# Patent

## Title
Protein for constructing protein complex from Clostridium thermocellum, and use thereof

## Abstract

It is an object to provide a protein having a dockerin, which is suited to production in yeasts and other eukaryotic microorganism in which sugar chain modification is predicted, and which provides excellent cohesin-dockerin binding ability, along with a use thereof. The present invention uses, as a protein for constructing a protein complex using a scaffolding protein having a type I cohesin from Clostridium thermocellum, a protein having a dockerin having at least one dockerin-specific sequence which is a dockerin-specific sequence associated with cohesin binding in type I dockerins from C. thermocellum, and which either has no intrinsic predicted N-type sugar chain modification site or has aspartic acid substituted for the asparagine of an intrinsic predicted N-type sugar chain modification site.

## Background
CROSS-REFERENCE TO RELATED APPLICATION(S)
This application claims priority to Japanese Patent Application No. 2010-088952 filed on Apr. 7, 2010, the contents of which are hereby incorporated by reference into the present application.
TECHNICAL FIELD
The present application relates to a protein for constructing a protein complex from Clostridium thermocellum, and to a use thereof.
DESCRIPTION OF RELATED ART
In recent years there has been increased interest in biomass resources derived from plant photosynthesis as a substitute for limited petroleum supplies, and various attempts have been made to use biomass for energy and various kinds of materials. In order for biomass to be used effectively as an energy source or other raw material, it must be saccharified into a carbon source that is readily available to animals and microorganisms.
Using typical forms of biomass such as cellulose and hemicellulose requires good cellulases for saccharifying (decomposing) these materials. Attention has focused on cellulosomes, which are produced by certain bacteria, as a source of such cellulases. Cellulosomes are protein complexes formed on the cell surfaces of bacteria, and comprise cellulases and scaffolding proteins (scaffoldins) to which the cellulases bind. Scaffolding proteins have sites called cohesins, and cellulases are known to bind to these cohesins via their own dockerins. Cellulosomes are capable of providing a variety of cellulases in large quantities and at high densities on bacterial cell surfaces.
Artificial construction of cellulosomes by genetic engineering has been studied in recent years. In the context of cellulosome construction, various studies have been made of binding between cohesins and dockerins, which is the basis of cellulosome construction. For example, several amino acid residues have been deleted or alanine scanned from dockerins of Clostridium thermocellum to evaluate binding with cohesins and identify the residues necessary for binding ability (Non-patent Document 1). According to this document, a dockerin produced in E. coli maintains about 70% the amount of binding with a cohesin when asparagine in the amino acid sequence is replaced with alanine, but interactions with calcium ions contributing to structural stability are weakened. It has also been reported that when binding ability is eliminated by substituting AA (alanine-alanine) for ST (serine-threonine) in one of two repeating amino acid sequences making up two helixes in a dockerin of C. thermocellum, the other helix binds with a cohesin (Non-patent document 2). With respect to cohesins, when several amino acid residues of a cohesin of C. thermocellum were replaced and binding with dockerins was evaluated, it was found that binding with dockerins from C. thermocellum was eliminated by replacing certain threonines with leucine, and instead, the cohesin bound to a dockerin from Clostridium cellulolyticum with which it did not ordinarily interact (Non-patent Document 3).

    [Non-patent Document 1] A. Karpol et al., Biochem. J 410, 331-338 (2008)
    [Non-patent Document 2] A. L. Carvalho et al., PNAS 104(9), 3089-3094 (2007)
    [Non-patent Document 3] A. Mechaly et al., J. Biol. Chem. 276 (13), 9883-9888 (2001)


BRIEF SUMMARY OF INVENTION
Causing a yeast or the like to produce and excrete large quantities of cellulase is considered as desirable when constructing an artificial cellulosome. If a cellulosome can be constructed on the cell surface of a yeast or other eukaryotic microorganism, the glucose decomposed by the cellulosome can be used immediately by the yeast as a carbon source for efficient production of various useful substances. However, when a foreign protein from a bacteria or other prokaryote is produced with a yeast or other eukaryote, interaction between proteins can be affected by giant sugar chain modification.
According to the reports above, it appears that amino acid substitution of dockerin domains affects cohesin-dockerin binding, either by reducing binding ability (Non-patent Documents 1, 2) or altering binding specificity (Non-patent Document 3). However, there have been no reports on improving cohesin-dockerin binding ability. Moreover, the reports above pertain only to cohesins and dockerins produced in E. coli, in which sugar chain modification of proteins does not occur. Thus, at present there are no reports at all on how amino acid substitution of dockerin domains affects cohesin-dockerin binding in yeasts and other eukaryotic microorganisms, in which sugar chain modification does occur.
It is an object of the disclosures of this Description to provide a protein having a dockerin, wherein the protein is useful for producing a protein complex derived from Clostridium thermocellum in a yeast or other eukaryotic microorganism in which sugar chain modification is expected, and provides excellent cohesin-dockerin binding ability, along with a use thereof.
In a search for dockerins of C. thermocellum using DDBJ (www.ddbj.nig.ac.jp/index-j.html), the inventors in this case discovered 72 attributed dockerins on the genome of C. thermocellum, and after using UniProt (www.uniprot.org) and the like to identify specific sequences thought to be associated with cohesin-dockerin binding in these dockerins, we analyzed these specific sequences by multiple alignment and the like. As a result, the similarity of these 142 specific sequences exceeded 90%. It is therefore thought that all these specific sequences have binding ability with cohesins.
The inventors also discovered that of these specific sequences, 113 or about 80% of the relevant sequences have predicted sugar chain modification sites, while the remaining 29 sequences lack predicted sugar chain modification sites. The inventors then targeted two predicted sugar chain binding sites located near the scaffolding protein binding region of a dockerin from C. thermocellum, replacing the asparagines at these sites with alanine or aspartic acid. Sugar chain modification was eliminated by replacing asparagine with alanine in the dockerin, but cohesin binding ability was not improved. It is possible that the dockerin with asparagine replaced with alanine could not bind with cohesin because it does not assume a stable structure when produced in yeast. On the other hand, when a dockerin having the asparagine of the target site replaced with aspartic acid was produced in yeast, however, cohesin-dockerin binding increased, resulting in improved yeast saccharification ability.
From this, it was found that cohesin-dockerin binding ability can be increased and saccharification ability in eukaryotes in which sugar chain modification may occur can be improved if either predicted sugar chain modification sites are inherently lacking, or if when such a site is present, and an asparagine at the predicted site is replaced with aspartic acid to eliminate sugar chain modification, thereby improving cohesin-dockerin binding ability.
The disclosures of this description provide a protein for constructing a protein complex using a framework including a type I cohesin from C. thermocellum, wherein the protein has a dockerin containing at least one dockerin-specific sequence associated with cohesin binding in type I dockerins from C. thermocellum, and this dockerin satisfies either of the following conditions (a) and (b):
(a) having no intrinsic predicted N-type sugar chain modification site;
(b) having aspartic acid substituted for an asparagine of an intrinsic predicted N-type sugar chain modification site.
In a dockerin-specific sequence satisfying condition (a) above, the intrinsic predicted N-type sugar chain modification site may be an aspartic acid.
The protein disclosed in this Description may also have cellulolysis promotion activity, and this cellulolysis promotion activity may be cellulase activity. The cellulolysis promotion activity may also be conferred by an amino acid sequence from Clostridium thermocellum. 
The disclosures of this Description provide a eukaryotic microorganism having a protein complex using a scaffolding protein from Clostridium thermocellum in the cell surface, wherein the eukaryotic microorganism is provided with a scaffolding protein from Clostridium thermocellum and the protein disclosed in this description, which binds with this scaffolding protein.
The disclosures of this Description provide a method for producing a useful substance, having a step of saccharifying a cellulose-containing material using a process of fermenting a cellulose-containing material as a carbon source with the eukaryotic microorganism disclosed in this Description, which is a eukaryotic microoganism in which the aforementioned dockerin protein has cellulolysis promotion activity.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows a pAI-AGA1 vector prepared in Example 1.
FIG. 2 shows a pDL-CtCBDCohAGA2 vector having a Leu2 marker and ADH3 homologous region prepared in Example 2.
FIG. 3 shows an amino acid sequence having alanine or aspartic acid substituted for the No. 18 and No. 50 asparagines in the amino acid sequence of a Cel48S dockerin gene, and the corresponding genetic sequence.
FIG. 4 shows a pXU-Cel48Sdoc vector, pXU-Cel48S-N-A-doc vector and pXU-Cel48S-N-D-doc vector prepared in Example 3.
FIG. 5 shows the displayed amount of dockerin in a protein complex surface-displaying yeast containing an amino acid-substituted Cel48S dockerin.
FIG. 6 shows an amino acid sequence having aspartic acid substituted for the No. 18 and No. 54 asparagines in the amino acid sequence of a Xyn10C dockerin gene, and a corresponding genetic sequence.
FIG. 7 shows a pXU-Xyn10Cdoc vector and pXU-Xyn10C-N-D-doc vector prepared in Example 5.
FIG. 8 shows the displayed amount of dockerin in a protein complex surface-displaying yeast containing an amino acid-substituted Xyn10C dockerin.
FIG. 9 shows a pXU-Cel8A-Cel48Sdoc and pXU-Cel8A-Cel48S-N-Ddoc vector prepared in Example 7.
FIG. 10 shows the displayed amount of cellulase in a protein complex surface-displaying yeast containing amino acid-substituted dockerin-type cellulase.
FIG. 11 shows CMC decomposition activity of a protein complex surface-displaying yeast containing amino acid-substituted dockerin-type cellulase.



DETAILED DESCRIPTION OF INVENTION
The disclosures of this Description relate to a protein for constructing a protein complex using a scaffolding protein having type I cohesin from C. thermocellum, to a eukaryotic microorganism provided with a protein complex comprising this protein, and to a method for producing a useful substance using this eukaryotic microorganism.
The protein disclosed in this description has at least one dockerin-specific sequence which is a sequence associated with cohesin binding ability in a type I dockerin from C. thermocellum, and which either has no intrinsic predicted N-type sugar chain modification site or has aspartic acid substituted for asparagine at an intrinsic predicted N-type sugar chain modification site. Sugar chain modification is thus eliminated even if the protein disclosed in this Description is produced in a yeast or other eukaryotic microorganism in which sugar chain modification is expected to occur. As a result, the protein disclosed in this Description has excellent binding ability with type I cohesins of scaffolding proteins from C. thermocellum, and can be used to construct a protein complex in which this protein is bound densely and/or in large amounts.
The eukaryotic microorganism disclosed in this description may be provided in the cell surface with a protein complex in which the protein disclosed in this Description is accumulated densely and/or in large amounts. It is thus possible to obtain a eukaryotic microorganism in which the function of the protein of the invention is enhanced. Because the protein of the invention has excellent cohesin binding ability even when produced in a eukaryotic microorganism, this protein and the aforementioned scaffolding protein may both be produced by the eukaryotic microorganism disclosed in this Description. This eukaryotic microorganism may be a yeast.
The method of producing a useful substance disclosed in this Description comprises a step of fermenting a cellulose-containing material as a carbon source using the eukaryotic microorganism disclosed in this Description, in which the aforementioned protein is a protein having cellulolysis promotion activity. Because the eukaryotic microorganism disclosed in this Description has enhanced cellulolysis promotion activity, it can efficiently ferment a cellulose-containing material as a carbon source.
(Protein for Constructing Protein Complex Using Scaffolding Protein Having Type 1 Cohesin from C. Thermocellum)
The protein disclosed in this Description is a protein especially suited to constructing a protein complex using a scaffolding protein having a type I cohesin from C. thermocellum. This protein may have a dockerin comprising at least one dockerin-specific sequence that is associated with cohesin binding in a type I dockerin from C. thermocellulum, and that fulfills the condition of either (a) having no intrinsic predicted N-type sugar chain modification site or (b) having aspartic acid substituted for an asparagine of an intrinsic predicted N-type sugar chain modification site.
C. thermocellum is known as a cellulosome-producing microorganism. C. thermocellum also has cellulase activity, and produces proteins containing type I dockerins. Based on the results of a search of the C. thermocellum genome in DDBJ (www.ddbj.nig.ac.ip/index-j.html), the 72 amino acid sequences shown by Seq. Nos. 1 to 72 in Table 1 below can be given as examples of type I dockerin amino acid sequences from C. thermocellum. The locus (sequence) names shown in Table 1 are the names of each dockerin. Thus, the amino acid sequences specified by dockerin names in the locus column of Tables 2 to 21 derive from the amino acid sequences of dockerins having the same name in Table 1.









TABLE 1





SEQ
locus



ID
(SEQ.



NO :
 Name)
Amino Acid Sequence







 1
Cthe0015
DVNADGKDSTDLTLLKRYLLRSATLEEKLNADTDGNGTVNSTDLNYLKKYLRVI


 


 2
Cthe0032
DLNNDGNNSTDYMLKKYLKVLERMNVPEKAADLNGDGSINSTDLTLKRFIMKAI


 


 3
Cthe0043
DLNGDGNNSTDFTMLKRAILGNPAPGTNLAAGDLNRDGNTNSTDLMLRRYLLKLI


 


 4
Cthe0044
DNLDGKINSTDLSALKRHILRITTLSGKQLENADVNNDGSVNSTDASILKKYAKAI


 


 5
Cthe0109
DFNSDSSVNSTDLMILNRAVLGLG


 


 6
Cthe0190
ELNGDGKNSSDLNMMKRYLLRLIDGLNDTACADLNGDGKNSSDYSLKRYLLRMI


 


 7
Cthe0191
DLNGDAKINSTDLNMMKRYLLQMIDRFGVDDESCADLNGDGKITSSDYNLLKRYILHLI


 


 8
Cthe0211
DVNGDGHVNSSDYSLFKRYLLRVIDRFPVGDQSVADVNRDGRDSTDLTMLKRYLRAI


 


 9
Cthe0239
GDYNGDGAVNSTDLLACKRYLLYALKPEQNVIAGDLDGNGKINSTDYAYLKRYLLKQI


 


10
Cthe0246
DLNADGKNSTDYNLGKRLILRTISELPISNGSVAFDLNGDSKVDSTDLTALKRYLLGVI


 


11
Cthe0258
DVNGDSKNADVLLMKKYILKVINDLPSDGVKAADVNADGQINSDFTWLKKYMLKAV


 


12
Cthe0269
DVNGDGNVNSTDLTMLKRYLLKSVTNINREAADVNRDGAINSSDMTILKRYLIKSI


 


13
Cthe0270
DLNGDGKVNSSDLAILKRYMLRAISDFPIPEGRKLADLNRDGNVNSTDYSILKRYILKAI


 


14
Cthe0274
CDVGDLNVDGSINSVDITYMKRYLLRSISVLPYQENERIRIPAADTNGDGAINSSDMVLLKRYVLRSI


 


15
Cthe0405
DVNGDGNVNSTDVVWLRRFLLKLVEDFPVPSGKQAADMNDDGNINSTDMIALKRKVLKIP


 


16
Cthe0412
DCNGDGKVNSTDAVALKRYILRSGISINTDNADVNADGRVNSTDLAILKRYLKEI


 


17
Cthe0413
DCNDDGKVNSTDVAVMKRYLKKENVNINLDNADVNADGKVNSTDFSILKRYYMKNI


 


18
Cthe0433
DLNGDGRVNSSDLALMKRYVVKQIEKLNVPVKAADLNGDDKVNSTDYSVLKRYLLRSI


 


19
Cthe0435
DVNADGVVNISDYVLMKRYILRIIADFPADDDMWVGDVNGDNVINDIDCNYLKRYLLHMI


 


20
Cthe0438
DLNGDNNINSSDYTLLKRYLLHTI


 


21
Cthe0536
DVNGDGRVNSSDVALLKRYLLGLVENINKEAADVNVSGTVNSTDLAIMKRYVLRSI


 


22
Cthe0543
DVNFDGRINSTDYSRLKRYVIKSLEFTDPEEHQKFIAAADVDGNGRINSTDLYVLNRYILKLI


 


23
Cthe0578
DNLDGKINSSDVTLLKRYIVKSIDVFPTADPERSLIASDVNGDGRVNSTDYSYLKRYVLKII


 


24
Cthe0640
DLNGDNNVNSTDLTLLKRYLTRVINDFPHPDGSVNADVNGDGKINSTDYSAMRIYILRII


 


25
Cthe0661
DVNGDLKVNSTDFSMLRRYLLKTIDNFPTENGKQAADLNGDGRINSSDLTMLKRYLLMEV


 


26
Cthe0624
DLNNDSKVNAVDIMMLKRYILGIIDNINLTADIYFDGVVNSSDYNIMKRYLLKAI


 


27
Cthe0625
DLNGDGVVNSTDSVILKRHIIKFSEITDPVKLKAADLNGDGNINSSDVSLMKRYLLRII


 


28
Cthe0660
DLNGDGKINSTDISLMKRYLLKQIVDLPVEDDIKAADINKDGKVNSTDMSILKRVILRNY


 


29
Cthe0729
DSNSDCKVNSTDLTLMKRYLLQQSISYINLINADLNGDGKINSSDYTLLKRYLLGYI


 


30
Cthe0745
DINNDKTVNSTDVTYLKRFLLKQINSLPNQKAADVNLDGNINSTDLVILKRYVLRGI


 


31
Cthe0797
DVNGDGKINSTDCTMLKRYILRGIEEFPSPSGIIAADVNADLKINSTDLVLMKKYLLRSI


 


32
Cthe0798
DVNLDGQVNSTDFSLLKRYILKVVDNSINVTNADMNINDGNNSTDISILKRILLRN


 


33
Cthe0821
DINRDGKINSTDLGMLNRHILKLVILDDNLKLAAADIDGNGNINSTDYSWLKKYILKVI


 


34
Cthe0825
DVNDDGKVNSTDLTLLKRYVLKAVSTLPSSKAEKNADVNRDGRVNSSDVTILSRYLIRVI


 


35
Cthe0912
DVNGDGTINSTDLTMLKRSVLRAITLTDDAKARADVDKNGSINSTDVLLLSRYLLRVI


 


36
Cthe0918
DLNRNGIVNDEDYILLKNYLLRGNKLVIDLNVADVNKDGKVNSTDCLFLKKYLGLI


 


37
Cthe1271
DTNSDGKINSTDVTALKRHLLRVTQLTGDNLANADVNGDGNVNSTDLLLLKRYILGEI


 


