAATCACTATCGAAT                           ACTTTGATCCGTTTCGAAGTCATGAAAGAGGGCGGATCTGA                           CTTTTGAGTTGTACGGTACT               424   YDL052c   SLC1   MSVIGRFLYYLRSVLVLLALAGCG   865   ATGAGTGTGATAGGTAGGTTCTTGTATTACTTGAGGTCCGT   864   aaatacaacaATGAGT   1734   gacagtcctgACAACA   1735                   FYGVIASILCTLIGHQHLAQWITA       GTTGGTCGTACTGGCGCTTGCAGGCTGTGGCTTTTACGGTG       GTATAGGTAGGTT       GCGCAGTAATCCA                   RC       TAATCGCCTCTATCCTTTGCACGTTAATCGGTAAGCAACAT                           TTGGCTCAGTGATTACTGCGCGTTGT               425   YRD032c   PST2   MPHVAIIIYTLGHVAATAEKEKKG   867   ATGCCAAGAGTAGCTATCATCATTTACACACTATATGGTCA   866   aaatacaacaATGCCA   1736   gacagtcctgATCATA   1737                   IEAAGGSADIYQVEETLSPEVVKA       CGTTGCTGCCACCGCAGAGGCAGAAAAGAAGGGAATTGAAG       AGAGTAGCTATCAT       TTCTGTCAACGTAT                   LGGAPKDYPIATQDTLTEYD       CCGCTGGAGGCTCTGCAGACATTTATCAAGTCGAGGAAACG                           TTGTCTCCAGAAGTTGTTAAGGCGCTTGGCGGTGCTCCAAA                           GCCAGATTACCCAATTGCCACTCAAGATACGTTGACAGAAT                           ATGAT               426   YDR056c       MLVRLLRVILLASMVFCADILQLS   56   ATGCTTGTGCGGCTGTTGCGTGTGATTTATTGGCCAGCATG   55   aaatacaacaATGCTT   1738   gacagtcctgTTCAAT   1739                   YSDDAKDAIPLGTFEIDSTSDGHV       GTTTTCTGTGCTGATATTTTACAATTAAGCTATTCAGATGA       GTGCGGCTGTT       CTGGGCATTCAAAC                   IVTTNVIQDVEVSGEYCLNAQIE       TGCGAAAGACGCTATACCCTAGGAACATTTGAGATTGATAG                           TACATCCGATGGGAATGTTACAGTAACAACGTTAATATACA                           GGATGTTGAAGTTTCTGGAGAATACTGTTTGAATGCCCAGA                           TTGAA               427   YDR057w   YOS9   MQAKIIYALSAISALIPLGSSLLA   62   ATGCAAGCTAAAATTATATATGCTCTGAGCGCAATTTCTGC   61   aaatacaacaATGCAA   1740   gacagtcctgATATCC   1741                   PIEDPIVSHKYLISYIDEDDWSDR       GTTGATTCCGTTAGGATCATCACTATTAGCACCTATAGAAG       GCTAAAATTATAT       CGAGTTCATGACAG                   ILQWQSVRRSGY       ACCCCATAGTATCGAATAAGTACCTCATATCTTACATCGAT                           GAGGACGACTGGAGTGATAGGATATTACAAAATCAGTCTGT                           CATGAACTCGGGATAT               428   YDR196c       MLVVGLTGGICGKSTVSRRLRDXY   869   ATGCTGGTAGTGGGATTGACAGGTGGGATCGCTTGTGGTAA   868   aactacaacaATGCTG   1742   gacgtcctgTTTGTCC   1743                   KLPIVADKIARQVVEPGQNAYDQI       GAGCACAGTGTCGAGAAGACTCAGAGACAAATACAAACTAC       GTAGTGGGATTGA       TTGAAGTATAACA                   VLYFXDK       CCATTGTTGATGCGGACAAGATTGCTAGACAAGTGGTCGAA                           CCAGGACAGAATGCTTATGATCAAATTGTGTTATACTTCAA                           GGACAAA               429   YDR221w       MVSRFSLFLLLIEQSPLVASLQQS   871   ATGGTGAGCATGTTCTCATTATTTCTGCTATTAATTGAGCA   870   aaatacaacaATGGTA   1744   gacagtcctgCTCATC   1745                   QRHIVGVPWEKQHLYDSREPDLTK       ATCGCCGCTTGTAGGGTCATTGCAACAAAGTCAACGGCATA       GCATGTTCTCATT       TGAACCATCAGGAC                   KHCLMHEDIVLDISQINDGVCDCP       TAGTGGGTGTTCCCTGGGAAAAGCAGCACTTGTATGACTCG                   DGSDE       AATGAACCAGATTTGACTAAATGGCACTGTTTGAACCACGA                           AGATATCGTATTGGATATAAGCCAGATTAATGATGGGTTTG                           TGATTGTGCTGATGGTTCAGATGAG               430   YDR245w   MNN10   MSSVPUNSQLPISHKHLEYDEDEK   873   ATGTDCTAGTGTACCTTATAATTCCCAACTTCCTATATCCA   872   aatacaacaATGTCAG   1746   gacagtcctgGGAGTT   1747                   KSRGSKLGLKYKNIYKRKTLCSSL       ACCATCTAGAGTACGATGAAGATGAAAAGAAGAGCAGAGGC       TGTACCTTATAA       GATGCTAGAACCAG                   ARWRKLILLISLALFLFIWISDST       TCAAAACTAGGCCTGAAATATAAAATGATATACTGGAAAAC                   ISRNPSTTISFOGCNSNDKKLSNT       TTTATGCAGTTCGCTAGCGAGATGGAGAAAGCATAATACTA                   GSSIKS       TTAATATCTTTAGCTTTGTTTTTATTCATATGGATAAGCGA                           TTCCACCATAAGCAGAAATCCATCTACCACAAGTTTTCAAG                           GCCAAAATAGTAACGATAATAAGTTGAGTAATACTGGTTCT                           AGCATCAATCC               431   YDR294c   DPL1   MSGVSNKTVSIIHGNYGMPIHLLR   875   ATGAGTGGAGTATCAAATAAAACAGTATCAATTAATGGTTG   874   aaatacaacaATGAGT   1748   gacagtcctgGTTGTA   1749                   EEGDFADFMILTINELKIAIHGYR       GTATGGCATGCCAATTCATTTACTAAGGGAAGAAGGCGACT       GGAGTATCAAATAA       CCATGGGTATTTC                   NTPWYN       TTGCCCAGTTTATGATTCTAACCATCAACGAATTAAAAATA                           GCCATACATGGTTACCTCAGAAATACCCCATGGTACAAC               432   YDR304c   CPR5   MKLQFFSFITLFACLFTTAIFAKE   46   ATGAAGCTTCAATTTTTTTCCTTTATTACCTTATTTGCTTG   45   acatacaacaATGAAC   1750   gacagtcctgAATTCT   1751                   DTAEDPEITHKVYFDINKGDKQIG       TCTCTTCACAACAGCCATTTTTGCGAAAGAGGACACGGCAG       TTCAATTTTTTTTC       ACCAATTTGTTTAT                   RI       AAGATCCTGAGATCACACACAAGGTCTACTTGACATTAATC                           ACGGTGATAAACAAATTGGTAGAATT               433   YDR518w   EUG1   MDVTTRFISAIVSFCLFASFTLAE   50   ATGCAAGTGACCACAAGATTTATATCTGCGATAGTCTCGTT   49   aaatacaacaATGCAA   1752   gacagtcctgATGAGA   1753                   NSARATPGSDLLVLTEKKFKSFIE       TTGCCTGTTTGCTTCTTTCACGTTGGCTGAAAACAGCGCAA       GTGACCACAAGATT       TTCGATGAATGATT                   SH       GAGCTACGCCGGATCAGATTTACTCGTTCTAACAGAGAAGA                           AATTTAAATCATTCATCGAATCTCAT               434   YDR519w   FPR2   MKFNIYLFVTFFSTILAGSLSDLE   94   ATGATGTTTAATATTTACCTTTTCGTCACTTTTTTTTCCAC   93   aaatacaacaATGATG   1754   gacagtcctgTCTTGA   1755                   IGIIKRIPVEDCLIKARPGDKVKV       CATTCTTGCAGGTTCCCTGTCAGATTTGGAAATCGGTATTA       TTTAATATTTACCT       ATAACTGAGTCAA                   HYTGSLLESGTVFDSSYSR       TCAAGAGAATACCGGTAGAAGATTGCTTAATTAAGGCAATG                           CCAGGTGATAAAGTTAAGGTTCATTATACAGGATCTTTTAT                           TAGAATCGGGAACTGTATTTGACTCAAGTTATTCAAGA               435   YEL036c   ANP1   MKYNNRKLSFNPTTVSIAGTLLTV   877   ATGAAGTATAATAACAGAAAACTCTCGTTCAACCCTACCAC   876   aaatacaacaATGAAG   1756   gacagtcctgATCTTT   1757                   FFLTRLVLSFFSISLFQLVTFQGI       AGTAAGTATCGCTGGAACGTTGCTTACGGTGTTCTTTCTCA       TATAATAACAGAA       GTTGCCTTGGTAAT                   FKPYVPDFKNYTPSVEFYDLRNYQ       CAAGACTCGTGCTTTCGTTCTTCTCGATATCGCTATTFCAG                   GNKD       CTGGTAACTTTCCAAGGAATCTTCAAGCCCTATGTTCCAGA                           TTTTAAAATACTCCCAGAGCGTAGAGTTCTACGACCTACGA                           AATTACCAAGGCAACAAAGAT               436   YEL043w       MPVSVITTVLAGLNLSYRLYKFLT   879   ATGCCAGTCTCTGTAATAACCACGGTTTTAGCATGTCTGTG   878   aaatacaacaATGCCA   1758   gacagtcctgCTGGAAGTA   1759                   IPVSSIVSTLKIKTPPATKVSIDK       GCTCTCTTATAGGCTCTATAAGTTTCTCACTATTCCTGTGT       GTCTCTGTAATAAC       GTCCTCCG                   IATDSVTIHWENEPVKAEDNGS       CCAGCATCGTCTCCACTTTGAAGATCAAAACTCCACCGGCA                           ACAAAAAGTGTCTATCGACAAAATAGCCACGGATTCAGTGA                           CCATTCATTGGGAGAACGAACCTGTAAAAGCGGAGGAACAA                           TGCAGT               437   YER053c-a       MQDLEIFLSIFAFIFVFYGAHRTV   881   ATGCAAGATTTAGAGATTTTTTGAGTATTTCGCTTTCATTT   880   aaatacaacaATGCAA   1760   gacagtcctgCTGCAA   1761                   NNRKKSDVPYLQ       TCGTTTTCTACTTGGTGCTCATAGAACAGTCATGAACAGAA       GATTTAGAGATTTT       GTAAGGAACATCGC                           ACAAGAGCGATGTTCCTTACTTGCAG               438   YGL001c   ERG26   MSKIDSVLIIGGSGFLGLHIQQFF   883   ATGTCAAAGATAGATTCAGTTTAATTATCGGTGGTTCTGGT   882   aaatacaacaATGTCA   1762   gacagtcctgTTTACT   1763                   DINPKPDIHIFDVRDLPEKLSKQF       TTTCTTGGATTGCACTTAATTCAGCAATTTTTTGATATTAA       AAGATAGATTCAGT       TTCGTTAATTGCGT                   TFNVDDIKFHKGDLTSPDDMENAI       TCCTAAGCCAGACATCCACATTTTTGATGTTAGAGATCTCC                   NESK       CTGAAAAACTTTCAAAACAGTTTACTTTTAATGTAGACGAC                           ATAAAATTCATAAGGGTGATTTAACATCACCTGATGATATG                           AAAAACGCAATTAACGAAAAGTAAA               439   YGL027c   CWH41   HLISKSKMFKTFWILTSIVLLASA   885   ATGCTTATTTCAAAATCTAAGATGTTTAAACATTTTGGATA   884   aaatacaacaATGCTT   1764   gacagtcctgACTTTC   1765                   TVDISKLQEFEEYQKFTNESLLWA       CTAACCAGCATAGTTCTCCTGGCATCTGCCACCGTTGATAT       ATTTCAAAATCTAA       ATGGACATATCTGG                   PYRSNCYFGKRRYVHES       TAGTAAACTACAAGAATTCGAAGAATATCAAAAGTTCACGA                           ATGAATCTTTACTGTGGGCACCGTATAGATCCAATTGTTAC                           TTTGGTATGAGCCCCAGATATGTCCATGAAAGT               440   YGL038c   OCH1   MSRKLSHLIATRKSKTIVVTVLLI   887   ATGTCTAGGAAGTTGTCCCACCTGATCGCTACAAGGAAATC   886   aaatacaacaATGTCT   1786   gacagtcctgATCACG   1767                   YSLLTFHSNKRLLSQFYPSKDDFQ       AAAAACAATAGTCGTAACCGGTACTTCTTATTTATTCTTTG       AGGAAGTTGTCCCA       TAAATTATGCAATT                   TLLPTTSHSQDIHLKKQITVKKKK       TTGACATTTCACTTGTCAAACAAAAGGCTGCTTTCTCAGTT                   QRLHNRD       TTACCCTAGCAAAAGATGATTTCAAGCAAACTCTTCTCCCT                           ACGACTTCTCATTCACAAGATATAAATTTGAAGAACAAATT                           ACAGTTAACAAGAAAAAAAATCAATTGCATAATTTACGTGA                           T                  
 