38
Cthe1398
DLNGDNRINSTDLTLMKRYILKSIEDLPVEDDLWAADINGDGKINSTDYTYLKKYLLQAI


 


39
Cthe1400
DLNGDGRVNSTDYTLLKRYLLGAIQTFPYERGIKAADLNLDGRINSTDYTVLKRYLLNAI


 


40
Cthe1472
DLNFDNVAVNSTDLLMLKRYILKSLELGTSEQEEKFKKAADLNRDNKVDSTDLTILKRYLLKAI


 


41
Cthe1806
EVIDTKVIDSTDDIVKYEYQFDKKILCADKETEILYFTVVADEEEIYTSDNTRTLVLSVNNDSTDKTTVSGYISVDF


 


42
Cthe1838
DVNGDGRVNSSDLTLMKRYLLKSISDFPTPEGKIAADLNEDGKVNSTDLLALKKLVLREL


 


43
Cthe1890
DLNADGSINSTDLMIMKRVLLKQRTLDDITPADLNGDGKVTSTDYSLMKRYLLKEI


 


44
Cthe1963
DLNGDGNINSSDLQALKRHLLGISPLTGEALLRADVNRSGKVDSTDYSVLKRYILRII


 


45
Cthe2038
DIVLDGNINSLDMMKLKKYLIRETQFNYDELLRADVNSDGEVNSTDYAYLKRYILRII


 


46
Cthe2089
DVNDDGKVNSTDAVALKRYVLRSGISINTDNADLNEDGRVNSTDLGILKRYILKEI


 


47
Cthe2137
DVDGNGTVNSTDVNYMKRYLLRQIEEFPYEKALMAGDVDGNGNINSTDLSYLKKYILKLI


 


48
Cthe2139
DVNAGVINSSDIMVLKRFLLRTITLTEEMLLNADTNGDGAVNSSDFTLLKRYILRSI


 


49
Cthe2147
DVNGDFAVNSNDLTLIKRYVLKNIDEFPSSHGLKAADVDGDEKITSSDAALVKRYVLRAI


 


50
Cthe2179
DLNGDGNVNSTDSILMKRYLMKSVDLNEEQLKAADVNLDGRVNSTDRSILNRYLLKII


 


51
Cthe2193
DINDDGNINSTDLQMLKRHLLRSIRLTEKQLLNADTNRDGRVDSTDLALLKRYILRVI


 


52
Cthe2194
DLNGDGNINSTDLQILKKHLLRITLLTGKELSNADVTKDGKVDSTDLTLLKRYILRFV


 


53
Cthe2195
DLNDDGKVNSTDFQILKKHLLRITLLTGKNLSNADLNKDGKVDSSDLSLMKRYLLQII


 


54
Cthe2196
DLNNDGKVNSTDFQLLKMHVLRQELPAGTDLSNADVNRDGKVDSSDCTLLKRYILRVI


 


55
Cthe2197
DLNGDGKVNSTDLQLMKMHVLRQRQLTGTSLLNADVNRDGKVDSTDVALLKRYILRQI


 


56
Cthe2271
DVNLDGSVDSIDLALLYNTTYYAVPLPNRLQYIAADVNYDSSCTMLDFYMLEDYLLGRIS




SFPAGQTYTVYYGDLNGDQLVTTDQSLLGRINLTFRQYVSADVNGDGTVNGDGTVDGIDLAIITAYINGQI


 


57
Cthe2360
DLNGDGRVNSTDLLLMKKRIIREIDKFNVPDENADLNLDGKINSSDYTILKRYVLKSI


 


58
Cthe2549
DVNKDGRINSTDIMYLKGYLLRNSAFNLDEYGLMAADVDGNGSVSSLDLTYLKRYILRRI


 


59
Cthe2590
DLNQDGQVSSTDLVAMKRYLLKNFELSGVGLEAADLNSDGKVNSTDLVALKRFLLKEI


 


60
Cthe2760
DLNYDGKVNSTDYLVLKRYLLGTIDKESDPNFLKAADLNRDGRVNSTDMSLMKRYLLGII


 


61
Cthe2761
DVNGDGKVNSTDCSIVKRYLLKNIEDFPYEYGKEAGDVNGDGKVNSTDYSLLKRFVLRNI


 


62
CthE2811
DLNGDGKVNSTDLTIMKRYILKNFDKLAVPEEAADLNGDGRINSTDLSILHRYLLRII


 


63
Cthe2812
DLNGDQKVTSTDYTMLKRYLMKSIDRFNTSEQAADLNRDGKINSTDLTILKR


 


64
Cthe2872
DINSDGNVNSTDLGILKRIIVKNPPASANMDAADVNADGKVNSTDYTVLKRYLLRSI


 


65
Cthe2879
DINSDGSINSTDVTLLKRHLLRENILTGTAYSNADTDGDGKITSIDLSYLKRYVLRLI


 


66
Cthe2949
DLNGDGLVNSSDYSLLKRYILKQIDLTEEKLKAADLNRNGSVDSVDYSILKRFLLKTI


 


67
Cthe2950
DLNNDGRTNSTDYSLMKRYLLGSISFTNEQLKAADVNLDGKVNSSDYTVLRRFLLGSI


 


68
Cthe2972
VLGDLNGDKQVNSTDYTALKRHLLNITRLSGTALANADLNGDGKVDSTDLMILHRYLLGII


 


69
Cthe3012
DLNGDGNVNSTDSTLMSRYLLGIITTLPAGEKAADLNGDGKVNSTDYNILKRYLLKYI


 


70
Cthe3132
DLNGDGRVNSTDLAVMKRYLLKQVQISDIRPADLNGDGKANSTDYQLLKRYILKTI


 


71
Cthe3136
DDGNGEISSIDYAILKSHLINSNLTFKQLAAADVDGNGYVNSIDLAILQMYLLGKGGTSDI


 


72
Cthe3141
DVNGNGSIESTDCVWVKRYLLKQIDSFPNENGARAADVNGNGTIDSTDYQLLKRFILKVI









Similar amino acid sequences that are dockerin-specific sequences associated with cohesin binding can be discovered in these 72 type I dockerins. A dockerin-specific sequence may consist of a naturally-derived consensus sequence (relevant sequence) consisting of 24 amino acids. The total of 142 amino acid sequences shown by SEQ ID NOS. 73 to 214 in Table 2 below can be given as examples of relevant sequences intrinsic to the 72 type I dockerins. These amino acid sequences can be obtained from databases such as UniProt (www.uniprot.org), InterPro (www.ebi.ac.uk/interpro) and Pfam (pfam.sanger.ac.uk). N-terminal relevant sequences are described in the 1st column of Table 2, while C-terminal relevant sequences are described in the 2nd column.










TABLE 2





SEQ
locus




ID
(SEQ




NO:
Name)
-
Related Sequence







 73
Cthe0015
1st
DVNADGKIDSTDLTLLKRYLLRSA


 74

2nd
DTDGNGTVNSTDLNYLKKYILRVI


 


 75
Cthe0032
1st
DLNNDGNINSTDYMILKKYILKVL


 76

2nd
DLNGDGSINSTDLTILKRFIMKAI


 


 77
Cthe0043
1st
DLNGDGNINSTDFTMLKRAILGNP


 78

2nd
DLNRDGNTNSTDLMILRRYLLKLI


 


 79
Cthe0044
1st
DINLDGKINSTDLSALKRHILRIT


 80

2nd
DVNNDGSVNSTDASILKKYIAKAI


 


 81
Cthe0109
1st
DFNSDSSVNSTDLMILNRAVLGLG




2nd



 


 82
Cthe0190
1st
ELNGDGKINSSDLNMMKRYLLRLI


 83

2nd
DLNGDGKINSSDYSILKRYLLRMI


 


 84
Cthe0191
1st
DLNGDAKINSTDLNMMKRYLLQMI


 85
 
2nd
DLNGDGKISSDYNLLKRYILHLI


 


 86
Cthe0211
1st
DVNGDGHVNSSDYSLFKRYLLRVI


 87

2nd
DVNRDGRIDSTDLTMLKRYLIRAI


 


 88
Cthe0239
1st
DYNGDGAVNSTDLLACKRYLLYAL


 89

2nd
DLDGNGKINSTDYAYLKRYLLKQI


 


 90
Cthe0246
1st
DLNADGKINSTDYNLGKRLILRTI


 91

2nd
DLNGDSKVDSTDLTALKRYLLGVI


 


 92
Cthe0258
1st
DVNGDSKINAIDVLLMKKYILKVI


 93

2nd
DVNADGQINSIDFTWLKKYMLKAV


 


 94
Cthe0269
1st
DVNGDGNVNSTDLTMLKRYLLKSV


 95

2nd
DVNRDGAINSSDMTILKRYLIKSI


 


 96
Cthe0270
1st
DLNGDKVNSSDLAILKRYMLRAI


 97

2nd
DLNRDGNVNSTDYSILKRYILKAI


 


 98
Cthe0274
1st
DLNVDGSINSVDITYMKRYLLRSI


 99

2nd
DTNGDGAINSSDMVLLKRYVLRSI


 


100
Cthe0405
1st
DVNGDGNVNSTDVVWLRRFLLKLV


101

2nd
DMNDDGNINSTDMIALKRKVLKIP


 


102
Cthe0412
1st
DCNGDGKVNSTDAVALKRYILRSG


103

2nd
DVNADGRVNSTDLAILKRYILKEI


 


104
Cthe0413
1st
DCNDDGKVNSTDVAVMKRYLKKEN


105

2nd
DVNADGKVNSTDFSILKRYVMKNI


 


106
Cthe0433
1st
DLNGDGRVNSSDLALMKRYVVKQI


107

2nd
DLNGDDKVNSTDYSVLKRYLLRSI


 


108
Cthe0435
1st
DVNADGVVNISDYVLMKRYILRII


109

2nd
DVNGDNVINDIDCNYLKRYLLHMI


 


110
Cthe0438
1st
DLNGDNNINSSDYTLLKRYLLHTI




2nd



 


111
Cthe0536
1st
DVNGDGRVNSSDVALLKRYLLGLV


112

2nd
DVNVSGTVNSTDLAIMKRYVLRSI


 


113
Cthe0543
1st
DVNFDGRINSTDYSRLKRYVIKSL


114

2nd
DVDGNGRINSTDLYVLNRYILKLI


 


115
Cthe0578
1st
DINLDGKINSSDVTLLKRYIVKSI


116

2nd
DVNGDGRVNSTDYSYLKRYVLKII


 


117
Cthe0640
1st
DLNGDNNVNSTDLTLLKRYLTRVI


118

2nd
DVNGDGKINSTDYSAMRYILRII


 


119
Cthe0661
1st
DVNGDLKVNSTDFSMLRRYLLKTI


120

2nd
DLNGDGRINSSDLTMLKRYLLMEV


 


121
Cthe0624
1st
DLNNDSKVNAVDIMMLKRYILGII


122

2nd
DIYFDGVVNSSDYNIMKRYLLKAI


 


123
Cthe0625
1st
DLNGDGVVNSTDSVILKRHIIKFS


124

2nd
DLNGDGNINSSDVSLMKRYLLRII


 


125
Cthe0660
1st
DLNGDGKINSTDISLMKRYLLKQI


126

2nd
DINKDGKVNSTDMSILKRVILRNY


 


127
Cthe0729
1st
DSNSDCKVNSTDLTLMKRYLLQQS


128

2nd
DLNGDGKINSSDYTLLKRYLLGYI


 


129
Cthe0745
1st
DINNDKTVNSTDVTYLKRFLLKQI


130

2nd
DVNLDGNINSTDLVILKRYVLRGI


 


131
Cthe0797
1st
DVNGDGKINSTDCTMLKRYILRGI


132

2nd
DVNADLKINSTDLVLMKKYLLRSI


 


133
Cthe0798
1st
DVNLDGQVNSTDFSLLKRYLKVV


134

2nd
DMNNDGNINSTDISILKRILLRN


 


135
Cthe0821
1st
DINRDGKINSTDLGMLNRHILKLV


136

2nd
DIDGNGNINSTDYSWLKKYILKVI


 


137
Cthe0825
1st
DVNDDGKVNSTDLTLLKRYVLKAV


138

2nd
DVNRDGRVNSSDVTILSRYLIRVI


 


139
Cthe0912
1st
DVNGDGTINSTDLTMLKRSVLRAI


140

2nd
DVDKNGSINSTDVLLLSRYLLRVI


 


141
Cthe0918
1st
DLNRNGIVNDEDYILLKNYLLRGN


142

2nd
DVNKDGKVNSTDCLFLKKYILGLI


 


143
Cthe1271
1st
DTNSDGKINSTDVTALKRHLLRVT


144

2nd
DVNGDGNVNSTDLLLLKRYILGEI


 


145
Cthe1398
1st
DLNGDNRINSTDLTLMKRYILKSI


146

2nd
DINGDGKINSTDYTYLKKYLLQAI


 


147
Cthe1400
1st
DLNGDGRVNSTDYTLLKRYLLGAI


148

2nd
DLNLDGRINSTDYTVLKRYLLNAI


 


149
Cthe1472
1st
DLNFDNAVNSTDLLMLKRYILKSL


150

2nd
DLNRDNKVDSTDLTILKRYLLKAI


 


151
Cthe1806
1st
EVIDTKVIDSTDDIVKYEYQFDKK


152

2nd
TLVLSVNNDSTDKTTVSGYISVDF


 


153
Cthe1838
1st
DVNGDGRVNSSDLTLMKRYLLKSI


154

2nd
DLNEDGKVNSTDLLALKKLVLREL


 


155
Cthe1890
1st
DLNADGSINSTDLMIMKRVLLKQR


156

2nd
DLNGDGKVTSTDYSLMKRYLLKEI


 


157
Cthe1963
1st
DLNGDGNINSSDLQALKRHLLGIS


158

2nd
DVNRSGKVDSTDYSVLKRYILRII


 


159
Cthe2038
1st
DIVLDGNINSLDMMKLKKYLIRET


160

2nd
DVNSDGEVNSTDYAYLKRYILRII


 


161
Cthe2089
1st
DVNDDGKVNSTDAVALKRYVLRSG


162

2nd
DLNEDGRVNSTDLGILKRYILKEI


 


163
Cthe2137
1st
DVDGNGTVNSTDVNYMKRYLLRQI


164

2nd
DVDGNGNINSTDLSYLKKYILKLI


 


165
Cthe2139
1st
DVNADGVINSSDIMVLKRFLLRTI


166

2nd
DTNGDGAVNSSDFTLLKRVILRSI


 


167
Cthe2147
1st
DVNGDFAVNSNDLTLIKRYVLKNI


168

2nd
DVDGDEKITSSDAALVKRYVLRAI


 


169
Cthe2179
1st
DLNGDGNVNSTDSILMKRYLMKSV


170

2nd
DVNLDGRVNSTDRSILNRYLLKII


 


171
Cthe2193
1st
DINDDGNINSTDLQMLKRHLLRSI


172

2nd
DTNRDGRVDSTDLALLKRYILRVI


 


173
Cthe2194
1st
DLNGDGNINSTDLQILKKHLLRIT


174
 
2nd
DVTKDGKVDSTDLTLLKRYILRFV


 


175
Cthe2195
1st
DLNDDGKVNSTDFQILKKHLLRIT


176

2nd
DLNKDGKVDSSDLSLMKRYLLQII


 


177
Cthe2196
1st
DLNNDGKVNSTDFQLLKMHVLRQE


178

2nd
DVNRDGKVDSSDCTLLKRYILRVI


 


179
Cthe2197
1st
DLNGDGKVNSTDLQLMKMHVLRQR


180

2nd
DVNRDGKVDSTDVALLKRYILRQI


 


181
Cthe2271
1st
DVNLDGSVDSTDLALLYNTTYYAV


182

2nd
DVNGDGTVDGIDLAIITAYINGQI


 


183
Cthe2360
1st
DLNGDGRVNSTDLLLMKKRIIREI


184

2nd
DLNLDGKINSSDYTILKRVVLKSI


 


185
Cthe2549
1st
DVNKDGRINSTDIMYLKGVLLRNS


186

2nd
DVDGNGSVSSLDLTYLKRYILRRI


 


187
Cthe2590
1st
DLNQDGQVSSTDLVAMKRYLLKNF


188

2nd
DLNSDGKVNSTDLVALKRFLLKEI


 


189
Cthe2760
1st
DLNYDGKVNSTDYLVLKRYLLGTI


190

2nd
DLNRDGRVNSTDMSLMKRYLLGII


 


191
Cthe2761
1st
DVNGDGKVNSTDCSIVKRYLLKNI


192

2nd
DVNGDGKVNSTDYSLLKRFVLRNI


 


193
Cthe2811
1st
DLNGDGKVNSTDLTIMKRYILKNF


194

2nd
DLNGDGRINSTDLSILHRYLLRII


 


195
Cthe2812
1st
DLNGDQKVTSTDYTMLKRYLMKSI


196

2nd
DLNRDGKINSTDLTILKRYLLYSI


 


197
Cthe2872
1st
DINSDGNVNSTDLGILKRIIVKNP


198

2nd
DVNADGKVNSTDYTVLKRYLLRSI


 


199
Cthe2879
1st
DINSDGSINSTDVTLLKRHLLREN


200

2nd
DTDGDGKITSIDLSVLKRVVLRLI


 


201
Cthe2949
1st
DLNGDGLVNSSDYSLLKRYILKQI


202

2nd
DLNRNGSVDSVDYSILKRFLLKTI


 


203
Cthe2950
1st
DLNNDGRTNSTDYSLMKRYLLGSI


204

2nd
DVNLDGKVNSSDYTVLRRFLLGSI


 


205
Cthe2972
1st
DLNGDKQVNSTDYTALKRHLLNIT


206

2nd
DLNGDGKVDSTDLMILHRYLLGII


 


207
Cthe3012
1st
DLNGDGNVNSTDSTLMSRYLLGII


208
 
2nd
DLNGDGKVNSTDYNILKRYLLKVI


 


209
Cthe3132
1st
DLNGDGRVNSTDLAVMKRYLLKQV


210

2nd
DLNGDGKANSTDYQLLKRYILKTI


 


211
Cthe3136
1st
DIDGNGEISSIDYAILKSHLINSN


212

2nd
DVDGNGYVNSIDLAILQMYLLGKG


 