      The gene regions encoding secretory signal peptides shown in Table 3 were amplified via PCR from the  Saccharomyces cerevisiae  genomic DNA. The sequences of the synthetic primers for the gene regions encoding secretory signal peptides used in PCR are as shown in Table 3. Each forward primer comprises at its 5′ end a sequence complementary to a 10-bp region encompassing the 3′ end of the HSP12 promoter contained in the pCLuRA-s plasmid. Each reverse primer comprises at its 5′ end a sequence complementary to a 10-bp region encompassing the 5′ end of the mature CLuc gene.  
      PCR was carried out using 50 μl of a reaction solution comprising 300 nM of each primer, 1 mM MgSO 4 , 200 μM dNTP, 1 ng of yeast genomic DNA (derived from the  Saccharomyces cerevisiae  S288C strain, Invitrogen), and 1× buffer and KOD-Plus-1U included in the KOD-Plus-. The PCR cycle comprised: the first step at 94° C. for 2 minutes; 35 cycles of the second step at 94° C. for 15 seconds (denaturation), at 50° C. for 30 seconds (annealing), and at 68° C. for 1 minute (elongation); and the third step at 68° C. for 5 minutes. Thus, DNA fragments independently comprising at both of its ends 10-bp regions complementary to the HSP12 promoter and the mature CLuc gene contained in the pCLuRA-s plasmid and gene regions encoding secretory signal peptides were obtained. Hereafter, these DNA fragments are referred to as “1st PCR products.” 
      As the controls, a DNA fragment having at both of its ends 10-bp regions complementary to the HSP12 promoter and the mature CLuc gene and a gene region (nucleotide sequence: SEQ ID NO: 1768; amino acid sequence: SEQ ID NO: 1769) encoding the c-factor-derived secretory signal peptide (hereafter referred to as “DNA fragment E”) or a gene region (nucleotide sequence: SEQ ID NO: 1770; amino acid sequence: SEQ ID NO: 1771) encoding secretory signal peptide derived from yeast virus (M28 virus) toxin (K28 prepro-toxin) (hereafter referred to as “DNA fragment F”) used in conventional expression systems in yeast, as with the 1st PCR products, was prepared in the following manner.  
      DNA fragment E was amplified via PCR from the pCLuRA plasmid (see International Application Number: PCT/JP2006/311597, claiming the priority right of JP Patent Application No. 2005-169768).  
      The following synthetic primers were used. MF(ALPHA)1 Sig. F comprises at its 5′ end a sequence complementary to a 10-bp region encompassing the 3′ end of the HSP12 promoter contained in the pCLuRA-s plasmid. Also, MF(ALPHA)1 Sig. R comprises at its 5′ end a sequence complementary to a 10-bp region encompassing the 5′ end of the mature CLuc gene.  
                              MF(ALPHA)1 Sig. F:               aaatacaaca-ATGAGATTTCCTTCAATTTT   (SEQ ID NO: 1772)               MF(ALPHA)1 Sig. R:       gacagtcctg-AGCTTCAGCCTCTCTTTTCT   (SEQ ID NO: 1773)          
 
      PCR was carried out using 50 μl of a reaction solution comprising 300 nM of each primer, 1 mM MgSO 4 , 200 μM dNTP, 1 ng of the pCLuRA plasmid, and 1× buffer and KOD-Plus-1U included in the KOD-Plus-. The PCR cycle comprised: the first step at 94° C. for 2 minutes; 30 cycles of the second step at 94° C. for 15 seconds (denaturation), at 50° C. for 30 seconds (annealing), and at 68° C. for 1 minute (elongation); and the third step at 68° C. for 5 minutes. Thus, a DNA fragment E having at both of its ends 10-bp regions complementary to the HSP12 promoter and the mature CLuc gene contained in the pCLuRA-s plasmid and the gene regions encoding α-factor-derived secretory signal peptides, as with the 1st PCR product, was obtained.  
      DNA fragment F was prepared in the following manner.  
      A low-temperature-inducible expression vector, the aforementioned pLTex321 s vector (see Patent Document 2), was cleaved with XhoI and SphI, a DNA fragment was fractionated via agarose gel electrophoresis, and a vector fragment of approximately 7.3 kbp was obtained. This vector fragment is hereafter referred to as “DNA fragment G.” 
      Subsequently, the following synthetic DNAs were prepared in order to introduce tags for purifying the expressed proteins, i.e., the 6× His tag and the V5 antigen tag, into DNA fragment G.  
      V5-H tag F:  
                          (SEQ ID NO: 1774)                         TCGAGGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGT                   ACCGGTCATCATCACCATCACCATTGAGCATG          
 
      V5-H tag R:  
                          (SEQ ID NO: 1775)                         CTCAATGGTGATGGTGATGATGACCGGTACGCGTAGAATCGAGACCGAGG                   AGAGGGTTAGGGATAGGCTTACCC          
 
      These synthetic DNAs were annealed, the double-stranded synthetic DNA was ligated to DNA fragment G using the DNA Ligation Kit ver. 2.1, and the ligation product was introduced into  E. coli  DH5α.  
      The resulting transformant was cultured overnight, plasmids were extracted using the GenElute Plasmid Miniprep Kit, and a transformant having a plasmid of interest was identified based on the restriction enzyme cleavage pattern and via nucleotide sequence analysis. The pLTex321sV5H vector was prepared from the transformant.  
      The resulting vector, pLTex321sV5H, was cleaved with SmaI and EcoRI, a DNA fragment was fractionated via agarose gel electrophoresis, and a vector fragment of approximately 7.4 kbp was obtained. This vector fragment is hereafter referred to as “DNA fragment H.” 
      Subsequently, the following synthetic DNAs were prepared in order to introduce the gene region encoding the preprotoxin-derived secretory signal peptide into DNA fragment H.  
      K28 PPT Sig. F:  
                          (SEQ ID NO: 1776)                         ATGGAATCTGTTTCTTCTTTGTTTAATATTTTTTCTACTATTATGGTTAA                   TTATAAATCTTTGGTTTTGG          
 
      K28 PPT Sig. R:  
                          (SEQ ID NO: 1777)                         GGAATTCCTGCAGCCCGGGCAAATTAGAAACAGACAACAAAGCCAAAACC                   AAAGATTTATAATTAACCAT          
 
      These synthetic DNAs were annealed, and the 3′ ends of the strands of the annealed double-stranded synthetic DNA were subjected to elongation using the Klenow fragment (TOYOBO). Thereafter, the resulting DNA fragment was cleaved with EcoRI, the cleavage product was ligated to DNA fragment H using the DNA Ligation Kit ver. 2.1, and the resultant was introduced into  E. coli  DH5α.  
      The resulting transformant was cultured overnight, plasmids were extracted using the GenElute Plasmid Miniprep Kit, and a transformant having a plasmid of interest was identified based on the restriction enzyme cleavage pattern and via nucleotide sequence analysis. The pLTex321sV5H K28 vector was prepared from the transformant.  
      Further, the 3′ end of the gene region encoding the preprotoxin-derived secretory signal peptide, which had been introduced into the pLTex321sV5H K28 vector, was elongated via PCR.  
      The following synthetic primers were used. K28 Sig. F is a 24-bp region encompassing the 5′ end of the gene region encoding the preprotoxin-derived secretory signal peptide in the pLTex321sV5H K28 vector. K28 Sig. R (36) comprises a DNA sequence complementary to 25-bp region encompassing the 3′ end of the gene region encoding the preprotoxin-derived secretory signal peptide in the pLTex321sV5H K28 vector, a DNA sequence encoding 5 amino acid residues to be elongated, and a DNA sequence containing SmaI, PstI, and EcoRI restriction enzyme cleavage sites.  
      K28 Sig. F: ATGGAATCTGTTTCTTCTTTGTTT (SEQ ID NO: 1778)  
      K28 Sig. R (36): GGAATTCCTGCAGCCCGGG-ACCTCTAGCATATTT-CAAATTAGAAACAGACAACAAAGCC (SEQ ID NO: 1779)  
      PCR was carried out using 50 μl of a reaction solution comprising 300 nM of each primer, 1 mM MgSO 4 , 200 μM dNTP, 1 ng of the pLTex321sV5H K28 vector, and 1× buffer and KOD-Plus-1U included in the KOD-Plus-. The PCR cycle comprised: the first step at 94° C. for 2 minutes; 30 cycles of the second step at 94° C. for 15 seconds (denaturation), at 50° C. for 30 seconds (annealing), and at 68° C. for 30 seconds (elongation); and the third step at 68° C. for 5 minutes.  
      The DNA fragment obtained via such PCR was treated with EcoRI, ligated to DNA fragment H using the DNA Ligation Kit ver. 2.1, and introduced into  E. coli  DH5α.  
      The resulting transformant was cultured overnight, plasmids were extracted using the GenElute Plasmid Miniprep Kit, and a transformant having a plasmid of interest was identified based on the restriction enzyme cleavage pattern and via nucleotide sequence analysis. The pLTex321sV5H K28L vector was prepared from the transformant.  
      The pLTex321sV5H K28L vector was used as a template, and a DNA fragment (DNA fragment F) containing a gene region encoding the preprotoxin-derived secretory signal peptide was amplified via PCR. The following synthetic primers were used. K28L Sig. F comprises at its 5′ end a sequence complementary to a 10-bp region encompassing the 3′ end of the HSP12 promoter contained in the pCLuRA-s plasmid. Also, K28L Sig. R comprises at its 5′ end a sequence complementary to a 10-bp region encompassing the 5′ end of the mature CLuc gene.  
                              K28L Sig. F:               aaatacaaca-ATGGAATCTGTTTCTTCTTT   (SEQ ID NO: 1780)               K28L Sig. R:       gacagtcctg-ACCTCTAGCATATTTCAAAT   (SEQ ID NO: 1781)          
 
      PCR was carried out using 50 μl of a reaction solution comprising 300 nM of each primer, 1 mM MgSO 4 , 200 μM dNTP, 1 ng of the pLTex321sV5H K28L vector, and 1× buffer and KOD-Plus-1U included in the KOD-Plus-. The PCR cycle comprised: the first step at 94° C. for 15 seconds; 30 cycles of the second step at 94° C. for 15 seconds (denaturation), at 50° C. for 30 seconds (annealing), and at 68° C. for 1 minute (elongation); and the third step at 68° C. for 5 minutes. Thus, a DNA fragment (DNA fragment F) having at both of its ends 10-bp regions complementary to the HSP12 promoter and the mature CLuc gene contained in the pCLuRA-s plasmid and the gene region encoding the preprotoxin-derived secretory signal peptide, as with the 1st PCR product, was obtained.  
      In order to elongate the regions complementary to the pCLuRA-s plasmids at both ends of the DNA fragment containing the gene region encoding each secretory signal peptide, amplification was further carried out via PCR.  
      The following synthetic primers were used. 2nd PCR F comprises a sequence complementary to a 50-bp region encompassing the 3′ end of the HSP12 promoter region. 2nd PCR R comprises a sequence complementary to a 50-bp region encompassing the 5′ end of the mature CLuc gene.  
      2nd PCR F:  
                          (SEQ ID NO: 1782)                         TTCGATAATCTCAAACAAACAACTCAAAACAAAAAAAACT-AAATACAAC                   A          
 
      2nd PCR R:  
                          (SEQ ID NO: 1783)                         CAGGAAGTTGGAACTGTGTTTGGTGGATCAGGTTCGTAAG-GACAGTCCT                   G          
 
      PCR was carried out using 50 μl of a reaction solution comprising 300 nM of each primer, 1 mM MgSO 4 , 200 μM dNTP, a 10-fold diluted 1st PCR product, 1 μl of DNA fragment E or DNA fragment F, and 1× buffer and KOD-Plus-1U included in the KOD-Plus-. The PCR cycle comprised: the first step at 94° C. for 2 minutes; 35 cycles of the second step at 94° C. for 15 seconds (denaturation), at 50° C. for 30 seconds (annealing), and at 68° C. for 1 minute (elongation); and the third step at 68° C. for 5 minutes. Thus, a DNA fragment having at both of its ends 50-bp regions complementary to the HSP12 promoter and the mature CLuc gene contained in the pCLuRA-s plasmid and the gene region encoding each of 440 types of secretory signal peptides, a DNA fragment comprising a gene region encoding the α-factor-derived secretory signal peptide, or a DNA fragment comprising a gene region encoding the preprotoxin-derived secretory signal peptide was obtained. Hereafter, these DNA fragments are referred to as “2nd PCR products.” 
     Example 3  
     Construction of Secretory Signal Peptide Library  
      Using the 2nd PCR products obtained in Example 2 and the reporter vector, pCLuRA-s, prepared in Example 1, the secretory signal peptide library using the  Saccharomyces cerevisiae  host was constructed in the following manner.  
      The pCLuRA-s plasmid was cleaved with BamHI and HindIII, a DNA fragment was fractionated via agarose gel electrophoresis, and a DNA fragment of approximately 7.3 kbp was obtained. Hereafter, this DNA fragment is referred to as “DNA fragment I.” 
      As the host of this library the  Saccharomyces cerevisiae  BY4743 PEP4Δ PRB1Δ strain was used. The BY4743 PEP4Δ PRB1Δ strain was prepared by producing the BY4741 PEP4Δ PRB1Δ strain and the BY4742 PEP4Δ PRB1Δ strain in which the PEP4 and PRB1 genes encoding major protease in the  Saccharomyces cerevisiae  BY4741 strain (Invitrogen) and the BY4742 strain (Invitrogen) had been disrupted by the method of Hegemann et al. (http://mips.gsf.de/proj/yeast/info/tools/hegemann/loxp_kanmx.html), and coupling these strains.  
      Using, as a template, the pUG6 plasmid (http://mips.gsf.de/proj/yeast/info/tools/hegemann/loxp_kanmx.html), a DNA fragment that is necessary for disrupting the PEP4 gene was first amplified via PCR. The following synthetic primers were used. dPEP4 kan F has at its 5′ end a sequence complementary to a 40-bp upstream region of the PEP4 gene and at its 3′ end a sequence complementary to a 19-bp upstream region of the loxP-kanMX-loxP module region of pUG6. dPEP4 kan R has at its 5′ end a sequence complementary to a 40-bp downstream region of the PEP4 gene and at its 3′ end a sequence complementary to a 22-bp downstream region of the loxP-kanMX-loxP module region of pUG6.  
      dPEP4 kan F:  
                          (SEQ ID NO: 1784)                         ATTTAATCCAAATAAAATTCAAACAAAAACCAAAACTAAC-CAGCTGAAG                   CTTCGTACGC          
 
      dPEP4 kan R:  
                          (SEQ ID NO: 1785)                         GGCAGAAAAGGATAGGGCGGAGAAGTAAGAAAAGTTTAGC-GCATAGGCC                   ACTAGTGGATCTG          
 
      PCR was carried out using 50 μl of a reaction solution comprising 300 nM of each primer, 1 mM MgSO 4 , 200 μM dNTP, 1 ng of the pUG6 plasmid, and 1× buffer and KOD-Plus-1U included in the KOD-Plus-. The PCR cycle comprised: the first step at 94° C. for 15 seconds; 30 cycles of the second step at 94° C. for 15 seconds (denaturation), at 50° C. for 30 seconds (annealing), and at 68° C. for 2 minutes (elongation); and the third step at 68° C. for 5 minutes. Thus, a DNA fragment having at both of its ends sequences complementary to 40-bp upstream and downstream regions of the PEP4 gene and containing the loxP-kanMX-loxP module region was obtained. This DNA fragment was used to disrupt the PEP4 genes of the BY4741 strain and the BY4742 strain by the method of Hegemann et al. to obtain the BY4741 PEP4Δ strain and the BY4742 PEP4Δ strain.  
      Further, a DNA fragment, which is necessary for disrupting the PRB1 gene, was prepared via PCR. The following synthetic primers were used. dPRB1 kan F has at its 5′ end a sequence complementary to a 40-bp upstream region of the PRB1 gene and at its 3′ end a sequence complementary to a 19-bp upstream region of the loxP-kanMX-loxP module region of pUG6. dPRB1 kan R has at its 5′ end a sequence complementary to a 40-bp downstream region of the PRB1 gene and at its 3′ end a sequence complementary to a 22-bp downstream region of the loxP-kanMX-loxP module region of pUG6.  
      dPRB 1 kan F:  
                          (SEQ ID NO: 1786)                         CAATAAAAAAACAAACTAAACCTAATTCTAACAAGCAAAG-CAGCTGAAG                   CTTCGTACGC          
 
      dPRB 1 kan R:  
                          (SEQ ID NO: 1787)                         AAGAAAAAAAAAAGCAGCTGAAATTTTTCTAAATGAAGAA-GCATAGGCC                   ACTAGTGGATCTG          
 