213
Cthe3141
1st
DVNGNGSIESTDCVWVKRYLLKQI


214

2nd
DVNGNGTIDSTDYQLLKRFILKVI









While a homology search of the doekerins shown in Table 1 revealed that the “homology” among these amino acid sequences does not exceed 90%, there is 90% or more “similarity” among the relevant sequences shown in Table 2. This suggests that the dockerins shown in Table 1 all have similar functions. It is therefore presumed that the relevant sequences shown in Table 2 are responsible for these functions.
In the dockerins shown in Table 1 or in other words in the relevant sequences shown in Table 2, the predicted N-type sugar chain modification sites are known to be N positions in N-X-T or N-X-S (in which N is asparagine, X is an amino acid other than proline, T is threonine and S is serine), which are consensus sequences that undergo N-type sugar chain modification in yeasts and other eukaryotic microorganisms (A. Herscovics et al., The FASEB Journal (6): 540-550 (1993)). An N-X-T/S of a dockerin or its relevant sequence can be found by suitable application of one of the databases described above or the like. A site corresponding to a predicted N-type sugar chain modification site in a dockerin or its relevant sequence may also correspond to N even when the amino acid sequence does not include one of the aforementioned consensus sequences. A site corresponding to a predicted N-type sugar chain modification site may be discovered by comparing an amino acid sequence that may contain this site by multiple alignment with the amino acid sequence of a known dockerin or its relevant sequence. If the amino acid sequence of a relevant sequence consists of about 24 or fewer amino acids, the predicted N-type sugar chain modification site in a dockerin, or a site corresponding to such a site, is typically the 9th amino acid from the N terminal.
The protein of the invention preferably has a dockerin comprising at least one dockerin-specific sequence having no predicted N-type sugar chain modification site. It also preferably has at least one dockerin-specific sequence in which the amino acid of a site corresponding to a predicted N-type sugar chain modification site is aspartic acid (D). It is thought that N-type sugar chain modification by yeasts and other eukaryotic microorganisms is eliminated when there is no N-type sugar chain modification site or when a site corresponding to a predicted sugar chain modification site is occupied by aspartic acid. A dockerin-specific sequence in which a site corresponding to a predicted sugar chain modification site is occupied by aspartic acid may be intrinsic to the original dockerin, or may have a N-type sugar chain modification site at which aspartic acid (D) has been substituted for asparagine (N).
Examples of one embodiment of this dockerin-specific sequence include dockerin-specific sequences having aspartic acid substituted for asparagine in the dockerins disclosed in Table 1 and the relevant sequences in these dockerins disclosed in Table 2 when these have intrinsic predicted N-type sugar chain modification sites. It is sufficient that the protein of the invention have a dockerin containing at least one such dockerin-specific sequence. Examples of relevant sequences having candidate N→D substitution sites include the following 113 relevant sequences. Consequently, preferred dockerin-specific sequences are sequences in which D is substituted for N (N-X-T/S) in the relevant sequences below.











TABLE 3







locus

Amino Acid Sequence









Cthe0015
2nd
DTDGNGTVNSTDLNYLKKYILRVI





(SEQ ID NO: 74)



 



Cthe0032
1st
DLNNDGNINSTDYMILKKYILKVL





(SEQ ID NO: 75)



 



Cthe0032
2nd
DLNGDGSINSTDLTILKRFIMKAI





(SEQ ID NO: 76)



 



Cthe0043
1st
DLNGDGNINSTDFTMLKRAILGNP





(SEQ ID NO: 77)



 



Cthe0043
2nd
DLNRDGNTNSTDLMILRRYLLKLI





(SEQ ID NO: 78)



 



Cthe0044
1st
DINLDGKINSTDLSALKRHILRIT





(SEQ ID NO: 79)



 



Cthe0044
2nd
DVNNDGSVNSTDASILKKYIAKAI





(SEQ ID NO: 80)



 



Cthe0109
1st
DFNSDSSVNSTDLMILNRAVLGLG





(SEQ ID NO: 81)



 



Cthe0190
1st
ELNGDGKINSSDLNMMKRYLLRLI





(SEQ ID NO: 82)



 



Cthe0190
2nd
DLNGDGKINSSDYSILKRYLLRMI





(SEQ ID NO: 83)



 



Cthe0191
1st
DLNGDAKINSTDLNMMKRYLLQMI





(SEQ ID NO: 84)



 



Cthe0211
1st
DVNGDGHVNSSDYSLFKRYLLRVI





(SEQ ID NO: 86)



 



Cthe0239
1st
DYNGDGAVNSTDLLACKRYLLYAL





(SEQ ID NO: 88)



 



Cthe0239
2nd 
DLDGNGKINSTDYAYLKRYLLKQI





(SEQ ID NO: 89)



 



Cthe0246
1st
DLNADGKINSTDYNLGKRLILRTI





(SEQ ID NO: 90)



 



Cthe0269
1st
DVNGDGNVNSTDLTMLKRYLLKSV





(SEQ ID NO: 94)



 



Cthe0269
2nd 
DVNRDGAINSSDMTILKRYLIKSI





(SEQ ID NO: 95)



 



Cthe0270
1st
DLNGDGKVNSSDLAILKRYMLRAI





(SEQ ID NO: 96)



 



Cthe0270
2nd
DLNRDGNVNSTDYSILKRYILKAI





(SEQ ID NO: 97)



 



Cthe0274
2nd
DTNGDGAINSSDMVLLKRYVLRSI





(SEQ ID NO: 99)



 



Cthe0405
1st
DVNGDGNVNSTDVVWLRRFLLKLV





(SEQ ID NO: 100)



 



Cthe0405
2nd
DMNDDGNINSTDMIALKRKVLKIP





(SEQ ID NO: 101)



 



Cthe0412
lst
DCNGDGKVNSTDAVALKRYILRSG





(SEQ ID NO: 102)



 



Cthe0412
2nd
DVNADGRVNSTDLAILKRYILKEI





(SEQ ID NO: 103)



 



Cthe0413
1st
DCNDDGKVNSTDVAVMKRYLKKEN





(SEQ ID NO: 104)



 



Cthe0413
2nd
DVNADGKVNSTDFSILKRYVMKNI





(SEQ ID NO: 105)



 



Cthe0433
1st
DLNGDGRVNSSDLALMKRYVVKQI





(SEQ ID NO: 106)



 



Cthe0433
2nd
DLNGDDKVNSTDYSVLKRYLLRSI





(SEQ ID NO: 107)



 



Cthe0435
1st
DVNADGVVNISDYVLMKRYILRII





(SEQ ID NO: 108)



 



Cthe0438
1st
DLNGDNNINSSDYTLLKRYLLHTI





(SEQ ID NO: 110)



 



Cthe0536
1st
DVNGDGRVNSSDVALLKRYLLGLV





(SEQ ID NO: 111)



 



Cthe0536
2nd
DVNVSGTVNSTDLAIMKRYVLRSI





(SEQ ID NO: 112)



 



Cthe0543
1st
DVNFDGRINSTDYSRLKRYVIKSL





(SEQ ID NO: 113)



 



Cthe0543
2nd
DVDGNGRINSTDLYVLNRYILKLI





(SEQ ID NO: 114)



 



Cthe0578
1st
DINLDGKINSSDVTLLKRYIVKSI





(SEQ ID NO: 115)



 



Cthe0578
2nd
DVNGDGRVNSTDYSYLKRYVLKII





(SEQ ID NO: 116)



 



Cthe0624
1st
DLNNDSKVNAVDIMMLKRYILGII





(SEQ ID NO: 121)



 



Cthe0624
2nd
DIYFDGVVNSSDYNIMKRYLLKAI





(SEQ ID NO: 122)



 



Cthe0625
1st
DLNGDGVVNSTDSVILKRHIIKFS





(SEQ ID NO: 123)



 



Cthe0625
2nd
DLNGDGNINSSDVSLMKRYLLRII





(SEQ ID NO: 124)



 



Cthe0640
1st
DLNGDNNVNSTDLTLLKRYLTRVI





(SEQ ID NO: 117)



 



Cthe0640
2nd
DVNGDGKINSTDYSAMIRYILRII





(SEQ ID NO: 118)



 



Cthe0660
1st
DLNGDGKINSTDISLMKRYLLKQI





(SEQ ID NO: 125)



 



Cthe0660
2nd
DINKDGKVNSTDMSILKRVILRNY





(SEQ ID NO: 126)



 



Cthe0661
1st
DVNGDLKVNSTDFSMLRRYLLKTI





(SEQ ID NO: 119)



 



Cthe0661
2nd
DLNGDGRINSSDLTMLKRYLLMEV





(SEQ ID NO: 120)



 



Cthe0729
1st
DSNSDCKVNSTDLTLMKRYLLQQS





(SEQ ID NO: 127)



 



Cthe0729
2nd
DLNGDGKINSSDYTLLKRYLLGYI





(SEQ ID NO: 128)



 



Cthe0745
1st
DINNDKTVNSTDVTYLKRFLLKQI





(SEQ ID NO: 129)



 



Cthe0745
2nd
DVNLDGNINSTDLVILKRYVLRGI





(SEQ ID NO: 130)



 



Cthe0797
1st
DVNGDGKINSTDCTMLKRYILRGI





(SEQ ID NO: 131)



 



Cthe0797
2nd
DVNADLKINSTDLVLMKKYLLRSI





(SEQ ID NO: 132)



 



Cthe0798
1st
DVNLDGQVNSTDFSLLKRYILKVV





(SEQ ID NO: 133)



 



Cthe0798
2nd
DMNNDGNINSTDISILKRILLRN





(SEQ ID NO: 134)



 



Cthe0821
1st
DINRDGKINSTDLGMLNRHILKLV





(SEQ ID NO: 135)



 



Cthe0821
2nd
DIDGNGNINSTDYSWLKKYILKVI





(SEQ ID NO: 136)



 



Cthe0825
1st
DVNDDGKVNSTDLTLLKRYVLKAV





(SEQ ID NO: 137)



 



Cthe0825
2nd
DVNRDGRVNSSDVTILSRYLIRVI





(SEQ ID NO: 138)



 



Cthe0912
1st
DVNGDGTINSTDLTMLKRSVLRAI





(SEQ ID NO: 139)



 



Cthe0912
2nd
DVDKNGSINSTDVLLLSRYLLRVI





(SEQ ID NO: 140)



 



Cthe0918
2nd
DVNKDGKVNSTDCLFLKKYILGLI





(SEQ ID NO: 142)



 



Cthe1271
1st
DTNSDGKINSTDVTALKRHLLRVT





(SEQ ID NO: 143)



 



Cthe1271
2nd
DVNGDGNVNSTDLLLLKRYILGEI





(SEQ ID NO: 144)



 



Cthe1398
1st
DLNGDNRINSTDLTLMKRYILKSI





(SEQ ID NO: 145)



 



Cthe1398
2nd
DINGDGKINSTDYTYLKKYLLQAI





(SEQ ID NO: 146)



 



Cthe1400
1st
DLNGDGRVNSTDYTLLKRYLLGAI





(SEQ ID NO: 147)



 



Cthe1400
2nd
DLNLDGRINSTDYTVLKRYLLNAI





(SEQ ID NO: 148)



 



Cthe1472
1st
DLNFDNAVNSTDLLMLKRYILKSL





(SEQ ID NO: 149)



 



Cthe1806
2nd
TLVLSVNNDSTDKTTVSGYISVDF





(SEQ ID NO: 152)



 



Cthe1838
1st
DVNGDGRVNSSDLTLMKRYLLKSI





(SEQ ID NO: 153)



 



Cthe1838
2nd
DLNEDGKVNSTDLLALKKLVLREL





(SEQ ID NO: 154)



 



Cthe1890
1st
DLNADGSINSTDLMIMKRVLLKQR





(SEQ ID NO: 155)



 



Cthe1963
1st
DLNGDGNINSSDLQALKRHLLGIS





(SEQ ID NO: 157)



 



Cthe1963
2nd
DVNRSGKVDSTDYSVLKRYILRII





(SEQ ID NO: 158)



 



Cthe2038
2nd
DVNSDGEVNSTDYAYLKRYILRII





(SEQ ID NO: 160)



 



Cthe2089
1st
DVNDDGKVNSTDAVALKRYVLRSG





(SEQ ID NO: 161)



 



Cthe2089
2nd
DLNEDGRVNSTDLGILKRYILKEI





(SEQ ID NO: 162)



 



Cthe2137
1st
DVDGNGTVNSTDVNYMKRYLLRQI





(SEQ ID NO: 163)



 



Cthe2137
2nd
DVDGNGNINSTDLSYLKKYILKLI





(SEQ ID NO: 164)



 



Cthe2139
1st
DVNADGVINSSDIMVLKRFLLRTI





(SEQ ID NO: 165)



 



Cthe2139
2nd
DTNGDGAVNSSDFTLLKRYILRSI





(SEQ ID NO: 166)



 



Cthe2179
1st
DLNGDGNVNSTDSILMKRYLMKSV





(SEQ ID NO: 169)



 



Cthe2179
2nd
DVNLDGRVNSTDRSILNRYLLKII





(SEQ ID NO: 170)



 



Cthe2193
1st
DINDDGNINSTDLQMLKRHLLRSI





(SEQ ID NO: 171)



 



Cthe2194
1st
DLNGDGNINSTDLQILKKHLLRIT





(SEQ ID NO: 173)



 



Cthe2195
1st
DLNDDGKVNSTDFQILKKHLLRIT





(SEQ ID NO: 175)



 



Cthe2196
1st
DLNNDGKVNSTDFQLLKMHVLRQE





(SEQ ID NO: 177)



 



Cthe2197
1st
DLNGDGKVNSTDLQLMKMHVLRQR





(SEQ ID NO: 179)



 



Cthe2360
1st
DLNGDGRVNSTDLLLMKKRIIREI





(SEQ ID NO: 183)



 



Cthe2360
2nd
DLNLDGKINSSDYTILKRYVLKSI





(SEQ ID NO: 184)



 



Cthe2549
1st
DVNKDGRINSTDIMYLKGYLLRNS





(SEQ ID NO: 185)



 



Cthe2590
2nd
DLNSDGKVNSTDLVALKRFLLKEI





(SEQ ID NO: 188)



 



Cthe2760
1st
DLNYDGKVNSTDYLVLKRYLLGTI





(SEQ ID NO: 189)



 



Cthe2760
2nd
DLNRDGRVNSTDMSLMKRYLLGII





(SEQ ID NO: 190)



 



Cthe2761
1st
DVNGDGKVNSTDCSIVKRYLLKNI





(SEQ ID NO: 191)



 



Cthe2761
2nd
DVNGDGKVNSTDYSLLKRFVLRNI





(SEQ ID NO: 192)



 



Cthe2811
1st
DLNGDGKVNSTDLTIMKRYILKNF





(SEQ ID NO: 193)



 



Cthe2811
2nd 
DLNGDGRINSTDLSILHRYLLRII





(SEQ ID NO: 194)



 



Cthe2812
2nd
DLNRDGKINSTDLTILKRYLLYSI





(SEQ ID NO: 196)



 



Cthe2872
1st
DINSDGNVNSTDLGILKRIIVKNP





(SEQ ID NO: 197)



 



Cthe2872
2nd
DVNADGKVNSTDYTVLKRYLLRSI





(SEQ ID NO: 198)



 



Cthe2879
1st
DINSDGSINSTDVTLLKRHLLREN





(SEQ ID NO: 199)



 



Cthe2949
1st
DLNGDGLVNSSDYSLLKRYILKQI





(SEQ ID NO: 201)



 



Cthe2949
2nd
DLNRNGSVDSVDYSILKRFLLKTI





(SEQ ID NO: 202)



 



Cthe2950
1st
DLNNDGRTNSTDYSLMKRYLLGSI





(SEQ ID NO: 203)



 



Cthe2950
2nd
DVNLDGKVNSSDYTVLRRFLLGSI





(SEQ ID NO: 204)



 



Cthe2972
1st
DLNGDKQVNSTDYTALKRHLLNIT





(SEQ ID NO: 205)



 



Cthe3012
1st
DLNGDGNVNSTDSTLMSRYLLGII





(SEQ ID NO: 207)



 



Cthe3012
2nd
DLNGDGKVNSTDYNILKRYLLKYI





(SEQ ID NO: 208)



 



Cthe3132
1st
DLNGDGRVNSTDLAVMKRYLLKQV





(SEQ ID NO: 209)



 



Cthe3132
2nd
DLNGDGKANSTDYQLLKRYILKTI





(SEQ ID NO: 210)



 



Cthe3141
1st
DVNGDNSIESTDCVWVKRYLLKQI





(SEQ ID NO: 213)



 



Cthe3141
2nd
DVNGNGTIDSTDYQLLKRFILKVI





(SEQ ID NO: 214)










The protein of the invention may be provided with a dockerin comprising one or two such dockerin-specific sequences, but typically, aspartic acid is substituted for asparagine at a predicted sugar chain modification site in the relevant sequence. The dockerins shown in the table below are examples of such dockerins. In these tables, the dockerins are specified by means of their relevant sequences. Thus, a preferred dockerin can have a dockerin-specific sequence in which D is substituted for N in (N-X-T/S) in one or two relevant sequences of any of the dockerins in the table below.