      PCR was carried out using 50 μl of a reaction solution comprising 300 nM of each primer, 1 mM MgSO 4 , 200 μM dNTP, 1 ng of the pUG6 plasmid, and 1× buffer and KOD-Plus-1U included in the KOD-Plus-. The PCR cycle comprised: the first step at 94° C. for 15 seconds; 30 cycles of the second step at 94° C. for 15 seconds (denaturation), at 50° C. for 30 seconds (annealing), and at 68° C. for 2 minutes (elongation); and the third step at 68° C. for 5 minutes. Thus, a DNA fragment having at both of its ends sequences complementary to 40-bp upstream and downstream regions of the PRB1 gene and the loxP-kanMX-loxP module region was obtained.  
      This DNA fragment was used to disrupt the PRB1 genes of the BY4741 PEP4Δ strain and the BY4742 PEP4Δ strain by the method of Hegemann et al. to obtain the BY4741 PEP4Δ PRB1Δ strain and the BY4742 PEP4Δ PRB1Δ strain.  
      The BY4741 PEP4Δ PRB1Δ strain was mated to the BY4742 PEP4Δ PRB1Δ strain by a conventional technique to obtain the BY4743 PEP4Δ PRB1Δ diploid strain.  
      Transformation was carried out using the 2nd PCR products of the BY4743 PEP4Δ PRB1Δ strain and DNA fragment I in the following manner.  
      The BY4743 PEP4Δ PRB1Δ strain was cultured in a YPD medium (1% yeast extract, 2% peptone, and 2% glucose) at 30° C. to a stationary phase, the strain was recovered from 250 μl of the culture solution via centrifugation, the strain was washed with 100 μl of sterile water, and the strain was further recovered via centrifugation.  
      The obtained strain was suspended in 60 μl of a yeast transformation solution (33.3% polyethylene glycol (PEG) 4,000, 100 mM of lithium acetate, 250 μg/ml of carrier DNA (Clontech), 10 ng of DNA fragment I, and 2 μl of each 10-fold diluted 2nd PCR product) and then cultured at 30° C. for 30 minutes. Thereafter, 6 μl of dimethyl sulfoxide (DMSO) was added, and culture was further carried out at 42° C. for an additional 1 hour. Thereafter, 1 ml of the SD+HL medium (0.67% yeast nitrogen base without amino acid, 2% glucose, 0.002% L-histidine HCl, and 0.01% L-leucine) was inoculated with 10 μl of the culture solution, and culture was carried out at 30° C. for 3 days. Thus, homologous recombination of DNA fragment I and each 2nd PCR product took place in the host BY4743 PEP4Δ PRB1Δ strain. Under the control (downstream) of the HSP12 promoter, the BY4743 PEP4Δ PRB1Δ strain sustaining a reporter plasmid in which the gene region encoding each secretory signal peptide has been fused to the 5′ terminus of the mature CLuc gene was selectively allowed to grow by introducing a uracil requirement. Thus, the secretory signal peptide library utilizing the  Saccharomyces cerevisiae  host was constructed.  
      Some of the resulting strains were subjected to sequence analysis of reporter plasmids, and the generation of the plasmid of interest was confirmed in the yeast cell.  
     Example 4  
     Evaluation of the Secretion Ability of the Secretory Signal Peptide  
      With the use of the secretory signal peptide library using the  Saccharomyces cerevisiae  host constructed in Example 3, the secretion ability of the secretory signal peptide was evaluated with the use of mature CLuc (secreted luciferase) in the following manner.  
      One ml of SD+HL+PPB medium (0.67% yeast nitrogen base without amino acid, 2% glucose, 0.002% L-histidine HCl, 0.01% L-leucine, and 200 mM potassium phosphate buffer (pH 6.0)) was inoculated with the  Saccharomyces cerevisiae  (the secretory signal peptide library) sustaining a reporter plasmid containing the gene region encoding each secretory signal peptide prepared in Example 3, and culture was conducted at 30° C. for 3 days. This culture solution was designated as the preculture, and part thereof was used in the following culture.  
      One ml of SD+HL+PPB medium was inoculated with the culture solution (10 μl) obtained via the preculture and culture was conducted at 30° C. to the logarithmic growth phase.  
      Using the culture solution that had been cultured until the logarithmic growth phase, activity of the mature CLuc (secreted luciferase) secreted in the culture was assayed to evaluate the secretion ability of each secretory signal peptide.  
      Activity of luciferase was assayed in the following manner.  
      To the culture solution (20 μl), 80 μl of a 2.5 μM luciferin solution was added, and, 2 seconds thereafter, the luminescence level was assayed for 5 seconds. Simultaneously, 200 μl of the culture solution was used to assay the absorbance thereof at 600 nm, and the luminescence level was divided by the obtained value to normalize the luciferase activity value based on the absorbance.  
      The results are shown in Table 4. Table 4 shows the relative secretion efficiency of the secretory signal peptides (indicated in terms of systematic and common names of the genes from which these secretory signal peptides have been derived) at moderate culture temperature (30° C.) in relation to that of the α-factor-derived secretory signal peptide. The secretory signal peptides used in conventional secretory expression systems are shaded. The result of assaying the secretion efficiency of the α-factor-derived secretory signal peptide is shown in the column indicated as “α-factor” in terms of common names. The result of assaying the secretion efficiency of the preprotoxin-derived secretory signal peptide is shown in the column indicated as “K28L” in terms of common names.  
               TABLE 4                          Table 4: Secretion efficiency of each of secretory signal peptides at       moderate culture temperature (30° C.)                                         Secretion                   efficiency relative                   to α-factor-           Systematic gene   Common gene   derived secretory           name   name   signal peptide                                             YDR420w   HKR1   5.64           YBR187w       3.37           YGL126w   SCS3   3.27           YHR139c   SPS100   2.69           YGR014w   MSB2   2.66           YBR078w   ECM33   2.48           YNL300w   TOS6   2.46           YLR084c   RAX2   2.27           YMR008c   PLB1   2.04           YCR061w       2.00           YDR055w   PST1   1.91           YLR110c   CCW12   1.85           YNL160w   YGP1   1.77           YLR250w   SSP120   1.68           YBR042c   CST26   1.65           YDR134c       1.64           YBR296c   PHO89   1.54           YLR034c   SMF3   1.51           YBR243c   ALG7   1.49           YLR332w   MID2   1.44           YDR077w   SED1   1.41           YPL234c   TFP3   1.34           YDR261c   EXG2   1.32           YBR070c   SAT2   1.31           YGR189c   CRH1   1.27           YKL096w-a   CWP2   1.25           YLR300w   EXG1   1.25           YIL140w   AXL2   1.25           YHR181w   SVP26   1.24           YEL001c       1.23           YCL043c   PDI1   1.19           YGL032c   AGA2   1.19           YNL237w   YTP1   1.17           YIL090w       1.07           YDR518w   EUG1   1.07           YEL040w   UTR2   1.02           YOR190w   SPR1   1.02               α-factor   1.00           YHR079c   IRE1   0.98           YBR293w       0.96           YNR044w   AGA1   0.92           YFL041w   FET5   0.92           YNL066w   SUN4   0.89           YCR028c   FEN2   0.87           YNL327w   EGT2   0.86           YNR067c   DSE4   0.85           YKL209c   STE6   0.78           YNL190w       0.78           YAL058w   CNE1   0.77           YPR124w   CTR1   0.77           YCR017c   CWH43   0.76           YDR205w   MSC2   0.76           YJL078c   PRY3   0.74           YCR011c   ADP1   0.71           YNL322c   KRE1   0.71           YKL163w   PIR3   0.69           YER145c   FTR1   0.68           YER113c       0.65           YDR056c       0.64           YDR304c   CPR5   0.63           YIL162w   SUC2   0.62           YLR286c   CTS1   0.62           YER118c   SHO1   0.61           YMR006c   PLB2   0.61           YOL011w   PLB3   0.61           YPL189w   GUP2   0.60           YDR057w   YOS9   0.59           YDR144c   MKC7   0.58           YNL219c   ALG9   0.56           YLR050c       0.54           YKL096w   CWP1   0.53           YMR307w   GAS1   0.53           YCL027w   FUS1   0.53           YNL238w   KEX2   0.52           YGL038c   OCH1   0.52           YMR058w   FET3   0.51           YDR349c   YPS7   0.51           YLR207w   HRD3   0.50           YFL051c       0.50           YJL159w   HSP150   0.50           YAR071w   PHO11   0.47               K28L   0.47           YDL072c   YET3   0.43           YBR093c   PHO5   0.41           YDR276c   PMP3   0.40           YAL053w       0.40           YHR110w   ERP5   0.40           YIL039w       0.39           YMR200w   ROT1   0.38           YDR519w   FPR2   0.38           YCR021c   HSP30   0.37           YFL026w   STE2   0.37           YOR008c   SLG1   0.37           YGL089c   MF(ALPHA)2   0.37           YPL187w   MF(ALPHA)1   0.36           YJR118c   ILM1   0.36           YNL291c   MID1   0.35           YDR367w       0.35           YEL004w   YEA4   0.34           YNL194c       0.34           YAL063c   FLO9   0.32           YHR143w   DSE2   0.32           YNR030w   ECM39   0.31           YBR183w   YPC1   0.29           YGL020c   MDM39   0.28           YIL027c   KRE27   0.28           YDR536w   STL1   0.27           YGR279c   SCW4   0.26           YGL027c   CWH41   0.26           YKL164c   PIR1   0.26           YBR092c   PHO3   0.26           YFR041c   ERJ5   0.25           YKL221w   MCH2   0.25           YPL130w   SPO19   0.25           YAL007c   ERP2   0.25           YBR229c   ROT2   0.25           YJL193w       0.24           YLR155c   ASP3-1   0.24           YDR221w       0.24           YDL212w   SHR3   0.21           YEL036c   ANP1   0.19           YLR413w       0.17           YLR120c   YPS1   0.17           YAR050w   FLO1   0.17           YJL174w   KRE9   0.16           YMR171c       0.16           YDL093w   PMT5   0.16           YKL187c       0.16           YGR282c   BGL2   0.15           YKL051w   SFK1   0.15           YBR067c   TIP1   0.15           YMR119w   ASI1   0.15           YJL158c   CIS3   0.15           YJL051w       0.15           YLR037c   DAN2   0.15           YLR390w-a   CCW14   0.14           YGL028c   SCW11   0.14           YDR534c   FIT1   0.14           YBR301w   DAN3   0.14           YNR018w       0.14           YEL002c   WBP1   0.14           YPR091c       0.13           YLR121c   YPS3   0.13           YIL147c   SLN1   0.12           YOL105c   WSC3   0.12           YMR149w   SWP1   0.12           YCL045c       0.11           YMR305c   SCW10   0.11           YBL017c   PEP1   0.10           YBR210w   ERV15   0.09           YLR214w   FRE1   0.09           YML116w   ATR1   0.09           YML012w   ERV25   0.09           YNL283c   WSC2   0.09           YOR010c   TIR2   0.09           YJL222w   VTH2   0.09           YJR150c   DAN1   0.08           YBR205w   KTR3   0.08           YNL142w   MEP2   0.08           YKL174c       0.07           YGR213c   RTA1   0.07           YMR243c   ZRC1   0.07           YLR414c       0.07           YCR069w   CPR4   0.07           YBR036c   CSG2   0.07           YER011w   TIR1   0.07           YKL220c   FRE2   0.06           YOL019w   TOS7   0.06           YKL178c   STE3   0.06           YLR087c   CSF1   0.06           YJL002c   OST1   0.06           YEL027w   CUP5   0.05           YCR034w   FEN1   0.05           YKL039w   PTM1   0.05           YHR140w       0.05           YBR054w   YRO2   0.05           YKL034w   TUL1   0.05           YOR149c   SMP3   0.05           YPR201w   ARR3   0.05           YBR241c       0.05           YLR242c   ARV1   0.05           YNR055c   HOL1   0.05           YMR266w   RSN1   0.05           YOR011w   AUS1   0.04           YIL005w   EPS1   0.04           YJR004c   SAG1   0.04           YOL030w   GAS5   0.04           YDR038c   ENA5   0.04           YDR245w   MNN10   0.04           YBR004c   FMP44   0.04           YPL096c-a   ERI1   0.04           YJL012c-a       0.04           YHL016c   DUR3   0.04           YLR459w   GAB1   0.04           YDR384c   ATO3   0.03           YDR040c   ENA1   0.03           YAL067c   SEO1   0.03           YEL017c-a   PMP2   0.03           YGL255w   ZRT1   0.03           YIL015w   BAR1   0.03           YKR102w   FLO10   0.03           YOR104w   PIN2   0.02           YJL171c       0.02           YDL052c   SLC1   0.02           YIR028w   DAL4   0.02           YHL042w       0.02           YBR040w   FIG1   0.02           YHR101c   BIG1   0.02           YIL170w   HXT12   0.02           YDL018c   ERP3   0.02           YOR085w   OST3   0.02           YBR021w   FUR4   0.02           YJR013w   GPI14   0.02           YER053c-a       0.02           YLR220w   CCC1   0.02           YHL035c   VMR1   0.02           YDR033w   MRH1   0.02           YIR019c   MUC1   0.02           YDL210w   UGA4   0.02           YER072w   VTC1   0.02           YEL065w   SIT1   0.02           YCR024c-a   PMP1   0.02           YHL036w   MUP3   0.02           YGL203c   KEX1   0.02           YJL062w   LAS21   0.02           YIL013c   PDR11   0.02           YDL035c   GPR1   0.01           YCR044c   PER1   0.01           YMR238w   DFG5   0.01           YAL018c       0.01           YML123c   PHO84   0.01           YOL013c   HRD1   0.01           YBR199w   KTR4   0.01           YKL046c   DCW1   0.01           YEL043w       0.01           YKR039w   GAP1   0.01           YKL004w   AUR1   0.01           YDR046c   BAP3   0.01           YDR072c   IPT1   0.01           YJR040w   GEF1   0.01           YCR037c   PHO87   0.01           YLL061w   MMP1   0.01           YJR151c   DAN4   0.01           YKR040c       0.01           YLL015w   BPT1   0.01           YML038c   YMD8   0.01           YGR121c   MEP1   0.01           YPL232w   SSO1   0.01           YDR331w   GPI8   0.01           YPL265w   DIP5   0.01           YGL008c   PMA1   0.01           YBR069c   TAT1   0.01           YDR090c       0.01           YGR191w   HIP1   0.01           YGR055w   MUP1   0.01           YOL084w   PHM7   0.01           YPR138c   MEP3   0.01           YEL063c   CAN1   0.01           YCR098c   GIT1   0.01           YJR158w   HXT16   0.01           YFL011w   HXT10   0.01           YKL065c   YET1   0.01           YGL002w   ERP6   0.01           YGL139w       0.01           YCL025c   AGP1   0.01           YDL199c       0.01           YBL089w   AVT5   0.01           YDL245c   HXT15   0.01           YCR023c       0.01           YBL042c   FUI1   0.01           YGR065c   VHT1   0.01           YMR319c   FET4   0.01           YNL318c   HXT14   0.01           YHR096c   HXT5   0.01           YFL050c   ALR2   0.01           YMR183c   SSO2   0.01           YDR093w   DNF2   0.01           YDR011w   SNQ2   0.01           YLR023c   IZH3   0.01           YOL103w   ITR2   0.01           YDR342c   HXT7   0.01           YHR094c   HXT1   0.01           YJR152w   DAL5   0.01           YBR068c   BAP2   0.01           YBR096w       0.01           YJL214w   HXT8   0.01           YNL280c   ERG24   0.01           YJL219w   HXT9   0.01           YKR093w   PTR2   0.01           YLR237w   THI7   0.01           YHR092c   HXT4   0.01           YKR050w   TRK2   0.01           YJR160c   MPH3   0.01           YDR343c   HXT6   0.01           YDL247w   MPH2   0.01           YER060w   FCY21   0.01           YOR328w   PDR10   0.01           YNL048w   ALG11   0.01           YJL129c   TRK1   0.01           YBR038w   CHS2   0.01           YDR345c   HXT3   0.01           YGR260w   TNA1   0.01           YBR086c   IST2   0.01           YOR153w   PDR5   0.01           YGR032w   GSC2   0.01           YBR140c   IRA1   0.00           YGL054c   ERV14   0.00           YER056c   FCY2   0.00           YJL093c   TOK1   0.00           YKR053c   YSR3   0.00           YEL069c   HXT13   0.00           YGR281w   YOR1   0.00           YIL047c   SYG1   0.00           YBR023c   CHS3   0.00           YCL038c   ATG22   0.00           YDL138w   RGT2   0.00           YDR497c   ITR1   0.00           YKL100c       0.00           YEL031w   SPF1   0.00           YBR041w   FAT1   0.00           YER166w   DNF1   0.00           YDR032c   PST2   0.00           YLR081w   GAL2   0.00           YFL017c   GNA1   0.00           YGR026w       0.00           YKL083w       0.00           YBR294w   SUL1   0.00           YGL077c   HNM1   0.00           YDR461w   MFA1   0.00           YER008c   SEC3   0.00           YPL221w   BOP1   0.00           YNR039c   ZRG17   0.00           YHR149c   SKG6   0.00           YOL156w   HXT11   0.00           YGL001c   ERG26   0.00           YDL194w   SNF3   0.00           YLR138w   NHA1   0.00           YJL094c   KHA1   0.00           YNL087w   TCB2   0.00           YER060w-a   FCY22   0.00           YOR016c   ERP4   0.00           YNR013c   PHO91   0.00           YPL092w   SSU1   0.00           YAL022c   FUN26   0.00           YNL268w   LYP1   0.00           YPR003c       0.00           YBR298c   MAL31   0.00           YOL075c       0.00           YMR011w   HXT2   0.00           YCR048w   ARE1   0.00           YNL270c   ALP1   0.00           YGR138c   TPO2   0.00           YOL122c   SMF1   0.00           YDR294c   DPL1   0.00           YMR274c   RCE1   0.00           YOL158c   ENB1   0.00           YDR126w   SWF1   0.00           YOR002w   ALG6   0.00           YLL052c   AQY2   0.00           YOL020w   TAT2   0.00           YNL192w   CHS1   0.00           YGL084c   GUP1   0.00           YDR264c   AKR1   0.00           YKR106w       0.00           YCR089w   FIG2   0.00           YPR194c   OPT2   0.00           YDR062w   LCB2   0.00           YPL274w   SAM3   0.00           YIL120w   QDR1   0.00           YIL121w   QDR2   0.00           YOR273c   TPO4   0.00           YKL217w   JEN1   0.00           YBR295w   PCA1   0.00           YLL028w   TPO1   0.00           YOL130w   ALR1   0.00           YPR156c   TPO3   0.00           YGR197c   SNG1   0.00           YGL161c   YIP5   0.00           YGL012w   ERG4   0.00           YKR105c       0.00           YPL249c   GYP5   0.00           YDL123w   SNA4   0.00           YNL145w   MFA2   0.00           YBR106w   PHO88   0.00           YOL092w       0.00           YLR353w   BUD8   0.00           YDR508c   GNP1   0.00           YBR043c   QDR3   0.00           YFL055w   AGP3   0.00           YJL117w   PHO86   0.00           YDR039c   ENA2   0.00           YBR132c   AGP2   0.00           YNL065w   AQR1   0.00           YBR180w   DTR1   0.00           YNR072w   HXT17   0.00           YOR348c   PUT4   0.00           YGR289c   MAL11   0.00           YGL198w   YIP4   0.00           YPL036w   PMA2   0.00           YGR227w   DIE2   0.00           YCR010c   ADY2   0.00           YGR217w   CCH1   0.00           YJR092w   BUD4   0.00           YGL010w       0.00           YDR233c   RTN1   0.00           YLL043w   FPS1   0.00           YOR034c   AKR2   0.00           YOR161c   PNS1   0.00           YNL159c   ASI2   0.00           YMR296c   LCB1   0.00           YCL069w       0.00           YPL176c   SSP134   0.00           YPL058c   PDR12   0.00           YNR019w   ARE2   0.00           YML072c   TCB3   0.00           YOR307c   SLY41   0.00           YDR406w   PDR15   0.00           YBR008c   FLR1   0.00           YKR051w       0.00           YIL030c   SSM4   0.00           YLR342w   FKS1   0.00           YHL003c   LAG1   0.00           YER181c       0.00           YOL081w   IRA2   0.00           YKR067w   GPT2   0.00           YOR301w   RAX1   0.00           YBR024w   SCO2   0.00           YER140w       0.00           YOR086c   TCB1   0.00           YOL002c   IZH2   0.00           YKR103w   NFT1   0.00           YKL212w   SAC1   0.00           YCR087w       0.00           YKR088c   TVP38   0.00           YNL260c       0.00           YNR056c   BIO5   0.00           YDL054c   MCH1   0.00           YPR198w   SGE1   0.00           YDR196c       0.00           YNR021w       0.00           YFL048c   EMP47   0.00           YLR452c   SST2   0.00                      
 