TABLE 4





locus
Amino Acid Sequence



















Cthe0032
1st
DLNNDGNINSTDYMILKKYILKVL
2nd
DLNGDGSINSTDLTILKRFIMKAI




(SEQ ID NO: 75)

(SEQ ID NO: 76)


 


Cthe0043
1st
DLNGDGNINSTDFTMLKRAILGNP
2nd
DLNRDGNTNSTDLMILRRYLLKLI




(SEQ ID NO: 77)

(SEQ ID NO: 78)


 


Cthe0044
1st
DINLDGKINSTDLSALKRHILRIT
2nd
DVNNDGSVNSTDASILKKYIAKAI




(SEQ ID NO: 79)

(SEQ ID NO: 80)


 


Cthe0190
1st
ELNGDGKINSSDLNMMKRYLLRLI
2nd
DLNGDGKINSSDYSILKRYLLRMI




(SEQ ID NO: 81)

(SEQ ID NO: 82)


 


Cthe0239
1st
DYNGDGAVNSTDLLACKRYLLYAL
2nd
DLDGNGKINSTDYAYLKRYLLKQI




(SEQ ID NO: 88)

(SEQ ID NO: 89)


 


Cthe0269
1st
DVNGDGNVNSTDLTMLKRYLLKSV
2nd
DVNRDGAINSSDMTILKRYLIKSI




(SEQ ID NO: 94)

(SEQ ID NO: 95)


 


Cthe0270
1st
DLNGDGKVNSSDLAILKRYMLRAI
2nd
DLNRDGNVNSTDYSILKRYILKAI




(SEQ ID NO: 96)

(SEQ ID NO: 97)


 


Cthe0405
1st
DVNGDGNVNSTDVVWLRRFLLKLV
2nd
DMNDDGNINSTDMIALKRKVLKIP




(SEQ ID NO: 100)

(SEQ ID NO: 101)


 


Cthe0412
1st
DCNGDGKVNSTDAVALKRYILRSG
2nd
DVNADGRVNSTDLAILKRYILKEI




(SEQ ID NO: 102)

(SEQ ID NO: 103)


 


Cthe0413
1st
DCNDDGKVNSTDVAVMKRYLKKEN
2nd
DVNADGKVNSTDFSILKRYVMKNI 




(SEQ ID NO: 104)

(SEQ ID NO: 105)


 


Cthe0433
1st
DLNGDGRVNSSDLALMKRYVVKQI
2nd
DLNGDDKVNSTDYSVLKRYLLRSI




(SEQ ID NO: 106)

(SEQ ID NO: 107)


 


Cthe0536
1st
DVNGDGRVNSSDVALLKRYLLGLV
2nd
DVNVSGTVNSTDLAIMKRYVLRSI




(SEQ ID NO: 111)

(SEQ ID NO: 112)


 


Cthe0543
1st
DVNFDGRINSTDYSRLKRYVIKSL
2nd
DVDGNGRINSTDLYVLNRYILKLI




(SEQ ID NO: 113)

(SEQ ID NO: 114)


 


Cthe0578
1st
DINLDGKNSSDVTLLKRYIVNKSI
2nd
DVNGDGRVNSTDYSYLKRYVLKII




(SEQ ID NO: 115)

(SEQ ID NO: 116)


 


Cthe0624
1st
DLNNDSKVNAVDImMLKRYILGII
2nd
DIYFDGVVNSSDYNIMKRYLLKAI




(SEQ ID NO: 121)

(SEQ ID NO: 122)


 


Cthe0625
1st
DLNGDGVVNSTDSVILKRHIIKFS
2nd
DLNGDGNINSSDVSLMKRYLLRII




(SEQ ID NO: 123)

(SEQ ID NO: 124)


 


Cthe0640
1st
DLNGDNNVNSTDLTLLKRYLTRVI
2nd
DVNGDGKINSTDYSAMIRYILRII




(SEQ ID NO: 117)

(SEQ ID NO: 118)


 


Cthe0660
1st
DLNGDGKINSTDISLMKRYLLKQI
2nd
DINKDGKVNSTDMSILKRVILRNY




(SEQ ID NO: 125)

(SEQ ID NO: 126)


 


Cthe0661
1st
DVNGDLKVNSTDFSMLRRYLLKTI
2nd
DLNGDGRINSSDLTMLKRYLLMEV




(SEQ ID NO: 119)

(SEQ ID NO: 120)


 


Cthe0729
1st
DSNSDCKVNSTDLTLMKRYLLQQS
2nd
DLNGDGKINSSDYTLLKRYLLGYI




(SEQ ID NO: 127)

(SEQ ID NO: 128)


 


Cthe0745
1st
DINNDKTVNSTDVTYLKRFLLKQI
2nd
DVNLDGNINSTDLVILKRYVLRGI




(SEQ ID NO: 129)

(SEQ ID NO: 130)


 


Cthe0797
1st
DVNGDGKINSTDCTMLKRYILRGI
2nd
DVNADLKINSTDLVLMKKYLLRSI




(SEQ ID NO: 131)

(SEQ ID NO: 132)


 


Cthe0798
1st
DVNLDGQVNSTDFSLLKRYILKVV
2nd
DMNNDGNINSTDISILKRILLRN




(SEQ ID NO: 133)

(SEQ ID NO: 134)


 


Cthe0821
1st
DINRDGKINSTDLGMLNRHILKLV
2nd
DIDGNGNINSTDYSWLKKYLKVI




(SEQ ID NO: 135)

(SEQ ID NO: 136)


 


Cthe0825
1st
DVNDDGKVNSTDLTLLKRYVLKAV
2nd
DVNRDGRVNSSDVTLSRYLIRVI




(SEQ ID NO: 137)

(SEQ ID NO: 138)


 


Cthe0912
1st
DVNGDGTINSTDLTMLKRSVLRAI
2nd
DVDKNGSINSTDVLLLSRYLLRVI




(SEQ ID NO: 139)

(SEQ ID NO: 140)


 


Cthe1271
1st
DTNSDGKINSTDVTALKRHLLRVT
2nd
DVNGDGNVNSTDLLLLKRYILGEI




(SEQ ID NO: 143)

(SEQ ID NO: 144)


 


Cthe1398
1st
DLNGDNRINSTDLTLMKRYILKSI
2nd
DINGDGKINSTDYTYLKKYLLQAI




(SEQ ID NO: 145)

(SEQ ID NO: 146)


 


Cthe1400
1st
DLNGDGRVNSTDYTLLKRYLLGAI
2nd
DLNLDGRINSTDYTVLKRYLLNAI




(SEQ ID NO: 147)

(SEQ ID NO: 148)


 


Cthe1838
1st
DVNGDGRVNSSDLTLMKRYLLKSI
2nd
DLNEDGKVNSTDLLALKKLVLREL




(SEQ ID NO: 153)

(SEQ ID NO: 154)


 


Cthe2089
1st
DVNDDGKVNSTDAVALKRYVLRSG
2nd
DLNEDGRVNSTDLGILKRYILKEI




(SEQ ID NO: 161)

(SEQ ID NO: 162)


 


Cthe2137
1st
DVDGNGTVNSTDVNYMKRYLLRQI
2nd
DVDGNGNINSTDLSYLKKYILKLI




(SEQ ID NO: 163)

(SEQ ID NO: 164)


 


Cthe2139
1st
DVNADGVINSSDIMVLKRFLLRTI
2nd
DTNGDGAVNSSDFTLLKRYILRSI




(SEQ ID NO: 165)

(SEQ ID NO: 166)


 


Cthe2179
1st
DLNGDGNVNSTDSILMKRYLMKSV
2nd
DVNLDGRVNSTDRSILNRYLLKII




(SEQ ID NO: 169)

(SEQ ID NO: 170)


 


Cthe2360
1st
DLNGDGRVNSTDLLLMKKRIIREI
2nd
DLNLDGKINSSDYTILKRYVLKSI




(SEQ ID NO: 183)

(SEQ ID NO: 184)


 


Cthe2549
1st
DVNKDGRINSTDIMYLKGYLLRNS
2nd
DVDGNGSVSSLDLTYLKRYILRRI




(SEQ ID NO: 185)

(SEQ ID NO: 186)


 


Cthe2760
1st
DLNYDGKVNSTDYLVLKRYLLGTI
2nd
DLNRDGRVNSTDMSLMKRYLLGII




(SEQ ID NO: 189)

(SEQ ID NO: 190)


 


Cthe2761
1st
DVNGDGKVNSTDCSIVKRYLLKNI
2nd
DVNGDGKVNSTDYSLLKRFVLRNI




(SEQ ID NO: 191)

(SEQ ID NO: 192)


 


Cthe2811
1st
DLNGDGKVNSTDLTIMKRYILKNF
2nd
DLNGDGRINSTDLSILHRYLLRII




(SEQ ID NO: 193)

(SEQ ID NO: 194)


 


Cthe2872
1st
DINSDGNVNSTDLGILKRIIVKNP
2nd
DVNADGKVNSTDYTVLKRYLLRSI




(SEQ ID NO: 197)

(SEQ ID NO: 198)


 


Cthe2949
1st
DLNGDGLVNSSDYSLLKRYILKQI
2nd
DLNRNGSVDSVDYSILKRFLLKTI




(SEQ ID NO: 201)

(SEQ ID NO: 202)


 


Cthe2950
1st
DLNNDGRTNSTDYSLMKRYLLGSI
2nd
DVNLDGKVNSSDYTVLRRFLLGSI




(SEQ ID NO: 203)

(SEQ ID NO: 204)


 


Cthe3012
1st
DLNGDGNVNSTDSTLMSRYLLGII
2nd
DLNGDGKVNSTDYNILKRYLLKYI




(SEQ ID NO: 207)

(SEQ ID NO: 208)


 


Cthe3132
1st
DLNGDGRVNSTDLAVMKRYLLKQV
2nd
DLNGDGKANSTDYQLLKRYILKTI




(SEQ ID NO: 209)

(SEQ ID NO: 210)









The dockerins shown in Table 4 each have two relevant sequences in the dockerin, and each relevant sequence has a predicted N-type sugar chain modification site. A preferred dockerin can be obtained with any of these dockerins by making a dockerin-specific sequence in which aspartic acid is substituted for asparagine at the predicted N-type sugar chain modification site of one or both of the two relevant sequences.








TABLE 5





locus
Amino Acid Sequence



















Cthe0109
1st
DFNSDSSVNSTDLMILNRAVLGLG
2nd





(SEQ ID NO: 81)




 


Cthe0191
1st
DLNGDAKINSTDLNMMKRYLLQMI
2nd
DLNGDGKITSSDYNLLKRYILHLI




(SEQ ID NO: 84)

(SEQ ID NO: 85)


 


Cthe0211
1st
DVNGDGHVNSSDYSLFKRYLLRVI
2nd
DVNRDGRIDSTDLTMLKRYLIRAI




(SEQ ID NO: 86)

(SEQ ID NO: 87)


 


Cthe0246
1st
DLNADGKINSTDYNLGKRLILRTI
2nd
DLNGDSKVDSTDLTALKRYLLGVI




(SEQ ID NO: 90)

(SEQ ID NO: 91)


 


Cthe0435
1st
DVNADGVVNISDYVLMKRYILRII
2nd
DVNGDNVINDIDCNYLKRYLLHMI




(SEQ ID NO: 108)

(SEQ ID NO: 109)


 


Cthe0438
1st
DLNGDNNINSSDYTLLKRYLLHTI
2nd





(SEQ ID NO: 110)




 


Cthe1472
1st
DLNFDNAVNSTDLLMLKRYILKSL
2nd
DLNRDNKVDSTDLTILKRYLLKAI




(SEQ ID NO: 149)

(SEQ ID NO: 150)


 


Cthe1890
1st
DLNADGSINSTDLMIMKRVLLKQR
2nd
DLNGDGKVTSTDYSLMKRYLLKEI




(SEQ ID NO: 155)

(SEQ ID NO: 156)


 


Cthe1963
1st
DLNGDGNINSSDLQALKRHLLGIS
2nd
DVNRSGKVDSTDYSVLKRYILRII




(SEQ ID NO: 157)

(SEQ ID NO: 158)


 


Cthe2193
1st
DINDDGNINDSTDLQMLKRHLLRSI
2nd
DTNRDGRVDSTDLALLKRYILRVI




(SEQ ID NO: 171)

(SEQ ID NO: 172)


 


Cthe2194
1st
DLNGDGNINSTDLQILKKHLLRIT
2nd
DVTKDGKVDSTDLTLLKRYILRFV




(SEQ ID NO: 173)

(SEQ ID NO: 174)


 


Cthe2195
1st
DLNDDGKVNSTDFQILKKHLLRIT
2nd
DLNKDGKVDSSDLSLMKRYLLQII




(SEQ ID NO: 175)

(SEQ ID NO: 176)


 


Cthe2196
1st
DLNNDGKVNSTDFQLLKMHVLRQE
2nd
DVNRDGKVDSSDCTLLKRYILRVI




(SEQ ID NO: 177)

(SEQ ID NO: 178)


 


Cthe2197
1st
DLNGDGKVNSTDLQLMKMHVLRQR
2nd
DVNRDGKVDSTDVALLKRYILRQI




(SEQ ID NO: 179)

(SEQ ID NO: 180)


 


Cthe2879
1st
DINSDGSINSTDVTLLKRHLLREN
2nd
DTDGDGKITSIDLSYLKRYVLRLI




(SEQ ID NO: 199)

(SEQ ID NO: 200)


 


Cthe2972
1st
DLNGDKQVNSTDYTALKRHLLNIT
2nd
DLNGDGKVDSTDLMILHRYLLGII




(SEQ ID NO: 205)

(SEQ ID NO: 206)









The dockerins shown in Table 5 each have one or two relevant sequences in the dockerin, and have a predicted N-type sugar chain modification site in the N-terminal relevant sequence. A preferred dockerin can be obtained with these dockerins by making a dockerin-specific sequence in which aspartic acid is substituted for asparagine at the predicted N-type sugar chain modification site of this relevant sequence.








TABLE 6





locus
Amino Acid Sequence



















Cthe0015
1st
DVNADGKIDSTDLTLLKRYLLRSA
2nd
DTDGNGTVNSTDLNYLKKYILRVI




(SEQ ID NO: 73)

(SEQ ID NO: 74)


 


Cthe0274
1st
DLNVDGSINSVDITYMKRYLLRSI
2nd
DINGDGAINSSDMVLLKRYVLRSI




(SEQ ID NO: 98)

(SEQ ID NO: 99)


 


cthe0918
1st
DLNRNGIVNDEDYILLKNYLLRGN
2nd
DVNKDGKVNSTDCLFLKKYILGLI




(SEQ ID NO: 141)

(SEQ ID NO: 142)


 


Cthe1806
1st
EVIDTKVIDSTDDIVKYEYQFDKK
2nd
TLVLSVNNDSTDKTTVSGYISVDF




(SEQ ID NO: 151)

(SEQ ID NO: 152)


 


Cthe2038
1st
DIVLDGNINSLDMMKLKKYLIRET
2nd
DVNSDGEVNSTDYAYLKRYILRII




(SEQ ID NO: 159)

(SEQ ID NO: 160)


 


Cthe2590
1st
DLNQDGQVSSTDLVAMKRYLLKNF
2nd
DLNSDGKVNSTDLVALKRFLLKEI




(SEQ ID NO: 187)

(SEQ ID NO: 188)


 


Cthe2812
1st
DLNGDQKVTSTDYTMLKRYLMKSI
2nd
DLNRDGKINSTDLTILKRYLLYSI




(SEQ ID NO: 195)

(SEQ ID NO: 196)









The dockerins shown in Table 6 each have two relevant sequences in the dockerin, and have a predicted N-type sugar chain modification site in the C-terminal relevant sequence. A preferred dockerin can be obtained with these dockerins by making a dockerin-specific sequence in which aspartic acid is substituted for asparagine in the predicted N-type sugar chain modification site of this relevant sequence.
The C. thermocellum type I dockerins shown in the following table, the binding ability of which with cohesins has been confirmed from existing literature and the like, are considered when selecting dockerin-specific sequences including preferred dockerins in the protein of the invention. In the following table, the dockerins are each specified by two relevant sequences. A preferred dockerin comprising a dockerin-specific sequence with aspartic acid substituted for asparagine at a predicted N-type sugar chain modification site in a relevant sequence can be obtained if this relevant sequence has 90% or more amino acid sequence similarity with any of the relevant sequences contained in these dockerins.









TABLE 7





locus
protein
Related Sequence




















Cthe0269
Cel8A
1st
DVNGDGNVNSTDLTMLKRYLLKSV
2nd
DVNRDGAINSSDMTILKRYLIKSI





(SEQ ID NO: 94)

(SEQ ID NO: 95)


 


Cthe0412
Ce19K
1st
DCNGDGKVNSTDAVALKRYILRSG
2nd
DVNADGRVNSTDLAILKRYILKEI





(SEQ ID NO: 102)

(SEQ ID NO: 103)


 


Cthe0413
Cbh9A
1st
DCNDDGKVNSTDVAVMKRYLKKEN
2nd
DVNADGKVNSTDFSILKRYVMNKNI





(SEQ ID NO: 104)

(SEQ ID NO: 105)


 


Cthe0578
Ce19R
1st
DINLDGKINSSDVTLLKRYIVKSI
2nd
DVNGDGRVNSTDYSYLKRYVLKII





(SEQ ID NO: 115)

(SEQ ID NO: 116)


 


Cthe0825
Cel9D
1st
DVNDDGKVNSTDLTLLKRYVLKAV
2nd
DVNRDGRVNSSDVTILSRYLIRVI





(SEQ ID NO: 137)

(SEQ ID NO: 138)


 


Cthe1838
Xyn10C
1st
DVNGDGRVNSSDLTLMKRYLLKSI
2nd
DLNEDGKVNSTDLLALKKLVLREL





(SEQ ID NO: 153)

(SEQ ID NO: 154)


 


Cthe2089
Cel48S
1st
DVNDDGKVNSTDAVALKRYVLRSG
2nd
DLNEDGRVNSTDLGILKRYILKEI





(SEQ ID NO: 161)

(SEQ ID NO: 162)


 


Cthe2147
Ce150
1st
DVNGDFAVNSNDLTLIKRYVLKNI
2nd
DVDGDEKITSSDAALVKRYVLRAI





(SEQ ID NO: 167)

(SEQ ID NO: 168)



















TABLE 8










Similarity of Amino Acid Sequence




















Cel8A
Ce19K
Cbh9A
Cel9R
Cel9D
Xyn10C
Cel48S
Ce150

























locus

Related Sequence
1st
2nd
1st
2nd
1st
2nd
1st
2nd
1st
2nd
1st
2nd
1st
2nd
1st
2nd





Cthe-
1st
DFNSDSSVNSTDLMILNRAVLGLG
85
85
84
85
93
 85
85
76
85
85
85
90
80
90
80
80


0109

(SEQ ID NO: 81)



















2nd
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-


 


Cthe-
1st
DLNGDNNNSSDYTLLKRYLLHTI
95
91
90
95
94
100
91
95
95
91
95
83
91
95
91
95


0438

(SEQ ID NO: 110)



















2nd
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-









The dockerins shown in Table 8 each have one relevant sequence on the N-terminal side, and this relevant sequence has 90% or greater amino acid sequence similarity to one of the relevant sequences in the 8 dockerins shown in Table 7, which have confirmed cohesin binding ability. A preferred dockerin can be obtained by making a dockerin-specific sequence in which aspartic acid has been substituted for asparagine at a predicted N-type sugar chain modification site in this relevant sequence.