      In order to evaluate the secretion abilities of the secretory signal peptides at low temperature, the culture temperature for the culture solution was lowered to 15° C., and culture was further continued at 15° C. for 72 hours. Thereafter, luminescence and absorbance were assayed by the luciferase activity assay technique, and the normalized activity values were also obtained.  
      The results are shown in Table 5. Table 5 shows the relative secretion efficiency of secretory signal peptides (indicated in terms of systematic and common names of the genes from which these secretory signal peptides have been derived) at low culture temperature (15° C.) in relation to the α-factor-derived secretory signal peptide. The secretory signal peptides used in conventional secretory expression systems are shaded. The result of assaying the secretion efficiency of the α-factor-derived secretory signal peptide is shown in the column indicated as “α-factor” in terms of common names. The result of assaying the secretion efficiency of the preprotoxin-derived secretory signal peptide is shown in the column indicated as “K28L” in terms of common gene name.  
               TABLE 5                          Table 5: Secretion efficiency of each of secretory signal peptides       at low culture temperature (15° C.)                                         Secretion                   efficiency relative                   to α-factor-           Systematic gene   Common gene   derived secretory           name   name   signal peptide                                             YBR243c   ALG7   5.76           YNL237w   YTP1   5.67           YCL043c   PDI1   5.61           YKL096w   CWP1   5.55           YBR078w   ECM33   5.17           YLR250w   SSP120   5.05           YEL001c       4.76           YMR008c   PLB1   4.71           YNL238w   KEX2   4.70           YLR084c   RAX2   4.61           YGR189c   CRH1   4.01           YLR286c   CTS1   3.95           YMR006c   PLB2   3.78           YKL096w-a   CWP2   3.70           YCR028c   FEN2   3.64           YGL126w   SCS3   3.54           YMR200w   ROT1   3.46           YDR304c   CPR5   3.44           YLR110c   CCW12   3.33           YDR518w   EUG1   3.21           YEL040w   UTR2   3.16           YLR332w   MID2   3.00           YDR056c       2.97           YJL193w       2.83           YHR139c   SPS100   2.76           YGR014w   MSB2   2.69           YPL234c   TFP3   2.59           YGL032c   AGA2   2.58           YDR057w   YOS9   2.56           YDR055w   PST1   2.54           YHR110w   ERP5   2.50           YOL011w   PLB3   2.48           YDR134c       2.47           YBR296c   PHO89   2.42           YDR144c   MKC7   2.37           YDR077w   SED1   2.37           YDR276c   PMP3   2.35           YNL291c   MID1   2.35           YAL058w   CNE1   2.32           YLR300w   EXG1   2.31           YIL140w   AXL2   2.30           YBR187w       2.26           YBR070c   SAT2   2.19           YJR118c   ILM1   2.16           YOR190w   SPR1   2.14           YDR519w   FPR2   2.13           YIL090w       2.13           YER113c       2.06           YDR261c   EXG2   2.05           YDR420w   HKR1   2.04           YFL051c       2.03           YNL300w   TOS6   2.02           YGL038c   OCH1   1.96           YJL078c   PRY3   1.95           YNL219c   ALG9   1.88           YLR034c   SMF3   1.86           YBR229c   ROT2   1.84           YBR093c   PHO5   1.81           YFL041w   FET5   1.77           YJL174w   KRE9   1.70           YHR143w   DSE2   1.67           YNL327w   EGT2   1.66           YOR008c   SLG1   1.66           YER145c   FTR1   1.63           YNL160w   YGP1   1.61           YLR050c       1.57           YGL089c   MF(ALPHA)2   1.57           YER118c   SHO1   1.53           YAR071w   PHO11   1.53           YCR061w       1.53           YKL187c       1.51           YFL026w   STE2   1.48           YNR067c   DSE4   1.47           YIL039w       1.44           YDR349c   YPS7   1.42           YKL221w   MCH2   1.41           YIL027c   KRE27   1.40           YCL045c       1.38           YOL105c   WSC3   1.37           YEL027w   CUP5   1.35           YNR044w   AGA1   1.35           YMR119w   ASI1   1.34           YLR390w-a   CCW14   1.32           YLR207w   HRD3   1.31           YCL027w   FUS1   1.30           YNL283c   WSC2   1.24           YLR120c   YPS1   1.23           YLR413w       1.23           YDR367w       1.22           YAL007c   ERP2   1.22           YKL209c   STE6   1.20           YCR011c   ADP1   1.16           YNL194c       1.14           YHR181w   SVP26   1.14           YPL130w   SPO19   1.14           YEL004w   YEA4   1.13           YNL190w       1.10           YDR205w   MSC2   1.09           YIL162w   SUC2   1.09           YDR221w       1.04               α-factor   1.00           YJL051w       0.96           YBR205w   KTR3   0.95           YGL027c   CWH41   0.95           YGR282c   BGL2   0.90           YAL053w       0.86           YBR092c   PHO3   0.85           YPL189w   GUP2   0.85           YCR017c   CWH43   0.81           YJL222w   VTH2   0.80           YIL015w   BAR1   0.79           YJL159w   HSP150   0.78           YPR124w   CTR1   0.76           YBR042c   CST26   0.74           YKL163w   PIR3   0.73           YNR030w   ECM39   0.71           YNL322c   KRE1   0.70           YKR102w   FLO10   0.70           YLR414c       0.70           YEL002c   WBP1   0.70               K28L   0.70           YAR050w   FLO1   0.67           YCR021c   HSP30   0.64           YAL063c   FLO9   0.63           YMR171c       0.63           YBR210w   ERV15   0.63           YPR091c       0.62           YML012w   ERV25   0.61           YLR087c   CSF1   0.61           YDR534c   FIT1   0.61           YFR041c   ERJ5   0.59           YBR183w   YPC1   0.59           YBR293w       0.59           YJL002c   OST1   0.56           YMR307w   GAS1   0.56           YOR149c   SMP3   0.55           YDL072c   YET3   0.54           YLR121c   YPS3   0.53           YIL147c   SLN1   0.51           YLR214w   FRE1   0.51           YKL051w   SFK1   0.50           YGL020c   MDM39   0.48           YLR155c   ASP3-1   0.48           YDR536w   STL1   0.46           YNL066w   SUN4   0.46           YKL034w   TUL1   0.45           YDL212w   SHR3   0.45           YKL039w   PTM1   0.44           YCR069w   CPR4   0.43           YPL187w   MF(ALPHA)1   0.42           YMR305c   SCW10   0.39           YMR149w   SWP1   0.39           YHR079c   IRE1   0.38           YKL174c       0.36           YGR213c   RTA1   0.35           YML116w   ATR1   0.33           YBR241c       0.33           YJR004c   SAG1   0.33           YCR034w   FEN1   0.32           YGR279c   SCW4   0.32           YKL220c   FRE2   0.32           YKL164c   PIR1   0.32           YOL013c   HRD1   0.31           YEL036c   ANP1   0.31           YLR037c   DAN2   0.31           YDR245w   MNN10   0.31           YJR150c   DAN1   0.29           YOL019w   TOS7   0.28           YGL028c   SCW11   0.27           YLR459w   GAB1   0.26           YDL093w   PMT5   0.26           YCR044c   PER1   0.26           YJL171c       0.26           YBR301w   DAN3   0.26           YNR018w       0.24           YHR101c   BIG1   0.24           YBR067c   TIP1   0.24           YBR036c   CSG2   0.23           YNL142w   MEP2   0.22           YMR238w   DFG5   0.22           YHL042w       0.22           YGL139w       0.21           YML038c   YMD8   0.21           YJL158c   CIS3   0.20           YGL255w   ZRT1   0.20           YAL067c   SEO1   0.19           YLR242c   ARV1   0.19           YDR038c   ENA5   0.19           YIR019c   MUC1   0.18           YMR243c   ZRC1   0.18           YBL017c   PEP1   0.18           YOR011w   AUS1   0.17           YBR199w   KTR4   0.17           YOL030w   GAS5   0.18           YER011w   TIR1   0.16           YGL002w   ERP6   0.16           YJR013w   GPI14   0.16           YHR140w       0.16           YMR058w   FET3   0.15           YKL178c   STE3   0.15           YOR085w   OST3   0.15           YIL005w   EPS1   0.14           YOR010c   TIR2   0.14           YGL054c   ERV14   0.14           YNR055c   HOL1   0.13           YKL046c   DCW1   0.13           YIL170w   HXT12   0.12           YPR201w   ARR3   0.12           YDR040c   ENA1   0.12           YDL052c   SLC1   0.11           YOR104w   PIN2   0.11           YLL015w   BPT1   0.11           YIR028w   DAL4   0.11           YDL018c   ERP3   0.11           YJL012c-a       0.10           YKR040c       0.09           YBR054w   YRO2   0.09           YGL203c   KEX1   0.09           YKL100c       0.08           YHL035c   VMR1   0.08           YJL094c   KHA1   0.08           YBL089w   AVT5   0.07           YIL013c   PDR11   0.07           YHL036w   MUP3   0.07           YLR237w   THI7   0.07           YKR039w   GAP1   0.06           YKL083w       0.06           YDR072c   IPT1   0.06           YJR040w   GEF1   0.06           YDR384c   ATO3   0.06           YEL043w       0.06           YPL096c-a   ERI1   0.06           YDR331w   GPI8   0.05           YML123c   PHO84   0.05           YDR033w   MRH1   0.05           YEL065w   SIT1   0.05           YHL016c   DUR3   0.05           YER072w   VTC1   0.05           YBR096w       0.04           YDR046c   BAP3   0.04           YKL004w   AUR1   0.04           YBR021w   FUR4   0.04           YDL210w   UGA4   0.04           YLR220w   CCC1   0.04           YBR004c   FMP44   0.04           YKL065c   YET1   0.04           YBR040w   FIG1   0.04           YCL025c   AGP1   0.04           YER053c-a       0.03           YOR002w   ALG6   0.03           YMR266w   RSN1   0.03           YOL084w   PHM7   0.03           YDR345c   HXT3   0.03           YLL061w   MMP1   0.03           YPL221w   BOP1   0.03           YJR151c   DAN4   0.03           YDR090c       0.03           YJL062w   LAS21   0.03           YPR138c   MEP3   0.03           YAL018c       0.03           YCL038c   ATG22   0.03           YGR065c   VHT1   0.03           YDL035c   GPR1   0.03           YHR092c   HXT4   0.03           YNL280c   ERG24   0.02           YGL001c   ERG26   0.02           YCR037c   PHO87   0.02           YMR183c   SSO2   0.02           YER056c   FCY2   0.02           YLR138w   NHA1   0.02           YGR197c   SNG1   0.02           YDR011w   SNQ2   0.02           YOR153w   PDR5   0.02           YMR319c   FET4   0.02           YBR069c   TAT1   0.02           YKR105c       0.02           YER060w-a   FCY22   0.02           YGR026w       0.02           YOR016c   ERP4   0.02           YDR032c   PST2   0.02           YDR343c   HXT6   0.02           YER060w   FCY21   0.02           YDL194w   SNF3   0.02           YGL008c   PMA1   0.02           YDR093w   DNF2   0.02           YPL265w   DIP5   0.02           YNR039c   ZRG17   0.02           YDR342c   HXT7   0.02           YNL318c   HXT14   0.02           YDL245c   HXT15   0.02           YHR096c   HXT5   0.02           YNL145w   MFA2   0.02           YFL011w   HXT10   0.02           YJR158w   HXT16   0.02           YBL042c   FUI1   0.02           YHR094c   HXT1   0.02           YNR013c   PHO91   0.02           YDR126w   SWF1   0.02           YNL268w   LYP1   0.02           YPL092w   SSU1   0.01           YBR298c   MAL31   0.01           YBR038w   CHS2   0.01           YKR053c   YSR3   0.01           YDL247w   MPH2   0.01           YBR068c   BAP2   0.01           YIL120w   QDR1   0.01           YNR019w   ARE2   0.01           YHR149c   SKG6   0.01           YEL017c-a   PMP2   0.01           YMR011w   HXT2   0.01           YCR023c       0.01           YCL069w       0.01           YLR081w   GAL2   0.01           YER166w   DNF1   0.01           YGL077c   HNM1   0.01           YNL048w   ALG11   0.01           YGR121c   MEP1   0.01           YJL219w   HXT9   0.01           YBR180w   DTR1   0.01           YOL156w   HXT11   0.01           YDR508c   GNP1   0.01           YCR098c   GIT1   0.01           YOL075c       0.01           YPL232w   SSO1   0.01           YGR260w   TNA1   0.01           YLL052c   AQY2   0.01           YNR072w   HXT17   0.01           YGR055w   MUP1   0.01           YOL092w       0.01           YBR294w   SUL1   0.01           YLR023c   IZH3   0.01           YGL012w   ERG4   0.01           YDR062w   LCB2   0.01           YMR296c   LCB1   0.01           YDL138w   RGT2   0.01           YGR191w   HIP1   0.01           YEL031w   SPF1   0.01           YJL214w   HXT8   0.01           YKR050w   TRK2   0.01           YFL017c   GNA1   0.01           YJR152w   DAL5   0.01           YDL199c       0.01           YKR106w       0.01           YCR024c-a   PMP1   0.01           YOL122c   SMF1   0.01           YPL036w   PMA2   0.01           YOR328w   PDR10   0.01           YEL069c   HXT13   0.01           YGR032w   GSC2   0.01           YGL198w   YIP4   0.01           YOL103w   ITR2   0.01           YJL117w   PHO86   0.01           YDR039c   ENA2   0.01           YNL087w   TCB2   0.01           YBR043c   QDR3   0.01           YBR106w   PHO88   0.01           YCR048w   ARE1   0.01           YDR497c   ITR1   0.01           YNL270c   ALP1   0.01           YCR089w   FIG2   0.01           YJR160c   MPH3   0.01           YGR138c   TPO2   0.01           YAL022c   FUN26   0.01           YPL249c   GYP5   0.01           YOL158c   ENB1   0.01           YLR353w   BUD8   0.01           YNL192w   CHS1   0.01           YMR274c   RCE1   0.01           YPR194c   OPT2   0.01           YEL063c   CAN1   0.01           YJL129c   TRK1   0.01           YKL217w   JEN1   0.01           YJR092w   BUD4   0.01           YOR161c   PNS1   0.01           YFL050c   ALR2   0.01           YPR156c   TPO3   0.00           YOL020w   TAT2   0.00           YOR273c   TPO4   0.00           YOL130w   ALR1   0.00           YOR034c   AKR2   0.00           YDR264c   AKR1   0.00           YGR227w   DIE2   0.00           YKR093w   PTR2   0.00           YLL043w   FPS1   0.00           YBR295w   PCA1   0.00           YOR348c   PUT4   0.00           YOR307c   SLY41   0.00           YKR051w       0.00           YDL123w   SNA4   0.00           YGL084c   GUP1   0.00           YGR281w   YOR1   0.00           YBR041w   FAT1   0.00           YPL274w   SAM3   0.00           YLR342w   FKS1   0.00           YDR294c   DPL1   0.00           YBR140c   IRA1   0.00           YPR003c       0.00           YGL161c   YIP5   0.00           YGR217w   CCH1   0.00           YBR086c   IST2   0.00           YOR086c   TCB1   0.00           YHL003c   LAG1   0.00           YFL055w   AGP3   0.00           YER008c   SEC3   0.00           YNL159c   ASI2   0.00           YCR010c   ADY2   0.00           YIL121w   QDR2   0.00           YDR461w   MFA1   0.00           YGL010w       0.00           YPL058c   PDR12   0.00           YPL176c   SSP134   0.00           YML072c   TCB3   0.00           YIL030c   SSM4   0.00           YNL065w   AQR1   0.00           YDR406w   PDR15   0.00           YKR067w   GPT2   0.00           YKR103w   NFT1   0.00           YBR008c   FLR1   0.00           YER181c       0.00           YLL028w   TPO1   0.00           YNL260c       0.00           YKL212w   SAC1   0.00           YNR056c   BIO5   0.00           YBR132c   AGP2   0.00           YJL093c   TOK1   0.00           YBR023c   CHS3   0.00           YIL047c   SYG1   0.00           YOR301w   RAX1   0.00           YKR088c   TVP38   0.00           YDL054c   MCH1   0.00           YOL002c   IZH2   0.00           YER140w       0.00           YOL081w   IRA2   0.00           YDR196c       0.00           YDR233c   RTN1   0.00           YBR024w   SCO2   0.00           YCR087w       0.00           YLR452c   SST2   0.00           YFL048c   EMP47   0.00           YPR198w   SGE1   0.00           YGR289c   MAL11   0.00           YNR021w       0.00                      
 
      As shown in Table 4 and in Table 5, the secretion abilities of the secretory signal peptides at each culture temperature were evaluated by assaying the activities of the secreted luciferase secreted outside the yeast cells. As a result, all the secretory signal peptides were found to exhibit luciferase activity in the yeast culture solution. Among the 440 types of secretory signal peptides, 9 types of secretory signal peptides and 51 types of secretory signal peptides, which would exhibit the secretory ability more than 2 times higher than that of the α-factor derived secretory signal peptide used in conventional effective secretory protein expression systems in yeast were newly identified at moderate culture temperature (30° C.) and at low culture temperature (15° C.), respectively. These identified secretory signal peptides included those derived from membrane proteins as well as those derived from secretory proteins.  
     Example 5  
     Evaluation of Secretion Ability of Secretory Signal Peptide in Relation to Other Proteins  
      In Example 4, the secretion ability of the secretory signal peptide was evaluated in terms of the secreted luciferase (mature CLuc). In Example 5, the secretion abilities of the 16 types of secretory signal peptides shown in Table 6 among the secretory signal peptides shown in Table 3 were evaluated in terms of secretory proteins, i.e., human pancreatic amylase (cDNA: SEQ ID NO: 1788; amino acid sequence: SEQ ID NO: 1789), for which a simple method for activity assay has been established, in order to inspect the efficacy of the secretory signal peptides in secretory expression of other proteins.  Saccharomyces cerevisiae  was used as a host.  
      Table 6 shows the secretory signal peptides used in Example 5 in terms of systematic and common names of the genes from which the secretory signal peptides have been derived. Table 6 also shows the nucleotide sequence of a synthetic primer (SEQ ID NO: shown in the right column) used for amplifying the gene region encoding each secretory signal peptide from the genomic DNA of  Saccharomyces cerevisiae.   
               TABLE 6                          Secretory signal peptides used in Example 6                                                 Systematic gene   Common gene       SEQ       SEQ               name   name   Synthetic primer (Forward)   ID NO:   Synthetic primer (Reverse)   ID NO:               1   YCR028c   FEN2   ATGATGAAGGAATCGAAATCTATCAC   1790   gggGGTCAAATCGTTTCCGACCAT   1791                   2   YDR420w   HKR1   ATGGTCTCATGAAAATAAAAAAAATTT   1792   gggTTGCGTTGTCGTCGTCACA   1793               3   YGR014w   MSB2   ATGCAGTTTCCATTCGCTTG   1794   gggTGACACAGAAGTCTGGGAAGTGG   1795               4   YBR187w       ATGGGAAATATGATAAAGAAGGCA   1796   gggAGACTTCAAATGTGAAGAACTCCCAG   1797               5   YBR296c   PHO89   ATGGCTTTACATCAATTTGACTATATTT   1798   gggAGATCTAGAAGAGATCGACGACG   1799               6   YCR061w       ATGGTGCGTTTTGTTTCAATTT   1800   gggAGACCTATTCCCAGCCTGTGTA   1801               7   YNL237w   YPT1   ATGACAGCAGCTAATAAGAATATTGTCT   1802   gggAGATTTCGACCCAGCCTGCA   1803               8   YBR078w   ECM33   ATGCAATTCAAGAACGCTTTG   1804   gggGATTTTCTTGATAGAGTCAGCGG   1805               9   YLR084c   RAX2   ATGTTTGTTCATCGTCTCTGGAC   1806   gggATCATCAGAGGCATCTTCCAAC   1807               10    YNL300w   TOS6   ATGAAATTCTCTACTCTCTCCACCG   1808   gggTGTGCTGATCTCATGGGTGGT   1809               11    YBR243c   ALG7   ATGTTGCGACTTTTTTCACTGG   1810   gggTGCTATACCAAATCCCAGGG   1811               12    YGL126w   SCS3   ATGTCTAGCAAATGGTTTAATGCTATAC   1812   gggCCCTTTCTTTACCAACATAGCATTT   1813               13    YMR008c   PLB1   ATGAAGTTGCAGAGTTTGTTGGTT   1814   gggCTCCTTGGTGTATGCATCTCTTTT   1815               14    YHR139c   SPS100   ATGAAATTCACATCAGTGCT   1816   gggGGAAGACGATTCGCTTTGTA   1817               15    YKL096w   CWP1   ATGAAATTCTCCACTGCTTTGTC   1818   gggATCGCTCTCAGAACCTTCTTTG   1819               16    YCL043c   PDI1   ATGAAGTTTTCTGCTGGTGCC   1820   gggGTCGTGCGACTGAATGTACTCATT   1821                  
 