TABLE 9










Similarity of Amino Acid Sequence




















Cel8a
Cel9K
Cbh9A
Cel9R
Cel9D
Xyn10C
Cel48S
Cel50

























locus

Related Sequence
1st
2nd
1st
2nd
1st
2nd
1st
2nd
1st
2nd
1st
2nd
1st
2nd
1st
2nd





Cthe0239
1st
DYNGDGAVNSTDLLACKRYLLYAL
80
76
85
76
85
76
71
71
76
71
76
79
80
76
76
71




(SEQ ID NO: 88)



















2nd
DLDGNGKINSTDYAYLKRVLLKQI
87
79
90
91
85
95
83
95
91
83
87
83
90
91
83
91




(SEQ ID NO: 89)


















 


Cthe0435
1st
DVNADGVVNISDYVLMKRYILRII
83
83
77
83
80
83
79
83
83
83
83
77
81
79
83
83




(SEQ ID NO: 108)



















2nd
DVNGDNVINDIDCNYLKRYLLHMI
83
79
77
83
80
83
75
95
83
79
79
77
81
83
79
79




(SEQ ID NO: 109)


















 


Cthe2038
1st
DIVLDGNINSLDMMKLKKYLIRET
81
77
83
78
78
78
81
72
77
77
77
78
77
78
73
72




(SEQ ID NO: 159)



















2nd
DVNSDGEVNSTDYAYLKRYILRII
87
87
86
85
85
91
83
95
87
87
83
81
90
83
83
87




(SEQ ID NO: 160)


















 


Cthe2549
1st
DVNKDGRINSTDIMYLKGYLLRNS
82
82
79
79
79
82
82
81
86
86
82
86
87
86
73
77




(SEQ ID NO: 185)



















2nd
DVDGNGSVSSLDLTYILKRYLRRI
91
87
81
85
85
91
87
87
87
83
91
79
86
87
87
83




(SEQ ID NO: 186)









The dockerins shown in Table 9 each have two relevant sequences, and the relevant sequence on the C-terminal side has 90% or greater amino acid similarity to one of the relevant sequences in the 8 dockerins shown in Table 7, which have confirmed cohesin binding ability. A preferred dockerin can be obtained by making a dockerin-specific sequence in which aspartic acid has been substituted for asparagine at a predicted N-type sugar chain modification site in this relevant sequence.










TABLE 10










Similarity of Amino Acid Sequence




















Cel8A
Cel9K
Cbh9A
Cel9R
Cel9D
Xyn10C
Cel48S
Cel50

























locus

Related Sequence
1st
2nd
1st
2nd
1st
2nd
1st
2nd
1st
2nd
1st
2nd
1st
2nd
1st
2nd




























Cthe0015
1st
DVNADGKIDSTDLTLLKRVLLRSA
100
91
87
100
95
100
95
90
100
95
100
86
91
100
91
95




(SEQ ID NO: 73)



















2nd
DTDGNGTVNSTDLNYLKKYILRVI
91
87
81
87
90
91
87
91
95
87
87
81
86
83
87
91




(SEQ ID NO: 74)


















 


Cthe0032
1st
DLNNDGNINSTDYMILKKYILKVL
87
87
90
90
85
95
83
91
91
91
87
83
90
90
86
91




(SEQ ID NO: 75)



















2nd
DLNGDGSINSTDLTILKRFIMKAI
100
95
90
100
100
100
95
87
100
95
100
91
91
100
95
96




(SEQ ID NO: 76)


















 


Cthe0043
1st
DLNGDGNINSTDFTMLKRAILGNP
95
90
84
95
93
95
85
90
95
85
95
85
85
95
90
90




(SEQ ID NO: 77)



















2nd
DLNRDGNTNSTDLMILRRYLLKLI
87
91
88
87
85
87
87
83
87
95
87
86
86
87
83
83




(SEQ ID NO: 78)


















 


Cthe0044
1st
DINLDGKINSTDLSALKRHILRIT
90
86
88
90
100
90
95
86
90
91
90
86
86
90
81
86




(SEQ ID NO: 79)



















2nd
DVNNDGSVNSTDASILKKYIAKAI
91
91
82
91
90
91
95
83
91
95
91
79
86
91
87
95




(SEQ ID NO: 80)


















 


Cthe0190
1st
ELNGDGKINSSDLNMMKRYLLRLI
95
87
85
95
100
95
91
91
95
95
95
86
86
95
87
91




(SEQ ID NO: 82)



















2nd
DLNGDGKINSSDYSILKRYLLRMI
91
83
85
91
94
95
87
95
91
91
91
81
86
91
83
91




(SEQ ID NO: 83)


















 


Cthe0191
1st
DLNGDAKINSTDLNMMKRYLLQMI
95
87
85
95
100
95
91
91
95
95
95
86
86
95
87
91




(SEQ ID NO: 84)



















2nd
DLNGDGKITSSDYNLLKRYILHLI
91
83
85
91
94
95
87
95
91
91
91
81
86
91
83
91




(SEQ ID NO: 85)


















 


Cthe0211
1st
DVNGDGHVNSSDYSLFKRYLLRVI
91
83
81
91
90
95
87
95
95
91
91
81
86
91
83
91




(SEQ ID NO: 86)



















2nd
DVNRDGRIDSTDLTMLKRYLIRAI
95
95
82
95
86
95
95
83
95
100
95
83
86
95
87
91




(SEQ ID NO: 87)


















 


Cthe0246
1st
DLNADGKINSTDYNLGKRLILRTI
87
79
76
87
83
91
83
87
87
83
87
83
78
87
79
87




(SEQ ID NO: 90)



















2nd
DLNGDSKVDSTDLTALKRYLLGVI
87
79
94
87
100
87
83
87
91
87
87
90
95
87
79
87




(SEQ ID NO: 91)


















 


Cthe0269
1st
DVNGDGNVNSTDLTMLKRYLLKSV
100
95
86
100
90
100
95
87
100
91
100
87
91
100
95
95




(SEQ ID NO: 94)



















2nd
DVNRDGAINDDSMTILKRYLIKSI
95
100
78
91
81
91
91
79
91
91
91
79
82
91
91
87




(SEQ ID NO: 95)


















 


Cthe0270
1st
DLNGDGKVNSSDLAILKRYMLRAI
100
91
90
100
100
100
95
87
100
95
100
87
91
100
91
95




(SEQ ID NO: 96)



















2nd
DLNRDGNVNSTDYSILKRYILKAI
91
95
84
91
85
95
91
87
91
95
91
79
82
91
87
91




(SEQ ID NO: 97)


















 


Cthe0274
1st
DLNVDGSINSVDITYMKRYLLRSI
91
87
85
95
93
95
91
87
91
83
87
83
86
91
83
83




(SEQ ID NO: 98)



















2nd
DTNGDGAINSSDMVLLKRYVLRSI
100
95
82
95
90
91
91
79
95
87
95
83
86
87
95
91




(SEQ ID NO: 99)


















 


Cthe0405
1st
DVNGDGNVNSTDVVWLRRFLLKLV
91
87
90
91
86
83
87
91
91
91
91
95
95
87
87
91




(SEQ ID NO: 100)



















2nd
DMNDDGNINSTDMIALKRKVLKIP
90
86
90
86
93
86
86
82
90
86
90
95
90
86
86
81




(SEQ ID NO: 101)


















 


Cthe0412
1st
DCNGDGKVNSTDAVALKRYILRSG
86
78
100
86
100
82
94
81
86
86
86
90
95
85
78
91




(SEQ ID NO: 102)



















2nd
DVNADGRVNSTDLAILKRYILKEI
100
91
86
100
91
100
95
87
100
91
100
87
90
100
91
95




(SEQ ID NO: 103)


















 


Cthe0413
1st
DCNDDGKVNSTDVAVMKRYLKKEN
90
81
100
91
100
91
94
81
90
86
90
85
95
95
82
95




(SEQ ID NO: 104)



















2nd
DVNADGKVNSTDFSILKRYVMKNI
100
91
82
100
91
100
91
91
100
87
100
87
86
100
91
95




(SEQ ID NO: 105)


















 


Cthe0433
1st
DLNGDGRVNSSDLALMKRYVVKQI
95
87
90
100
95
100
91
87
100
91
95
87
90
100
91
95




(SEQ ID NO: 106)



















2nd
DLNGDDKVNSTDYSVLKRYLLRSI
95
87
90
95
94
100
91
91
95
87
95
87
91
95
87
95




(SEQ ID NO: 107)


















 


Cthe0536
1st
DVNGDGRVNSSDVALLKRYLLGLV
100
90
90
100
95
95
95
87
100
91
100
85
95
100
90
100




(SEQ ID NO: 111)



















2nd
DVNVSGTVNSTDLAIMKRYVLRSI
95
95
82
95
90
100
100
79
95
87
91
83
86
91
91
91




(SEQ ID NO: 112)


















 


Cthe0543
1st
DVNFDGRINSTDYSRLKRYYIKSL
91
83
84
87
83
91
91
87
87
83
87
79
82
87
79
87




(SEQ ID NO: 113)



















2nd
DVDGNGRINSTDLYVLNRYILKLI
91
83
86
91
86
91
87
87
91
91
91
90
90
91
83
87




(SEQ ID NO: 114)


















 


Cthe0578
1st
DINLDGKINSSDVTLLKRYIVKSI
95
91
94
95
94
91
100
83
95
91
95
83
91
95
87
95




(SEQ ID NO: 115)



















2nd
DVNGDGRVNSTDYSYLKRYVLKII
87
79
81
87
81
91
83
100
87
87
87
81
86
87
79
87




(SEQ ID NO: 116)


















 


Cthe0640
1st
DLNGDNNVNSTDLTLLKRYLTRVI
91
91
85
91
100
91
91
87
95
95
91
81
86
91
87
91




(SEQ ID NO: 117)



















2nd
DVNGDGKINSTDYSAMIRYILRII
83
75
81
83
85
87
79
91
83
83
83
81
86
83
75
83




(SEQ ID NO: 118)


















 


Cthe0661
1st
DVNGDLKVNSTDFSMLRRYLLKTI
95
87
78
95
81
95
87
91
95
87
95
83
82
95
95
91




(SEQ ID NO: 119)



















2nd
DLNGDGRINSSDLTMLKRYLLMEV
100
91
89
100
95
100
95
87
100
91
100
87
90
100
91
95




(SEQ ID NO: 120)


















 


Cthe0624
1st
DLNNDSKVNAVDIMMLKRYILGII
87
79
89
87
94
87
79
83
87
87
83
90
90
87
75
79




(SEQ ID NO: 121)



















2nd
DIYFDGVVNSSDYNIMKRYLLKAI
83
87
78
83
83
87
87
79
90
83
83
75
78
90
94
90




(SEQ ID NO: 122)


















 


Cthe0625
1st
DLNGDGVVNSTDSVILKRHIIKFS
90
90
90
90
85
86
86
82
90
86
90
81
90
86
90
95




(SEQ ID NO: 123)



















2nd
DLNGDGNINSSDVSLMKRYLLRII
95
91
90
95
100
91
91
91
95
95
95
86
90
95
91
95




(SEQ ID NO: 124)


















 


Cthe0660
1st
DLNGDGKINSTDISLMKRYLLKQI
95
87
85
100
95
100
91
87
100
91
95
87
86
100
91
95




(SEQ ID NO: 125)



















2nd
DINKDGKVNSTDMSILKRVILRNY
91
91
73
91
86
91
91
81
95
95
91
91
82
95
82
86




(SEQ ID NO: 126)


















 


Cthe0729
1st
DSNSDCKVNSTDLTLMKRYLLQQS
90
86
86
91
91
91
86
81
90
90
90
78
81
91
86
86




(SEQ ID NO: 127)



















2nd
DLNGDGKINSSDYTLLKRYLLGYI
95
85
89
95
94
91
90
87
95
90
95
80
90
95
79
95




(SEQ ID NO: 128)


















 


Cthe0745
1st
DINNDKTVNSTDVTYLKRFLLKQI
87
87
86
87
82
87
83
83
91
83
83
87
90
87
91
95




(SEQ ID NO: 129)



















2nd
DVNLDGNINSTDLVILKRYVLRGI
95
95
89
95
100
91
100
79
95
91
95
87
86
91
91
91




(SEQ ID NO: 130)


















 


Cthe0797
1st
DVNGDGKINSTDCTMLKRYILRGI
95
87
86
95
90
95
91
91
95
87
95
83
91
95
87
96




(SEQ ID NO: 131)



















2nd
DVNADLKINSTDLVLMKKYLLRSI
95
87
82
95
90
91
91
79
95
87
95
87
86
91
95
91




(SEQ ID NO: 132)


















 


Cthe0798
1st
DVNLDGQVNSTDFSLLKRYILKVV
91
91
83
91
88
91
91
91
95
91
91
81
81
91
87
91




(SEQ ID NO: 133)



















2nd
DMNNDGNINSTDSILKRILLRN
95
95
80
95
90
95
91
86
95
95
95
91
82
96
91
90




(SEQ ID NO: 134)


















 


Cthe0821
1st
DINRDGKINSTDLGMLNRHILKLV
91
91
77
91
86
91
91
87
91
100
91
81
81
91
83
87




(SEQ ID NO: 135)



















2nd
DIDGNGNINSTDYSWLKKYILKVI
87
83
81
87
81
91
83
100
91
87
87
81
86
87
83
91




(SEQ ID NO: 136)


















 


Cthe0825
1st
DVNDDGKVNSTDLTLLKRYVLKAV
100
91
86
100
90
100
95
87
100
95
100
87
91
100
91
95




(SEQ ID NO: 137)



















2nd
DVNRDGRVNSSDVTILSRYLIRVI
91
91
86
91
86
87
91
87
95
100
91
81
90
91
83
95




(SEQ ID NO: 138)


















 


Cthe0912
1st
DVNGDGTINSTDLTMLKRSVLRAI
95
91
82
91
94
95
91
79
95
87
91
87
86
91
91
91




(SEQ ID NO: 139)



















2nd
DVDKNGSINSTDVLLLSRYLLRVI
87
91
86
87
90
83
87
83
95
95
87
90
95
91
83
91




(SEQ ID NO: 140)


















 


Cthe0918
1st
DLNRNGIVNDEDYILLKNYLLRGN
86
91
84
81
77
86
86
81
86
90
86
77
82
81
81
82




(SEQ ID NO: 141)



















2nd
DVNKDGKVNSTDCLFLKKYILGLI
85
85
80
85
80
85
85
87
90
87
85
85
90
90
76
85




(SEQ ID NO: 142)


















 


Cthe1271
1st
DTNSDGKINSTDVTALKRHLLRVT
95
90
95
95
95
90
90
91
95
95
95
86
100
90
86
95




(SEQ ID NO: 143)



















2nd
DVNGDGNVNSTDLLLLKRYILGEI
91
87
85
91
90
91
87
79
91
90
91
87
90
91
87
87




(SEQ ID NO: 144)


















 


Cthe1398
1st
DLNGDNRINSTDLTLMKRYILKSI
100
91
90
100
95
100
95
87
100
91
100
87
91
100
91
95




(SEQ ID NO: 145)



















2nd
DINGDGKINSTDYTYLKKYLLQAI
91
83
86
91
85
95
87
95
91
87
91
83
91
91
83
91




(SEQ ID NO: 146)


















 


Cthe1400
1st
DLNGDGRVNSTDYTLLKRVLLGAI
91
83
89
91
94
95
87
87
91
87
91
80
90
91
83
91




(SEQ ID NO: 147)



















2nd
DLNLDGRINSTDYTVLKRYLLNAI
91
87
94
91
93
95
95
87
91
91
91
83
91
91
83
91




(SEQ ID NO: 148)


















 


Cthe1472
1st
DLNFDNAVNSTDLLMLKRYILKSL
91
91
84
87
83
87
91
75
87
83
87
83
82
87
87
83




(SEQ ID NO: 149)



















2nd
DLNRDNKVDSTDLTILKRYLLKAI
95
95
89
95
90
95
95
83
95
100
95
83
86
95
87
91




(SEQ ID NO: 150)


















 


Cthe1806
1st
EVIDTKVIDSTDDIVKVEYQFDKK
75
-
75
84
84
76
85
70
75
100
-
75
68
84
-
100




(SEQ ID NO: 151)



















2nd
TLVLSVNNDSTDKTTVSGYISVDF
75
91
83
83
80
83
70
73
75
83
75
100
83
83
75
100




(SEQ ID NO: 152)


















 


Cthe1838
1st
DVNGDGRVNSSDLTLMKRYLLKSI
100
91
86
100
90
100
95
87
100
91
100
87
91
100
91
95




(SEQ ID NO: 153)



















2nd
DLNEDGKVNSTDLLALKKLVLREL
87
79
90
87
85
87
83
81
87
81
87
100
90
87
79
83




(SEQ ID NO: 154)


















 


Cthe1890
1st
DLNADGSINSTDLMIMKRVLLKQR
90
86
85
91
85
91
86
81
90
86
90
95
86
91
86
86




(SEQ ID NO: 155)



















2nd
DLNGDGKVTSTDYSLMKRYLLKEI
95
87
85
95
90
100
91
91
95
87
95
83
86
95
87
95




(SEQ ID NO: 156)


















 


Cthe1963
1st
DLNGDGNINSSDLQALKRHLLGIS
90
85
89
95
100
90
85
82
90
82
90
90
90
90
85
90




(SEQ ID NO: 157)



















2nd
DVNRSGKVDSTDYSVLKRYILRII
87
87
81
87
85
91
87
91
87
95
87
81
86
87
79
87




(SEQ ID NO: 158)


















 


Cthe2089
1st
DVNDDGKVNSTDAVALKRYVLRSG
91
82
95
90
95
86
91
86
91
90
91
90
100
86
82
95




(SEQ ID NO: 161)



















2nd
DLNEDGRVNSTDLGILKRYILKEI
100
91
85
100
95
100
95
87
100
91
100
87
86
100
91
95




(SEQ ID NO: 162)


















 


Cthe2137
1st
DVDGNGTVNSTDVNYMKRYLLRQI
91
87
86
91
86
91
87
87
95
83
87
87
90
91
91
95




(SEQ ID NO: 163)



















2nd
DVNGNGNINSTDLSYLKKYILKLI
91
87
81
91
86
91
87
95
91
91
91
86
86
91
87
87




(SEQ ID NO: 164)


















 


Cthe2139
1st
DVNADGVINSSDIMVLKRFLLRTI
91
91
86
91
85
91
87
83
91
87
91
95
91
91
91
87




(SEQ ID NO: 165)



















2nd
DTNGDGAVNSSDFTLLKRYILRSI
100
95
82
95
85
95
87
87
95
83
95
79
86
91
95
91




(SEQ ID NO: 166)


















 


Cthe2179
1st
DLNGDGNVNSTDSILMKRYLMKSV
95
91
95
91
90
91
91
83
95
87
95
87
95
91
91
95




(SEQ ID NO: 169)



















2nd
DVNLDGRVNSTDRSILNRYLLKII
87
87
83
87
88
87
91
87
87
91
87
77
81
87
79
87




(SEQ ID NO: 170)


















 


Cthe2193
1st
DINDDGNINSTDLQMLKRHLLRSI
95
91
82
100
95
95
91
83
95
87
95
87
86
95
91
95




(SEQ ID NO: 171)



















2nd
DTNRDGRVDSTDLALLKRYILRVI
91
91
81
91
90
91
91
87
95
100
91
77
86
87
94
91




(SEQ ID NO: 172)


















 


Cthe2194
1st
DLNGDGNINSTDLQILKKHLLRIT
95
90
85
100
100
95
90
86
96
91
95
86
86
95
90
95




(SEQ ID NO: 173)



















2nd
DVTKDGKVDSTDLTLLKRYILRFV
91
91
88
95
95
95
91
87
100
95
91
86
90
100
86
90




(SEQ ID NO: 174)


















 


Cthe2195
1st
DLNDDGKVNSTDFQILKKHLLRIT
95
86
85
100
94
95
86
91
95
86
95
86
86
95
86
95




(SEQ ID NO: 175)



















2nd
DLNKDGKVDSSDLSLMKRYLLQII
91
91
83
91
100
91
91
87
95
100
91
86
86
95
83
87




(SEQ ID NO: 176)


















 


Cthe2196
1st
DLNNDGKVNSTDFQLLKMHVLRQE
95
90
85
100
90
95
86
90
95
90
95
86
86
95
86
95




(SEQ ID NO: 177)



















2nd
DVNRDGKVDSSDCTLLKRYILRVI
87
87
81
87
85
87
87
91
91
95
87
77
86
87
79
91




(SEQ ID NO: 178)


















 


Cthe2197
1st
DLNGDGKVNSTDLQLMKMHVLRQR
95
86
85
100
95
95
90
86
95
90
95
86
86
95
86
95




(SEQ ID NO: 179)



















2nd
DVNRDGKVDSTDVALLKRYILRQI
91
91
86
95
86
91
91
83
95
95
91
83
90
95
87
95




(SEQ ID NO: 180)


















 


Cthe2360
1st
DLNGDGRVNSTDLLLMKKRIIREI
91
83
85
91
85
91
87
79
91
83
91
91
86
91
83
87




(SEQ ID NO: 183)



















2nd
DLNLDGKINSSDYTILKRYVLKSI
91
87
89
91
88
95
95
87
91
87
91
79
86
91
83
91




(SEQ ID NO: 184)


















 


Cthe2590
1st
DLNQDGQVSSTDLVAMKRYLLKNF
91
95
90
95
95
91
91
81
95
95
91
95
95
91
87
87




(SEQ ID NO: 187)



















2nd
DLNSDGKVNSTDLVALKRFLLKEI
95
91
95
95
95
91
91
83
95
91
95
100
95
91
87
91




(SEQ ID NO: 188)


















 


Cthe2760
1st
DLNVDGKVNSTDYLVLKRYLLGTI
83
79
94
83
87
87
83
83
83
83
83
85
90
83
75
83




(SEQ ID NO: 189)



















2nd
DLNRDGRVNSTDMSLMKRYLLGII
87
87
82
87
94
87
87
83
87
95
87
80
80
87
79
83




(SEQ ID NO: 190)


















 


Cthe2761
1st
DVNGDGKVNSTDCSIVKRYLLKNI
95
87
82
95
86
95
91
91
95
87
95
83
86
95
87
95




(SEQ ID NO: 191)



















2nd
DVNGDGKVNSTDYSLLKRFVLRNI
95
87
82
95
86
100
91
91
95
87
95
87
86
95
87
95




(SEQ ID NO: 192)


















 


Cthe2811
1st
DLNGDGKVNSTDLTIMKRYILKNF
100
91
90
100
95
100
95
90
100
95
100
87
91
100
91
95




(SEQ ID NO: 193)



















2nd
DLNGDGRINSTDLSILHRYLLRII
95
87
85
95
100
95
91
91
95
91
95
86
86
95
87
91




(SEQ ID NO: 194)


















 


Cthe2812
1st
DLNGDQKVTSTDYTMLKRYLMKSI
91
83
85
91
90
95
87
87
91
83
91
79
86
91
87
95




(SEQ ID NO: 195)



















2nd
DLNRDGKINSTDLTILKRYLLYSI
91
91
88
91
94
91
91
85
91
100
91
80
85
91
83
87




(SEQ ID NO: 196)


















 


Cthe2872
1st
DINSDGNVNSTDLGILKRIIVKNP
95
95
78
95
86
95
91
86
95
95
95
91
82
95
91
90




(SEQ ID NO: 197)



















2nd
DVNADGKVNSTDYTVLKRYLLRSI
95
87
91
95
90
100
91
91
95
87
95
87
95
95
87
95




(SEQ ID NO: 198)


















 


Cthe2879
1st
DINSDGSINSTDVTLLKRHLLREN
100
100
90
100
91
95
95
90
100
100
100
86
95
100
95
100




(SEQ ID NO: 199)



















2nd
DTDGDGKITSIDLSYLKRYVLRLI
91
79
81
91
90
91
83
95
91
87
87
81
86
87
79
83




(SEQ ID NO: 200)


















 


Cthe2949
1st
DLNGDLVNSSDYSLLKRYILKQI
87
83
80
91
85
95
83
87
91
83
87
79
81
91
87
91




(SEQ ID NO: 201)



















2nd
DLNRNGSVDSVDYSILKRFLLKTI
91
91
84
91
85
95
87
91
91
91
87
83
82
91
83
87




(SEQ ID NO: 202)


















 


Cthe2950
1st
DLNNDGRTNSTDYSLMKRYLLGSI
91
87
84
91
94
95
87
87
91
87
91
80
85
91
83
91




(SEQ ID NO: 203)



















2nd
DVNLDGKVNSSDYTVLRRFLLGSI
87
83
94
87
93
91
91
83
87
83
87
85
90
87
79
87




(SEQ ID NO: 204)


















 


Cthe2972
1st
DLNGDKQVNSTDYTALKRHLLNII
86
81
89
86
88
90
81
91
86
82
86
80
90
86
86
90




(SEQ ID NO: 205)



















2nd
DLNGDGKVDSTDLMILHRVLLGII
87
79
89
87
94
87
83
83
87
83
87
90
90
87
79
83




(SEQ ID NO: 206)


















 


Cthe3012
1st
DLNGDGNVNSTDSTLMSRYLLGII
87
83
94
87
94
87
83
87
87
87
87
80
95
87
83
91




(SEQ ID NO: 207)



















2nd
DLNGDGKVNSTDYNILKRYLLKYI
91
83
85
91
90
95
87
91
91
87
91
81
86
91
83
91




(SEQ ID NO: 208)


















 


Cthe3132
1st
DLNGDGRVNSTDLAVMKRYLLKQV
95
87
95
100
95
100
91
87
100
91
95
91
95
100
91
95




(SEQ ID NO: 209)



















2nd
DLNGDGKANSTDYQLLKRYILKTI
91
79
85
95
90
95
83
91
91
87
91
83
86
91
83
91




(SEQ ID NO: 210)









The dockerins shown in Table 10 each have two relevant sequences, and each of the relevant sequences has 90% or greater amino acid similarity to one of the relevant sequences in the 8 dockerins shown in Table 7, which have confirmed cohesin binding ability. A preferred dockerin can be obtained by making a dockerin-specific sequence in which aspartic acid has been substituted for asparagine at a predicted N-type sugar chain modification site in one or both of these relevant sequences.
When the amino acid sequence of a dockerin has 90% or greater similarity to the amino acid sequence of any of the known dockerins having cohesin binding ability shown in Table 7, moreover, a preferred dockerin can be obtained by making a dockerin-specific sequence in which aspartic acid has been substituted for asparagine at a predicted N-type sugar chain modification site in this dockerin.









TABLE 11









Similarity of




Amino Acid Sequence












locus
Amino Acid Sequence
Cel8A
Cel9K
Cbh9A
Cel9R





Cthe0043
DLNGDGNINSTDFTHLKRAILGNPAPGTNLAAGDLNRDGNTNSTDLMILRRYLLKLI
91
83
83
76



(SEQ ID NO: 3)






 


Cthe0044
DNLDGKINSTDLSALKRHILRITTLSGKQLENADVNNDGSVNSTDASILKKYIAKAI
86
81
88
77



(SEQ ID NO: 4)






 


Cthe0109
DFNSDSSVNSTDLMILNRAVLGLG
85
85
85
76



(SEQ ID NO: 5)






 


Cthe0211
DVNGDHVNSSDYSLFKRYLLRVIDRFPVGDQSVADVNRDGRIDSTDLTMLKRYLIRAI
86
83
88
91



(SEQ ID NO: 8)






 


Cthe0269
DVNGDGNVNSTDLTMLKRYLLKSVTNINREAADVNRDGAINSSDMTILKRYLIKSI
100
87
89
77



(SEQ ID NO: 12)






 


Cthe0270
DLNGDGKVNSSDLAILKRYMLRAISDFPIPEGRKLADLNRDGNVNSTDYSILKRYILKAI
88
82
82
87



(SEQ ID NO: 13)






 


Cthe0405
DVNGDGNVNSTDVVWLRRFLLKLVEDFPVPSGKQAADMNDDGNINSTDMILKRKVLKIP
82
79
77
75



(SEQ ID NO: 15)






 


Cthe0412
DCNGDGKVNSTDAVALKRYILRSGISINTDNADVNADGRVNSTDLAILKRYILKEI
87
100
96
74



(SEQ ID NO: 16)






 


Cthe0413
DCNDDGKVNSTDVAVMKRYLKKENVNINLDNADVNADGKVNSTDFSILKRYVMKNI
89
96
100
79



(SEQ ID NO: 17)






 


Cthe0433
DLNGDGRVNSSDLALMKRYVVKQIEKLNVPVKAADLNGDDKVNSTDYSVLKRYLLRSI
87
87
92
80



(SEQ ID NO: 18)






 


Cthe0438
DLNGDNNINSSDYTLLKRYLLHTI
95
95
100
95



(SEQ ID NO: 20)






 


Cthe0536
DVNGDGRVNSSDVALLKRYLLGLVENINKEAADVNVSGTVNSTDLAIMKRYVLRSI
94
87
87
74



(SEQ ID NO: 21)






 


Cthe0578
DINLDGKINSSDVTLLKRYIVKSIDVFPTADPERSLISDVNGDGRVNSTDVSYLKRYVLKII
77
74
79
100



(SEQ ID NO: 23)






 


Cthe0625
DLNGDGVVNSTDSVILKRHIIKFSEITDPVKLKAADLNGDGNINSSDVSLMKRYLLRII
86
82
84
80



(SEQ ID NO: 27)






 


Cthe0660
DLNGDGKINSTDISLMKRYLLKQIVDLPVEDDIKAADINKDGKVNSTDMSILKRVILRNY
88
85
80
82



(SEQ ID NO: 28)






 


Cthe0661
DVNGDLKVNSTDFSMLRRYLLKTIDNFPTENGKQAADLNGDGRINSSDLTMLKRYLLMEV
86
87
84
79



(SEQ ID NO: 25)






 


Cthe0745
DINNDKTVNSTDVTYLKRPLLKQINSLPNQKAADVNLDGNINSTDLVILKRYVLRGI
91
84
82
74



(SEQ ID NO: 30)






 


Cthe0797
DVNGDGKINSTDCTMLKRYILRGIEEFPSPSGIIAADVNADLKINSTDLVLMKKYLLRSI
83
80
76
80



(SEQ ID NO: 31)






 


Cthe0798
DVNLDGQVNSTDFSLLKRYILKVVDINSINVTNADMNNDGNINSTDISILKRLLRN
91
86
88
80



(SEQ ID NO: 32)






 


Cthe0825
DVNDDGKVNSTDLTLLKRYVLKAVSTLPSSKAEKNADVNRDGRVNSSDVTILSRYLRVI
88
80
78
88



(SEQ ID NO: 34)






 


Cthe0912
DVNGDGTINSTDLTMLKRSVLRAITLTDDAKARADVDKNGSINSTDVLLLSRYLLRVI
87
82
79
74



(SEQ ID NO: 35)






 


Cthe1398
DLNGDNRINSTDLTLMKRYLKSIEDLPVEDDLWAADINGDGKINSTDYTYLKKYLLQAI
85
86
84
88



(SEQ ID NO: 38)






 


Cthe1838
DVNGDGRVNSSDLTLMKRYLLKSISDFPTPEGKIADLNEDGKVNSTDLLALKKLVLREL
83
81
78
83



(SEQ ID NO: 42)






 


Cthe2089
DVNDDGKVNSTDAVALKRYVLRSGISINTDNADLNEDGRVNSTDLGILKRYILKEI
89
98
94
74



(SEQ ID NO: 46)






 


Cthe2137
DVDGNGTVNSTDVNYMKRYLLRQIEEFPYEKALMAGDVDGNGNINSTDLSYLKKYILKLI
81
83
80
85



(SEQ ID NO: 47)






 


Cthe2179
DLNGDGNVNSTDSILMKRYLMKSVDLNEEQLKAADVNLDGRVNSTDRISLNRYLLKII
86
85
85
76



(SEQ ID NO: 50)






 


Cthe2193
DINDDGNINSTDLQMLKRHLLRSILTEKQLLNADTNRDGRVDSTDLALLKRYILRVI
81
79
87
91



(SEQ ID NO: 51)






 


Cthe2195
DLNDDGKVNSTDFQILKKHLLRITLLTGKNLSNADLNKDGKVDSSDLSLMKRYLLQII
86
81
86
90



(SEQ ID NO: 53)






 


Cthe2196
DLNNDGKVNSTDFQLLKMHVLRQELPAGTDLSNADVNRDGKVDSSDCTLLKRYILRVI
82
79
84
91



(SEQ ID NO: 54)






 


Cthe2761
DVNGDGKVNSTDCSIVKRYLLKNEDFPYEYGKEAGDVNGDGKVNSTDYSLLKRFVLRNI
86
80
83
80



(SEQ ID NO: 61)






 


Cthe2811
DLNGDGKVNSTDLTIMKRYILKNFDKLAVPEEAADLNGDGRNSTDLSILHRYLLRII
86
87
91
82



(SEQ ID NO: 62)






 


Cthe2812
DLNGDQKVTSTDYTMLKRYLMKSIDRFNTSEQAADLNRDGKINSTDLTILKR
88
84
86
75



(SEQ ID NO: 63)