      In order to introduce the gene regions encoding the aforementioned 16 types of secretory signal peptides into the pLTex321sV5H low-temperature-inducible expression vector prepared in Example 2, the gene regions were first amplified via PCR from the genomic DNA of  Saccharomyces cerevisiae.  The synthetic primers for the gene regions are shown in Table 6. Each reverse primer comprises at its 5′ end a 3-nucleotide (GGG) sequence of the SmaI cleavage site. Inclusion of such sequence in each reverse primer results in the generation of each DNA fragment encoding secretory signal peptides obtained by PCR described below. Introduction of the resulting DNA fragment into the SmaI-cleaved pLTex321sV5H vector results in the regeneration of the SmaI cleavage site.  
      PCR was carried out using 50 μl of a reaction solution comprising 300 nM of each primer, 1 mM MgSO 4 , 200 μM dNTP, 1 ng of the yeast genomic DNA, and 1× buffer and KOD-Plus-1U included in the KOD-Plus-. The PCR cycle comprised: the first step at 94° C. for 2 minutes; 35 cycles of the second step at 94° C. for 15 seconds (denaturation), at 50° C. for 30 seconds (annealing), and at 68° C. for 1 minute (elongation); and the third step at 68° C. for 5 minutes. In order to add a phosphate group to the 5′ ends of the DNA fragments obtained via PCR, DNA fragments were phosphorylated using 100 μl of a reaction solution comprising 1× buffer included in T4 Polynucleotide Kinase (TOYOBO), 1 mM of ATP, and 20 U of T4 Polynucleotide Kinase at 37° C. for 1 hour.  
      Thereafter, the DNA fragments were fractionated via agarose gel electrophoresis, and DNA fragments each having a chain length of interest were obtained. Among the 16 obtained types of DNA fragments, DNA fragments encoding secretory signal peptides derived from the YGRO14w gene and the YBRO78w gene contained the SmaI cleavage site and the XhoI cleavage site. Thus, the other 14 types of DNA fragments were designated as DNA fragments J, and DNA fragments encoding 2 types of secretory signal peptides derived from the YGRO14w gene and the YBRO78w gene were designated as DNA fragments K. These DNA fragments were used to construct expression plasmids containing the genes encoding fusion proteins of secretory signal peptides with mature α-amylase (a protein consisting of an amino acid sequence derived from the amino acid sequence of human pancreatic α-amylase as shown in SEQ ID NO: 1789 by deletion of the secretory signal peptide consisting of amino acids 1 to 15) in the following manner.  
      The pLTex321sV5H low-temperature-inducible expression vector was cleaved with SmaI, the resulting DNA fragments were subjected to the reaction using 100 μl of a reaction solution containing 1× buffer and 2 U of  E. coli  Alkaline Phosphatase included in  E. coli  Alkaline Phosphatase (TOYOBO) at 60° C. for 30 minutes, and a DNA fragment lacking a phosphate group at its 5′ end was obtained. The resultant is referred to as DNA fragment L.  
      Subsequently, DNA fragments J were ligated to DNA fragments L using the DNA Ligation Kit ver. 2.1, and the ligation products were introduced into  E. coli  DH5α. The resulting transformants were cultured overnight, plasmids were extracted using the GenElute Plasmid Miniprep Kit, and transformants having plasmids of interest (expression vectors) were identified based on the restriction enzyme cleavage patterns and via nucleotide sequence analysis. Expression vectors into which the gene regions encoding 14 types of secretory signal peptides have been introduced were prepared from these transformants. Further, the resulting expression vectors were cleaved with SmaI and with XhoI, the DNA fragments were fractionated via agarose gel electrophoresis, and the DNA fragments of interest were obtained. These fragments are designated as DNA fragments M.  
      The gene region encoding mature α-amylase that contains no secretory signal peptide was amplified via PCR from a plasmid containing the gene (AMY2A, cDNA: SEQ ID NO: 1788) encoding human pancreatic α-amylase.  
      The following synthetic primers were used. hAMY2A ORF F−Sig consists of a 25-bp region encompassing the 5′ end of the gene region encoding mature α-amylase. hAMY2A ORF R+XhoI comprises at its 5′ end the XhoI cleavage site, and downstream thereof, a sequence complementary to a 27-bp region including the termination cordon of the gene region encoding human pancreatic α-amylase.  
      hAMY2A ORF F−Sig: CAGTATTCCCCAAATACACAACAAG(SEQ ID NO: 1822)  
      hAMY2A ORF R+XhoI: GCGC-CTCGAG-TTACAATTTAGATTCAGCATGAATTGC (SEQ ID NO: 1823)  
      PCR was carried out using 50 μl of a reaction solution comprising 300 nM of each primer, 1 mM MgSO 4 , 200 μM dNTP, 1 ng of plasmid containing the gene encoding human pancreatic α-amylase (AMY2A), and 1× buffer and KOD-Plus-1U included in the KOD-Plus-. The PCR cycle comprised: the first step at 94° C. for 2 minutes; 35 cycles of the second step at 94° C. for 15 seconds (denaturation), at 50° C. for 30 seconds (annealing), and at 68° C. for 2 minutes (elongation); and the third step at 68° C. for 5 minutes.  
      The resulting PCR product was cleaved with XhoI, the DNA fragments were fractionated via agarose gel electrophoresis, and the DNA fragment of interest (approximately 1.5 kbp) was obtained. This fragment is referred to as DNA fragment N.  
      Subsequently, DNA fragments M were ligated to DNA fragments N using the DNA Ligation Kit ver. 2.1, and the ligation products were introduced into  E. coli  DH5α. The resulting transformants were cultured overnight, plasmids were extracted using the GenElute Plasmid Miniprep Kit, and transformants having plasmids (expression vectors) containing the genes encoding the fusion proteins of the secretory signal peptides with mature α-amylase were identified based on the restriction enzyme cleavage patterns and via nucleotide sequence analysis. Expression vectors for 14 types of fusion proteins (of secretory signal peptides with mature α-amylase) were prepared from the resulting transformants.  
      The expression vector for a fusion protein (a fusion protein of a secretory signal peptide with mature α-amylase) was prepared using the secretory signal peptides derived from the YGRO14w gene and the YBRO78w gene in the following manner.  
      The gene region encoding mature α-amylase was amplified via PCR using synthetic primers, hAMY2A ORF F−Sig+SmaI and the aforementioned hAMY2A ORF R+XhoI (SEQ ID NO: 1823).  
      The sequence of the synthetic primer used, hAMY2A ORF F−Sig+SmaI, is as shown below. hAMY2A ORF F−Sig+SmaI comprises at its 5′ end a 3-nucleotide (GGG) sequence of the SmaI cleavage site, and downstream thereof, a 25-bp region encompassing the 5′ end of the gene region encoding mature α-amylase.  
      hAMY2A ORF F−Sig+SmaI: GGG-CAGTATTCCCCAAATACACAACAAG (SEQ ID NO: 1824)  
      PCR was carried out using 50 μl of a reaction solution comprising 300 nM of each primer, 1 mM MgSO 4 , 200 μM dNTP, 1 ng of plasmid containing the gene encoding human pancreatic α-amylase (AMY2A), and 1× buffer and KOD-Plus-1U included in the KOD-Plus-. The PCR cycle comprised: the first step at 94° C. for 2 minutes; 35 cycles of the second step at 94° C. for 15 seconds (denaturation), at 50° C. for 30 seconds (annealing), and at 68° C. for 2 minutes (elongation); and the third step at 68° C. for 5 minutes. The resulting PCR product was cleaved with XhoI, the DNA fragments were fractionated via agarose gel electrophoresis, and the DNA fragments of interest (approximately 1.5 kbp) were obtained. These fragments are referred to as DNA fragments O.  
      The pLTex321sV5H low-temperature-inducible expression vector was cleaved with SmaI and XhoI, the DNA fragments were fractionated via agarose gel electrophoresis, and the DNA fragments of interest (approximately 7.4 kbp) were obtained. These fragments are referred to as DNA fragments P.  
      Subsequently, DNA fragments O were ligated to DNA fragments P using the DNA Ligation Kit ver. 2.1, and the ligation products were introduced into  E. coli  DH5α. The resulting transformants were cultured overnight, plasmids were extracted using the GenElute Plasmid Miniprep Kit, and transformants having plasmids of interest were identified based on the restriction enzyme cleavage patterns and via nucleotide sequence analysis. Plasmids were prepared from the transformants. The resulting plasmids were cleaved with SmaI, and the DNA fragments were subjected to a reaction using 100 μl of a reaction solution containing 1× buffer and 2 U of  E. coli  Alkaline Phosphatase included in  E. coli  Alkaline Phosphatase at 60° C. for 30 minutes. Thus, a DNA fragment from which a phosphate group at its 5′ end had been removed was obtained. Such a fragment is referred to as DNA fragment Q.  
      Further, DNA fragments K were ligated to DNA fragments Q using the DNA Ligation Kit ver. 2.1, and the ligation products were introduced into  E. coli  DH5α. The resulting transformants were cultured overnight, plasmids were extracted using the GenElute Plasmid Miniprep Kit, and transformants having plasmids (expression vectors) containing the genes encoding the fusion proteins of the secretory signal peptides with mature α-amylase were identified based on the restriction enzyme cleavage patterns and via nucleotide sequence analysis. Expression vectors for 2 types of fusion proteins (of secretory signal peptides with mature α-amylase) were prepared from the resulting transformants.  
      As the controls, expression vectors for the fusion proteins (a fusion protein of a secretory signal peptide with mature α-amylase) utilizing the α-factor-derived secretory signal peptide or preprotoxin (K28 prepro-toxin)-derived secretory signal peptide described in Example 3 were prepared in the following manner.  
      The gene region encoding the α-factor-derived secretory signal peptide was amplified via PCR from the pCLuRA plasmid (see International Patent Application No. PCT/JP2006/311597, claiming the priority right of JP Patent Application No. 2005-169768).  
      The following synthetic primers were used. MF(ALPHA)1 Sig. 89aa F comprises a 26-bp region encompassing the 5′ end of the gene region encoding the α-factor-derived secretory signal peptide. MF(ALPHA)1 Sig. 89aa R+SmaI comprises at its 5′ end a 3-nucleotide (GGG) sequence of the SmaI cleavage site, and downstream thereof, a sequence complementary to a 22-bp region encompassing the 3′ end of the gene region encoding the α-factor-derived secretory signal peptide.  
      MF(ALPHA)1 Sig. 89aa F: ATGAGATTTCCTTCAATTTTTACTGC (SEQ ID NO: 1825)  
      MF(ALPHA)1 Sig. 89aa R+SmaI: GGG-AGCTTCAGCCTCTCTTTTCTCG (SEQ ID NO: 1826)  
      PCR was carried out using 50 μl of a reaction solution comprising 300 nM of each primer, 1 mM MgSO 4 , 200 μM dNTP, 1 ng of the pCLuRA plasmid, and 1× buffer and KOD-Plus-1U included in the KOD-Plus-. The PCR cycle comprised: the first step at 94° C. for 2 minutes; 35 cycles of the second step at 94° C. for 15 seconds (denaturation), at 50° C. for 30 seconds (annealing), and at 68° C. for 1 minute (elongation); and the third step at 68° C. for 5 minutes. The resulting DNA fragments were subjected to a reaction using 100 μl of a reaction solution comprising 1× buffer, 1 mM of ATP, and 20 U of T4 polynucleotide kinase included in T4 polynucleotide kinase at 37° C. for 1 hour. Thus, a phosphate group was added to the 5′ end of the DNA fragment.  
      Thereafter, the DNA fragments were fractionated via agarose gel electrophoresis, and the DNA fragments of interest (270 bp) were obtained. These fragments are referred to as DNA fragments R.  
      Subsequently, DNA fragments R were ligated to DNA fragments L using the DNA Ligation Kit ver. 2.1, and the ligation products were introduced into  E. coli  DH5α. The resulting transformants were cultured overnight, plasmids were extracted using the GenElute Plasmid Miniprep Kit, and transformants having plasmids of interest (expression vectors) were identified based on the restriction enzyme cleavage patterns and via nucleotide sequence analysis. The expression vector (pLTex321sV5Hα) into which the gene region encoding the α-factor-derived secretory signal peptide had been introduced was prepared from the transformant.  
      The gene regions encoding mature α-amylase were introduced into the pLTex321sV5Hα expression vector comprising the gene region encoding the α-factor-derived secretory signal peptide and the pLTex321sV5H K28L expression vector comprising the gene region encoding the preprotoxin-derived secretory signal peptide prepared in Example 2 in the following manner.  
      These expression vectors were each cleaved with SmaI and XhoI, the DNA fragments were fractionated via agarose gel electrophoresis, and the DNA fragments of interest were obtained. The resulting DNA fragments were ligated to DNA fragments N using the DNA Ligation Kit ver. 2.1, and the ligation products were introduced into  E. coli  DH5α. The resulting transformants were cultured overnight, plasmids were extracted using the GenElute Plasmid Miniprep Kit, and transformants having plasmids (expression vectors) containing the genes encoding the fusion proteins of the secretory signal peptides with mature α-amylase were identified based on the restriction enzyme cleavage patterns and via nucleotide sequence analysis. Expression vectors for 2 types of fusion proteins (of secretory signal peptides with mature α-amylase) were prepared from the resulting transformants.  
      Using the expression vector containing the gene encoding each of the 16 types of fusion proteins of secretory signal peptides with mature α-amylase obtained via the above procedure shown in Table 6 and the control vectors, i.e., an expression vector comprising a gene encoding the fusion protein of the α-factor-derived secretory signal peptide with mature α-amylase and an expression vector containing a gene encoding the fusion protein of preprotoxin (K28 prepro-toxin)-derived secretory signal peptide with mature α-amylase, the  Saccharomyces cerevisiae  BY4743 PEP4Δ PRB1Δ strain was transformed using the EZ yeast transformation kit (Qbiogene), and transformants each sustaining a relevant expression vector were obtained using SD+HL plate medium (0.67% of yeast nitrogen base without amino acid, 2% of glucose, 0.002% of L-histidine HCl, 0.01% of L-leucine, and 2% of agar).  
      In order to evaluate the levels of secretory expression of human pancreatic α-amylase in the resulting transformants, the following experiment was carried out.  
      One ml of SD+HL+PPB medium was inoculated with the resulting transformants, and preculture was conducted at 30° C. for 3 days. Part of the culture preculture solution was used in the following culture.  
      Subsequently, one ml of SD+HL+PPB medium was inoculated with the culture solution (10 μl) obtained via the preculture and culture was conducted at 30° C. to the logarithmic growth phase. Thereafter, the culture temperature for the culture solution was lowered to 10° C., and culture was further continued at 10° C. for 168 hours.  
      After the culture had been conducted at a low temperature for 168 hours, the activity of human pancreatic α-amylase secreted in the culture solution was assayed to evaluate the secretion levels of the transformants. The human pancreatic α-amylase activity was assayed using 5 μl of the supernatant of the culture solution and the amylase assay reagent (Diacolor Liquid AMY, Ono Pharmaceutical Co., Ltd.). Simultaneously, 200 μl of the culture solution was used to assay the absorbance thereof at 600 nm, and the activity value was divided by the assayed value to normalize the human pancreatic α-amylase activity value based on the absorbance.  
      The results are shown in Table 7. Table 7 shows the secretory expression levels of human pancreatic α-amylase when using secretory signal peptides used in conventional expression systems in yeast (α-factor-derived secretory signal peptide and preprotoxin (K28 prepro-toxin)-derived secretory signal peptide) and the 16 types of secretory signal peptides found in Example 1 in terms of the secretion efficiency, in relation to the secretory expression levels resulting from the use of the α-factor-derived secretory signal peptide. In Table 7, each secretory signal peptide is indicated in terms of the systematic and common names of the genes from which the secretory signal peptides are derived. The result of the α-factor-derived secretory signal peptide is shown in the column indicated as “α-factor” in terms of common name. The result of preprotoxin-derived secretory signal peptide is shown in the column indicated as “K28L” in terms of common name.  
                   TABLE 7                          Results of secretory expression of human           pancreatic α-amylase using each of secretory       signal peptides                                             Secretion                       efficiency relative to α-           Systematic   Common   factor-derived secretory           gene name   gene name   signal peptide                                          1   YCR028c   FEN2   9.46                    2   YDR420w   HKR1   28.25                3   YGR014w   MSB2   8.31                4   YBR187w       29.98                5   YBR296c   PHO89   8.08                6   YCR061w       5.90                7   YNL237w   YTP1   29.89                8   YBR078w   ECM33   16.68                9   YLR084c   RAX2   6.22               10   YNL300w   TOS6   49.16               11   YBR243c   ALG7   11.30               12   YGL126w   SCS3   8.93               13   YMR008c   PLB1   39.68               14   YHR139c   SPS100   27.42               15   YKL096w   CWP1   11.73               16   YCL043c   PDI1   8.45               17       α-factor   1.00               18       K28L   0.02                  
 
      As shown in Table 7, 16 types of secretory signal peptides were used to inspect the secretory expression of human pancreatic α-amylase. As a result, with the use of any secretory signal peptide, these secretory signal peptides were found to exhibit higher secretory expression levels than α-factor-derived secretory signal peptide and preprotoxin-derived secretory signal peptide that had been used in the highly efficient secretory protein expression in conventional expression systems in yeast.  
     EFFECTS OF THE INVENTION  
      The present invention provides secretory signal peptides exhibiting higher ability for transportation to cell organelles, including cell membranes, endoplasmic reticulum, and Golgi bodies, and higher efficiency of extracellular secretion, than conventional secretory signal peptides that can be used for membrane and secretory protein expression systems, for example.  
      All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.