Similarity of




Amino Acid Sequence












locus
Amino Acid Sequence
Cel9D
Xyn10C
Cel48S
Cel50





Cthe0043
DLNGDGNINSTDFTHLKRAILGNPAPGTNLAAGDLNRDGNTNSTDLMILRRYLLKLI
86
83
84
80


Cthe0044
DNLDGKINSTDLSALKRHILRITTLSGKQLENADVNNDGSVNSTDASILKKYIAKAI
90
81
81
88


Cthe0109
DFNSDSSVNSTDLMILNRAVLGLG
85
90
90
80


Cthe0211
DVNGDHVNSSDYSLFKRYLLRVIDRFPVGDQSVADVNRDGRIDSTDLTMLKRYLIRAI
77
84
79
83


Cthe0269
DVNGDGNVNSTDLTMLKRYLLKSVTNINREAADVNRDGAINSSDMTILKRYLIKSI
88
83
89
85


Cthe0270
DLNGDGKVNSSDLAILKRYMLRAISDFPIPEGRKLADLNRDGNVNSTDYSILKRYILKAI
93
88
83
88


Cthe0405
DVNGDGNVNSTDVVWLRRFLLKLVEDFPVPSGKQAADMNDDGNINSTDMILKRKVLKIP
89
93
81
82


Cthe0412
DCNGDGKVNSTDAVALKRYILRSGISINTDNADVNADGRVNSTDLAILKRYILKEI
80
81
98
78


Cthe0413
DCNDDGKVNSTDVAVMKRYLKKENVNINLDNADVNADGKVNSTDFSILKRYVMKNI
78
78
94
81


Cthe0433
DLNGDGRVNSSDLALMKRYVVKQIEKLNVPVKAADLNGDDKVNSTDYSVLKRYLLRSI
88
85
87
88


Cthe0438
DLNGDNNINSSDYTLLKRYLLHTI
95
95
95
95


Cthe0536
DVNGDGRVNSSDVALLKRYLLGLVENINKEAADVNVSGTVNSTDLAIMKRYVLRSI
85
81
87
81


Cthe0578
DINLDGKINSSDVTLLKRYIVKSIDVFPTADPERSLISDVNGDGRVNSTDVSYLKRYVLKII
88
83
74
82


Cthe0625
DLNGDGVVNSTDSVILKRHIIKFSEITDPVKLKAADLNGDGNINSSDVSLMKRYLLRII
93
88
84
87


Cthe0660
DLNGDGKINSTDISLMKRYLLKQIVDLPVEDDIKAADINKDGKVNSTDMSILKRVILRNY
95
90
86
95


Cthe0661
DVNGDLKVNSTDFSMLRRYLLKTIDNFPTENGKQAADLNGDGRINSSDLTMLKRYLLMEV
86
82
88
90


Cthe0745
DINNDKTVNSTDVTYLKRPLLKQINSLPNQKAADVNLDGNINSTDLVILKRYVLRGI
88
81
84
86


Cthe0797
DVNGDGKINSTDCTMLKRYILRGIEEFPSPSGIIAADVNADLKINSTDLVLMKKYLLRSI
90
91
80
88


Cthe0798
DVNLDGQVNSTDFSLLKRYILKVVDINSINVTNADMNNDGNINSTDISILKRLLRN
89
84
85
84


Cthe0825
DVNDDGKVNSTDLTLLKRYVLKAVSTLPSSKAEKNADVNRDGRVNSSDVTILSRYLRVI
100
91
81
93


Cthe0912
DVNGDGTINSTDLTMLKRSVLRAITLTDDAKARADVDKNGSINSTDVLLLSRYLLRVI
91
82
86
85


Cthe1398
DLNGDNRINSTDLTLMKRYLKSIEDLPVEDDLWAADINGDGKINSTDYTYLKKYLLQAI
90
88
86
90


Cthe1838
DVNGDGRVNSSDLTLMKRYLLKSISDFPTPEGKIADLNEDGKVNSTDLLALKKLVLREL
91
100
83
86


Cthe2089
DVNDDGKVNSTDAVALKRYVLRSGISINTDNADLNEDGRVNSTDLGILKRYILKEI
81
83
100
80


Cthe2137
DVDGNGTVNSTDVNYMKRYLLRQIEEFPYEKALMAGDVDGNGNINSTDLSYLKKYILKLI
91
84
85
90


Cthe2179
DLNGDGNVNSTDSILMKRYLMKSVDLNEEQLKAADVNLDGRVNSTDRISLNRYLLKII
90
81
86
85


Cthe2193
DINDDGNINSTDLQMLKRHLLRSILTEKQLLNADTNRDGRVDSTDLALLKRYILRVI
79
81
86
86


Cthe2195
DLNDDGKVNSTDFQILKKHLLRITLLTGKNLSNADLNKDGKVDSSDLSLMKRYLLQII
80
83
85
83


Cthe2196
DLNNDGKVNSTDFQLLKMHVLRQELPAGTDLSNADVNRDGKVDSSDCTLLKRYILRVI
79
82
85
86


Cthe2761
DVNGDGKVNSTDCSIVKRYLLKNEDFPYEYGKEAGDVNGDGKVNSTDYSLLKRFVLRNI
90
86
81
90


Cthe2811
DLNGDGKVNSTDLTIMKRYILKNFDKLAVPEEAADLNGDGRNSTDLSILHRYLLRII
90
89
87
88


Cthe2812
DLNGDQKVTSTDYTMLKRYLMKSIDRFNTSEQAADLNRDGKINSTDLTILKR
90
81
84
83









The amino acid sequence of each of the dockerins shown in Table 11 has 90% or greater similarity to any amino acid sequence of the 8 dockerins shown in Table 7. A preferred dockerin can be obtained by substituting aspartic acid for asparagine in at least one predicted N-type sugar chain modification site of this amino acid sequence.
When a dockerin has a relevant sequence having 90% or greater homology with the amino acid sequence of a relevant sequence of any of the known dockerins with cohesin binding ability shown in Table 7, a preferred dockerin can be obtained by substituting aspartic acid for asparagine at a predicted N-type sugar chain modification site in that relevant sequence of the dockerin. A predicted N-type sugar chain modification site in a dockerin having 90% or greater homology with the amino acid sequence of such a known dockerin is also a preferred candidate for substitution. Only the dockerins shown in Table 7 are applicable to such dockerins. A preferred dockerin can be obtained by substituting aspartic acid for asparagine at a predicted N-type sugar modification site in a relevant sequence in one of these dockerins.
Another embodiment of a dockerin-specific sequence is a dockerin-specific sequence having no intrinsic predicted N-type sugar chain modification site in one of the dockerins disclosed in Table 1 or the relevant sequences of these dockerins disclosed in Table 2. It is sufficient for the protein of the invention to have a dockerin containing at least one such dockerin-specific sequence. The following 29 relevant sequences are examples of relevant sequences that are such dockerin-specific sequences.










TABLE 12





SEQ ID





NO.:
locus
-
Amino Acid Sequence







73
Cthe0015
1st
DVNADGKIDSTDLTLLKRYLLRSA


 


85
Cthe0191
2nd
DLNGDGKITSDYNLLKRYILHLI


 


87
Cthe0211
2nd
DVNRDGRIDSTDLTMLKRYLIRAI


 


91
Cthe0246
2nd
DLNGDSKVDSTDLTALKRYLLGVI


 


92
Cthe0258
1st
DVNGDSKINAIDVLLMKKYILKVI


 


93
Cthe0258
2nd
DVNADGQINSIDFTWLKKYMLKAV


 


98
Cthe0274
1st
DLNVDGSINSVDITYMKRYLLRSI


 


109
Cthe0435
2nd
DVNGDNVINDIDCNYLKRYLLHMI


 


141
Cthe0918
1st
DLNRNGIVNDEDYILLKNYLLRGN


 


150
Cthe1472
2nd
DLNRDNKVDSTDLTILKRYLLKAI


 


151
Cthe1806
1st
EVIDTKVIDSTDDIVKYEYQFDKK


 


156
Cthe1890
2nd
DLNGDGKVTSTDYSLMKRYLLKEI


 


159
Cthe2038
1st
DIVLDGNINSLDMMKLKKYLIRET


 


167
Cthe2147
1st
DVNGDFAVNSNDLTLIKRYVLKNI


 


168
Cthe2147
2nd
DVDGDEKITSSDAALVKRYVLRAI


 


171
Cthe2193
2nd
DTNRDGRVDSTDLALLKRYILRVI


 


174
Cthe2194
2nd
DVTKDGKVDSTDLTLLKRYILRFV


 


176
Cthe2195
2nd
DLNKDGKVDSSDLSLMKRYLLQII


 


178
Cthe2196
2nd
DVNRDGKVDSSDCTLLKRYILRVI


 


180
Cthe2197
2nd
DVNRDGKVDSTDVALLKRYILRQI


 


181
Cthe2271
1st
DVNLDGSVDSIDLALLYNTTYYAV


 


182
Cthe2271
2nd
DVNGDGTVDGIDLAIITAYINGQI


 


186
Cthe2549
2nd
DVDGNGSVSSLDLTYLKRYILRRI


 


187
Cthe2590
1st
DLNQDGQVSSTDLVAMKRYLLKNF


 


195
Cthe2812
1st
DLNGDQKVTSTDYTMLKRYLMKSI


 


200
Cthe2879
2nd
DTDGDGKITSIDLSYLKRYVLRLI


 


206
Cthe2972
2nd
DLNGDGKVDSTDLMILHRYLLGII


 


211
Cthe3136
1st
DIDGNGEISSIDYAILKSHLINSN


 











212
Cthe3136
2nd
DVDGNGYVNSIDLAILQMYLLGKG










The protein of the invention may be provided with a dockerin comprising one or two of the dockerin-specific sequences shown in Table 12, and typically a dockerin that inherently has such a dockerin-specific sequence is preferred. Examples of such dockerins are those shown in the following tables. In these tables, the dockerins are specified by their relevant sequences. In these dockerin-specific sequences, an amino acid at a site corresponding to a predicted N-type sugar chain modification site is preferably aspartic acid. A dockerin having one or two such dockerin-specific sequences is preferred.








TABLE 13





locus
Amino Acid Sequence



















Cthe0258
1st
DVNGDSKINAIDVLLMKKYILKVI
2nd
DVNADGQINSIDFTWLKKYMLKAV




(SEQ ID NO: 92)

(SEQ ID NO: 93)


 


Cthe2147
1st
DVNGDFAVNSNDLTLIKRYVLKNI
2nd
DVDGDEKITSSDAALVKRYVLRAI




(SEQ ID NO: 167)

(SEQ ID NO: 168)


 


Cthe2271
1st
DVNLDGSVDSIDLALLYNTTYYAV
2nd
DVNGDGTVDGIDLAIITAYINGQI




(SEQ ID NO: 181)

(SEQ ID NO: 182)


 


Cthe3136
1st
DIDGNGEISSIDYAILKSHLINSN
2nd
DVDGNGYVNSIDLAILQMYLLGKG




(SEQ ID NO: 211)

(SEQ ID NO: 212)


 


Cthe3141
1st
DVNGNGSIESTDCVWVKRYLLKQI
2nd
DVNGNGTIDSTDYQLLKRFILKVI




(SEQ ID NO: 213)

(SEQ ID NO: 214)









The dockerins shown in Table 13 have two relevant sequences in the dockerin, and no predicted N-type sugar chain modification site in either relevant sequence.
In the protein of the invention, the C. thermocellum type I dockerins shown in Table 7, the binding ability of which with cohesins has been confirmed from existing literature or the like, are considered when selecting a dockerin-specific sequence having no intrinsic predicted N-type sugar chain modification site in a preferred dockerin. When a dockerin has a relevant sequence with 90% or greater similarity to the amino acid sequence of any of the relevant sequences in these dockerins, it can be used as a preferred dockerin if the sequence is a natural dockerin-specific sequence with aspartic acid occupying a site corresponding to a predicted N-type sugar chain modification site in the relevant sequence.










TABLE 14










Similarity of Amino Acid Sequence




















Cel8A
Cel9K
Cbh9A
Cel9R
Cel9D
Xyn10C
Cel48S
Cel50

























locus

Amino Acid Sequence of repeated region
1st
2nd
1st
2nd
1st
2nd
1st
2nd
1st
2nd
1st
2nd
1st
2nd
1st
2nd




























Cthe0258
1st
DVNGDSKINAIDVLLMKKYILKVI
91
79
90
91
86
87
83
87
95
87
87
90
95
91
79
91




(SEQ ID NO: 92)



















2nd
DVNADGQINSIDFTWLKKYMLKAY
95
87
86
95
81
95
83
95
95
83
91
87
91
95
87
87




(SEQ ID NO: 93)


















 


Cthe2147
1st
DVNGDFAVNSNDLTLIKRYVLKNI
95
91
78
91
82
91
87
79
91
83
91
79
82
91
100
87




(SEQ ID NO: 167)



















2nd
DVDGDEKITSSDAALVKRYVLRAI
95
87
91
95
96
95
95
87
95
95
95
83
95
95
87
100




(SEQ ID NO: 168)


















 


Cthe2271
1st
DVNLDGSVDSIDLALLYNTTYYAV
93
87
83
93
100
93
93
81
93
87
87
81
81
93
81
81




(SEQ ID NO: 181)



















2nd
DVNGDGTVDGIDLAIITAYINGQI
95
85
80
83
90
87
85
80
95
80
85
80
85
83
79
85




(SEQ ID NO: 182)


















 


Cthe3136
1st
DIDGNGEISSIDYAILKSHLINSN
95
86
86
90
87
100
86
90
95
80
86
80
91
90
86
91




(SEQ ID NO: 211)



















2nd
DVDGNGYVNSIDLAILQMYLLGKG
95
85
79
95
90
95
85
85
95
80
90
80
83
95
85
85




(SEQ ID NO: 212)


















 


Cthe3141
1st
DVNGNGSIESTDCVWVKRYLLKQI
87
83
86
91
82
87
83
91
91
83
87
87
90
87
87
91




(SEQ ID NO: 213)



















2nd
DVNGNGTIDSTDYQLLKRFILKVI
87
83
81
87
86
91
83
87
91
83
83
86
86
83
83
95




(SEQ ID NO: 214)









The dockerins shown in Table 14 have two relevant sequences, and each of these relevant sequences has 90% or greater similarity to the amino acid sequence of one of the 8 dockerins shown in Table 7, the binding ability of which with cohesins has been confirmed from existing literature or the like. Moreover, one or both of these relevant sequences is a natural dockerin-specific sequence. Aspartic acid may also be substituted for asparagine at a predicted N-type sugar chain modification site in a relevant sequence that is not a natural dockerin-specific sequence.
When a dockerin has an amino acid sequence with 90% or greater similarity to the amino acid sequence of any of the aforementioned known dockerins, moreover, it can be used as a preferred dockerin if it has at least one natural dockerin-specific sequence in which a site corresponding to a predicted N-type sugar chain modification site is occupied by aspartic acid in an intrinsic relevant sequence of the dockerin.









TABLE 15









Similarity of




Amino Acid




Sequence










locus
Amino Acid Sequence
Cel8A
Cel9K





Cthe1806
EVITKVIDSTDDIVKYEYQFDKKILCADKETEILYFTVVADEEEIYTSDNTRTLVLSVNNDSTDKTTVSGY
67
75



(SEQ ID NO: 41)




 


Cthe2147
DVNGDFAVNSNDLTLIKRYVLKNIDEFPSSHGLKAADVDGDEKITSSDAALVKRYVLRAI
85
78



(SEQ ID NO: 49)




 


Cthe3136
DIDGNGEISSIDYAILKSHLINSNLTFKQLAAADVDGNGYVNSIDLAILQMYLLGKGGTSDI
87
88



(SEQ ID NO: 71)




 


Cthe3141
DVNGNGSIESTDCVWVKRYLLKQIDSFPNENGARAADVNGNGTIDSTDYQLLKRFILKVI
75
84



(SEQ ID NO: 72)














Similarity of




Amino Acid




Sequence










locus
Amino Acid Sequence
Cbh9A
Cel9R





Cthe1806
EVITKVIDSTDDIVKYEYQFDKKILCADKETEILYFTVVADEEEIYTSDNTRTLVLSVNNDSTDKTTVSGY
91
83


 


Cthe2147
DVNGDFAVNSNDLTLIKRYVLKNIDEFPSSHGLKAADVDGDEKITSSDAALVKRYVLRAI
81
82


 


Cthe3136
DIDGNGEISSIDYAILKSHLINSNLTFKQLAAADVDGNGYVNSIDLAILQMYLLGKGGTSDI
90
73


 


Cthe3141
DVNGNGSIESTDCVWVKRYLLKQIDSFPNENGARAADVNGNGTIDSTDYQLLKRFILKVI
80
90














Similarity of




Amino Acid




Sequence










locus
Amino Acid Sequence
Cel9D
Xyn10C





Cthe1806
EVITKVIDSTDDIVKYEYQFDKKILCADKETEILYFTVVADEEEIYTSDNTRTLVLSVNNDSTDKTTVSGY
75
70


 


Cthe2147
DVNGDFAVNSNDLTLIKRYVLKNIDEFPSSHGLKAADVDGDEKITSSDAALVKRYVLRAI
93
86


 


Cthe3136
DIDGNGEISSIDYAILKSHLINSNLTFKQLAAADVDGNGYVNSIDLAILQMYLLGKGGTSDI
80
73


 


Cthe3141
DVNGNGSIESTDCVWVKRYLLKQIDSFPNENGARAADVNGNGTIDSTDYQLLKRFILKVI
80
75














Similarity of




Amino Acid




Sequence










locus
Amino Acid Sequence
Cel48S
Cel50





Cthe1806
EVITKVIDSTDDIVKYEYQFDKKILCADKETEILYFTVVADEEEIYTSDNTRTLVLSVNNDSTDKTTVSGY
70
100


 


Cthe2147
DVNGDFAVNSNDLTLIKRYVLKNIDEFPSSHGLKAADVDGDEKITSSDAALVKRYVLRAI
80
100


 


Cthe3136
DIDGNGEISSIDYAILKSHLINSNLTFKQLAAADVDGNGYVNSIDLAILQMYLLGKGGTSDI
90
77


 


Cthe3141
DVNGNGSIESTDCVWVKRYLLKQIDSFPNENGARAADVNGNGTIDSTDYQLLKRFILKVI
76
91









The dockerins shown in Table 15 have amino acid sequences each having 90% or greater similarity to the amino acid sequence of any of the 8 dockerins shown in Table 7. They can be used as preferred dockerins because they have natural dockerin-specific sequences in which at least one site corresponding to a N-type sugar chain modification site in the amino acid sequence is occupied by aspartic acid. When there is another relevant sequence in which a predicted N-type sugar chain modification site is occupied by asparagine, aspartic acid can be substituted for that asparagine.
The protein of the invention can be provided with an active site in addition to the dockerin. The type of active site can be selected appropriately according to the use. The protein of the invention can also be an artificial protein in which a dockerin is suitably combined with an active site. A cellulase that is a constituent protein of a cellulosome and already has an intrinsic dockerin can also be used either as is or after modifications.
The protein of the invention can have cellulolysis promoting activity for example when it is used to saccharify a cellulose-containing material from biomass. That is, it can be provided with a cellulolysis-promoting active site. Examples of cellulolysis-promoting activity include cellulase activity, cellulose-binding activity, cellulose loosening activity and the like.
An active site in a known cellulase can be used appropriately as a cellulase active site. Examples of cellulases include endoglucanase (EC 3.2.1.74), cellobiohydrolase (EC 3.2.1.91) and β-glucosidase (EC 23.2.4.1, EC 3.2.1.21). Cellulases are classified into 13 families (5, 6, 7, 8, 9, 10, 12, 44, 45, 48, 51, 61, 74) of the GHF (glycoside hydrolase family) (www.cazy.org/fam/acc.gh.html) based on similarity of amino acid sequence. It is also possible to combine cellulases of the same or different kinds classified into different families.
A cellulase is not particularly limited but is preferably one that itself has strong cellulase activity. Examples of such cellulases include those derived from Phanerochaete, Trichoderma reesei and other Trichoderma, Fusarium, Tremetes, Penicillium, Humicola, Acremonium, Aspergillus and other filamentous bacteria as well as from Clostridium, Pseudomonas, Cellulomonas, Ruminococcus, Bacillus and other bacteria, Sulfolobus and other Archaea, and Streptomyces, Thermoactinomyces and other Actinomycetes. These cellulases or their active sites may also be artificially modified.
Because the protein of the invention is derived from C. thermocellum, its cellulolysis-promoting activity is preferably conferred by an amino acid sequence derived from Clostridium thermocellum. 
From the standpoint of effective use of biomass, the protein of the invention may be provided with a hemicellulase active site. A lignin decomposing enzyme such as lignin peroxidase, manganese peroxidase or laccase is also possible. Other examples include the cellulose loosening proteins expansin and swollenin, and cellulose-binding domains (proteins) that are constituents of cellulosomes and cellulases. Other examples include xylanase, hemicellulase and other biomass decomposing enzymes. All these proteins can improve the accessibility of the cellulase to cellulose.
This protein is preferably provided with the function of extracellular secretability in eukaryotic microorganisms. That is, it is preferably a protein that is produced as a secretory protein in eukaryotic microorganisms. Cellulase and other enzymes often have intrinsic signals for extracellular secretion. A known secretion signal can be used to confer extracellular secretability on a dockerin protein. The secretion signal is selected appropriately according to the type of eukaryotic microorganism. Secretion signals and the like will be explained below.
A person skilled in the art will be able to produce the protein of the invention by genetic recombination or the like in a suitable host microorganism, or obtain it by chemical synthesis.
As explained above, because the protein of the invention has a specific dockerin it has improved binding ability with type I cohesins from C. thermocellum, and may have improved accumulation and accumulated density on scaffolding proteins with such cohesins.
(Scaffolding Protein Having Type I Cohesin from C. thermocellum)
The protein of the present invention is suitable as a protein for constructing a complex with a scaffolding protein having a type I cohesin from C. thermocellum. A scaffolding protein having a type I cohesin from C. thermocellum can be provided with 1 or 2 or more type I cohesins from C. thermocellum. Cohesins are known as domains on type I and other scaffolding proteins that bind non-covalently to cellulases and the like with enzymatic activity in cellulosomes formed by cellulosome-producing microorganisms (Sakka et al., Protein, Nucleic Acid and Enzyme, Vol. 44, No. 10 (1999), pp. 41-50; Demain, A. L. et al., Microbiol. Mol. Biol. Rev., 69(1), 124-54 (2005); Doi, R. H. et al., J. Bacterol., 185(20), 5907-5914 (2003), etc.). A scaffolding protein from C. thermocellum for binding with the protein of the invention has at least a type I cohesin domain on a type I scaffolding protein. It may also be provided with a type II cohesin domain on a type II scaffolding protein and a type III cohesin domain on a type III scaffolding protein. A number of sequences of such different types of cohesin domains have been determined in various cellulosome-producing microorganisms. The amino acid sequences and DNA sequences of these various types of cohesins can be easily obtained from various protein databases and DNA sequence databases accessible via the NCBI HP (www.ncbi.nlm.nih.gov).
A scaffolding protein having a cohesin from C. thermocellum need not itself be a scaffolding protein from C. thermocellum as long as it has a type I cohesin from C. thermocellum, and may be an artificial protein. The scaffolding protein may have a natural type I cohesin from C. thermocellum, or may have a modified cohesin with one or two or more mutations (additions, insertions, deletions or substitutions) introduced in the amino acid sequence of such a cohesin as long as binding ability is retained. Multiple such cohesins or the like may also be provided at suitable intervals in the cohesin protein. The amino acid sequence of a type I scaffolding protein and such a sequence with suitable mutations introduced therein can be used for the total amino acid sequence of the cohesin protein, and for the amino acid sequences between cohesins if such are present.
The scaffolding protein may also have a cellulose binding domain (CBD) of a scaffolding protein selected from types I to III. CBDs are known as domains in scaffolding proteins that bind to cellulose substrates (see Sakka et al above). There may be one or two or more cellulose binding domains. Many amino acid sequences and DNA sequences of CBDs in the cellulosomes of various cellulosome-producing microorganisms have already been determined. These various CBD amino acid sequences and DNA sequences can be easily obtained from various protein databases and DNA sequences databases accessible through the NCBI HP (www.ncbi.nlm.nih.gov) and the like.
The scaffolding protein preferably has extracellular secretability or cell surface display properties in eukaryotic microorganisms. That is, it is preferably a protein that is produced as a secretory protein in eukaryotic microorganisms, or a protein that is displayed on the cell surfaces of eukaryotic microorganisms. A known secretory signal or surface display system can be used to give a cohesin protein extracellular secretability or cell surface display properties.
A person skilled in the art can produce a scaffolding protein having these various domains as necessary by genetic recombination or the like in a suitable host microorganism. Cohesin proteins having cohesin domains of these various scaffolding proteins can also be obtained by chemical synthesis.
As explained above, because the protein of the invention has a specific dockerin, it has improved binding with type I cohesins from C. thermocellum, and may have enhanced accumulation and accumulated density on scaffolding proteins with such cohesins.
(Protein Complex)
The disclosures of this Description also provide a protein complex comprising a scaffolding protein having a type I cohesin from C. thermocellum and the protein of the invention bound to this scaffolding protein. This protein complex has enhanced activity of the protein of the invention because the accumulated amount and/or accumulated density of the protein of the invention is greater.
(Eukaryotic Microorganism Provided with Protein Complex on Cell Surface)
The eukaryotic microorganism disclosed in this Description is provided on the cell surface with the protein complex disclosed in this Description. In this eukaryotic microorganism, the scaffolding protein and protein of the invention making up the protein complex may be supplied from outside the cell and self-assembled on the cell surface to construct the protein complex, but preferably the microorganism produces these proteins itself. This is because sugar chain modification by the sugar chain modification system is eliminated or controlled even when the protein of the invention is produced within an eukaryotic microorganism, resulting in improved cohesin binding.
When the protein of the invention has cellulase activity or other cellulolysis promotion activity, a protein complex comprising accumulated proteins having cellulase or other cellulolysis promotion activity can be constructed on the cell surface of the eukaryotic microorganism. Such a eukaryotic microorganism can use glucose obtained by decomposition and saccharification of a cellulose-containing material on its cell surface as a carbon source.
There are no particular limits on how the DNA coding for such a protein is retained within the host microorganism as long as it is able to express the protein. For example, it can be linked under the control of a promoter capable of operating in the eukaryotic microorganism, and with a suitable terminator located downstream therefrom. The promoter may be a constitutive promoter or an inducible promoter. In this state, the DNA may be incorporated into a host chromosome, or may be in the form of a 2μ plasmid held within the host nucleus or a plasmid held outside the nucleus. In general, a selection marker gene that is usable in the host is retained at the same time when introducing such exogenous DNA.
The dockerin proteins and cohesin proteins produced in the eukaryotic microorganism are preferably given extracellular secretability or cell surface display properties. The protein of the invention is preferably given extracellular secretability, while the scaffolding protein is preferably given cell surface display properties, by which it is excreted outside the cell and displayed on the cell surface. To give it extracellular secretability, a protein is assigned a secretory signal. Examples of excretory signals include secretory signals of the Rhizopus oryzae and C. albicans glucoamylase genes, yeast invertase leaders, α-factor leaders and the like. Using an agglutinating protein or a part thereof, the protein can be secreted in such a way that it is displayed on the surface of the eukaryotic microorganism. One example is a peptide consisting of 320 amino acid residues of the 5′ region of the SAG1 gene, which codes for the agglutinating protein α-agglutinin. Polypeptides and methods for displaying desired proteins on cell surfaces are disclosed in WO 01/79483, Japanese Patent Application Laid-open No. 2003-235579, WO 2002/042483 pamphlet, WO 2003/016525 pamphlet, Japanese Patent Application Laid-open No. 2006-136223, and the publications of Fujita et al (Fujita et al., 2004, Appl. Environ. Microbiol. 70:1207-1212 and Fujita et al., 2002, Appl. Environ. Microbiol. 68:5136-5141), and Murai et al., 1998, Appl. Environ. Microbiol. 64:4857-4861.
The eukaryotic microorganism is not particularly limited, and for example various known yeasts can be used. For purposes of ethanol fermentation and the like as discussed below, examples include Saccharomyces cerevisiae and other Saccharomyces yeasts, Schizosaccharomyces pombe and other Schizosaccharomyces yeasts, Candida shehatae and other Candida yeasts, Pichia stipitis and other Pichia yeasts, Hansenula yeasts, Trichosporon yeasts, Brettanomyces yeasts, Pachysolen yeasts, Yamadazyma yeasts, and Kluyveromyces marxianus, Kluyveromyces lactis and other Kluyveromyces yeasts. Of these, a Saccharomyces yeast is desirable from the standpoint of industrial utility and the like, and Saccharomyces cerevisiae is especially desirable.
A eukaryotic microorganism expressing an exogenous protein can be prepared according to the methods described in Molecular Cloning, 3rd Ed., Current Protocols in Molecular Biology and the like. Vectors and methods for constructing vectors for expressing the protein of the invention and scaffolding protein in a eukaryotic microorganism are similarly well-known to those skilled in the art. The vector can be in various forms according to the mode of use. For example, it can assume the form of a DNA fragment, or of a 2 micron plasmid or other suitable yeast vector. The eukaryotic microorganism disclosed in this description can be obtained by transforming a eukaryotic microorganism with such a vector. Various conventional known methods can be adopted for transformation, such as transformation methods, transfection methods, conjugation methods, protoplast methods, electroporation, lipofection, lithium acetate methods and the like.
(Method for Producing Useful Substance)
The method for producing a useful substance disclosed in this Description may comprise a step of saccharifying and fermenting a cellulose-containing material by means of a process whereby a cellulose-containing material is fermented as a carbon source using the eukaryotic microorganism disclosed in this Description, in which the protein of the invention has cellulolysis promotion activity. With this method, a cellulose-containing material can be directly decomposed and saccharified with a eukaryotic microorganism, and used as glucose or the like by the eukaryotic microorganism. A useful substance is produced by this fermentation step according to the useful substance production ability of the eukaryotic microorganism used.
The useful substance is a product obtained by fermentation of glucose or the like by the eukaryotic microorganism, and differs both according to the type of eukaryotic microorganism and the fermentation conditions. The useful substance is not particularly limited, and can be any produced by yeasts and other eukaryotic microorganisms using glucose. The useful substance may also be a compound that is not an intrinsic metabolite, but one that the yeast or other eukaryotic microorganism has been made capable of synthesizing by a genetically engineered substitution, addition or the like in one or two or more enzymes in the glucose metabolism system. Examples of useful substances include ethanol as well as C3-5 lower alcohols, lactic acid and other organic acids, fine chemicals obtained by addition of isoprenoid synthesis pathways (coenzyme Q10, vitamins and other raw materials and the like), glycerin, plastics, synthetic raw materials and the like obtained by modifications in the glycolytic system, and other materials used in biorefinery technology. The useful substance production step may be followed by a step of collecting a useful substance-containing fraction from the culture liquid, and a further step of refining or concentrating this fraction. The collection step and refining or other step can be selected appropriately according to the type of useful substance and the like.
Proteins of the invention retained as protein complexes on the surface of the eukaryotic microorganism preferably have two or more cellulolysis-promoting activities. For example, it is desirable to use two or more cellulases having endoglucanase and cellobiohydrolase or other activity, respectively.
A cellulose-containing material is a material containing cellulose, a β-glucan consisting of D-glucose units condensed through β-1,4 glycosidic bonds. The cellulose-containing material may be any containing cellulose, regardless of derivation or form. Consequently, the cellulose-containing material may include lignocellulose material, crystalline cellulose material, soluble cellulose material (amorphous cellulose material), insoluble cellulose material and various other cellulose materials and the like for example. Examples of lignocellulose materials include lignocellulose materials comprising complexes of lignin and the like in the wood and leaves of woody plants and the leaves, stalks, roots and the like of herbaceous plants. These lignocellulose materials may be rice straw, wheat straw, corn stalks, bagasse and other agricultural waste, collected wood, brush, dried leaves and the like and chips obtained by grinding these, sawdust, chips and other sawmill waste, forest thinnings, damaged wood and other forest waste, and construction waste and other waste products. Examples of crystalline cellulose materials and insoluble cellulose materials include crystalline or insoluble cellulose materials containing crystalline cellulose and insoluble cellulose after separation of lignin and the like from lignocellulose materials. Cellulose materials may also be derived from used paper containers, used paper, used clothes and other used fiber materials and pulp wastewater.
Prior to being brought into contact with a cellulase, cellulose-containing material may also be subjected to suitable pretreatment or the like in order to facilitate decomposition by the cellulase. For example, the cellulose can be partially hydrolyzed to render it amorphous or reduce its molecular weight under acidic conditions using an inorganic acid such as sulfuric acid, hydrochloric acid, phosphoric acid, nitric acid or the like. It can also be treated with supercritical water, alkali, pressurized hot water or the like to render it amorphous or reduce its molecular weight.
Cellulose-containing materials include polymers and derivatives of polymers of glucose units condensed through β-1,4-glycosidic bonds. The degree of glucose polymerization is not particularly limited. Derivatives include carboxymethylated, aldehyded, esterified and other derivatives. The cellulose may be either crystalline cellulose or amorphous cellulose.
As understood in the technical field, identity or similarity in this Description signifies a relationship between two or more proteins or polynucleotides, determined by comparing their sequences. In this field, “identity” signifies the degree of sequence invariance between proteins or polynucleotides as determined by alignment between proteins or polynucleotides or in some cases alignment between a series of such sequences. Similarity signifies the degree of correlation between protein or polynucleotide sequences as determined by alignment of protein or polynucleotide sequences, or in some cases alignment between a series of such sequences. More specifically, it is determined by the identity or conservation (substitution that maintains the physical characteristics of a sequence or specific amino acid in a sequence) of the sequence. In the BLAST sequence homology test results below, similarity is called similarity. The method of determining identity or similarity is preferably designed so as to show the longest possible alignment between sequences. Methods for determining identity or similarity are provided by available public programs. For example, they can be determined using the BLAST (Basic Local Alignment Search Tool) program of Altschul et al (for example, Altschul, S F, Gish W, Miller W, Myers E W, Lipman D J, J. Mol. Biol. 215:403-410 (1990); Altschul S F, Madden T L, Schaffer A A, Zhang J, Miller W, Lipman D J, Nucleic Acids Res. 25:3389-3402 (1997)). The conditions when using software such as BLAST are not particularly limited, but the default values are used by preference.
EXAMPLES
The present invention is explained in detail below using examples, but the present invention is not limited by these examples. The gene recombination operations below were performed in accordance with Molecular Cloning: A Laboratory Manual (T. Maniatis et al., Cold Spring Harbor Laboratory).
Example 1
A pAI-AGA1 vector (FIG. 1) was prepared having an AAP1 homologous region and a HOR7 promoter upstream and a Tdh3 terminator, His3 marker and AAP1 homologous region downstream from an aga1 gene, which was amplified and cloned by ordinary PCR methods. The yeast S. cerevisiae BY 4741 was transformed using this vector to obtain a BY-AGA1 yeast displaying large quantities of aga1 on the cell surface.
Example 2
CBD-cohesin was amplified and cloned by ordinary PCR methods from the C. thermocellum genome (SEQ ID NO. 215). A pDL-CtCBDCohAGA2 vector was then prepared having an ADH3 homologous region and HOR7 promoter upstream and a V5-tag, aga2, Tdh3 terminator, Leu2 marker and ADH3 homologous region downstream from the resulting gene (FIG. 2). The resulting vector was introduced into the BY-AGA1 yeast prepared in Example 1, to obtain a CtCBDcoh yeast displaying cohesin from C. thermocellum on the cell surface.
Example 3
The Cel48S dockerin gene was amplified and cloned by ordinary PCR methods from the C. thermocellum genome (SEQ ID NO. 216). Using the resulting Cel48S dockerin gene as a template, two primers, 48Sdock-N18A-Fw and 48Sdock-N50A-Rv (SEQ ID NOS. 217, 218) were used to obtain a gene having alanine substituted for the No. 18 and No. 50 asparagines. A gene having aspartic acid substituted for the No. 18 and No. 50 asparagines was obtained in the same way using the two primers 48Sdock-N18D-Fw and 48Sdock-N50D-Rv (SEQ ID NOS. 219, 220) with the Cel48S dockerin gene as the template (FIG. 3). A pXU-Cel48Sdoc vector, pXU-Cel48S-N-A-doc vector and pXU-Cel48S-N-Doc vector were prepared each having a HXT3 homologous region, HOR7 promoter and His-tag upstream and a Tdh3 terminator, Ura3 marker and HXT3 homologous region downstream from the respective gene (FIG. 4). The resulting vectors were introduced into the CtCBDcoh yeast obtained in Example 2 to obtain CtCBDcoh48Sdoc, CtCBDcoh48SdocN-A and CtCBDcoh48SdocN-D displaying cohesin from C. thermocellum on the cell surface and simultaneously producing a dockerin or amino acid-substituted dockerin.
Example 4
The three yeasts CtCBDcoh48Sdoc, CtCBDcoh48SdocN-A and CtCBDcoh48SdocN-D obtained in Example 3 were each cultured for 24 hours at 30° C. in YP+2% glucose medium, and the equivalent of OD 600=0.5, 62.5 μl was collected, washed with PBS solution, mixed with PBS+1 mg/ml BSA+anti-His-FITC solution, reacted for 30 minutes at 4° C., and washed twice with PBS solution, and the amount of dockerin displayed on the yeast cell surface was then evaluated by flow cytometry. The amount of Cel48S dockerin displayed was reduced by about half by substitution of alanine for asparagine. On the other hand, the amount of Cel48S dockerin displayed was increased by 3.3 times by substitution of aspartic acid for asparagine (FIG. 5).
Example 5
The Xyn10C dockerin gene was amplified and cloned by ordinary PCR methods from the C. thermocellum genome (SEQ ID NO. 221). Genes having aspartic acid substituted for the No. 18 and No. 54 asparagine were obtained using the two primers 10Cdock-N18D-Fw and 10Cdock-N50D-Rv (SEQ ID NOS. 222, 223) with this Xyn10C dockerin gene as the template (FIG. 6). A pXU-Xyn10Cdoc vector and pXU-Xyn10C-N-D-doc vector were prepared each having an HXT3 homologous region, HOR7 promoter and His-tag upstream and a Tdh3 terminator, Ura3 marker and HXT3 homologous region downstream from the respective genes (FIG. 7). The resulting vectors were each introduced into the CtCBDcoh yeast obtained in Example 2 to obtain CtCBDcoh10Cdoc and CtCBDcoh10CdocN-D displaying cohesin from C. thermocellum on the cell surface and simultaneously producing a dockerin or amino acid-substituted dockerin.
Example 6
The two yeasts CtCBDcoh10Cdoc and CtCBDcoh10CdocN-D obtained in Example 5 were each cultured for 24 hours at 30° C. in YP+2% glucose medium, and the equivalent of OD 600=0.5, 62.5 μl was collected, washed with PBS solution, mixed with PBS+1 mg/ml BSA+anti-His-FITC solution, reacted for 30 minutes at 4° C., and washed twice with PBS solution, and the amount of dockerin displayed on the yeast cell surface was then evaluated by flow cytometry. The amount of Xyn10C dockerin displayed was increased by 1.8 times by substitution of aspartic acid for asparagine (FIG. 8). The two asparagines targeted in the dockerin in this case are conserved in about 82% of the 142 dockerins attributed to the C. thermocellum genome. Since amino acid substitution had a similar effect in two different dockerins, Cel48S and Xyn10C, it appears that this is applicable to most enzyme groups of C. thermocellum. 
Example 7
The Cel8A cellulase gene was amplified and cloned by ordinary PCR methods from the C. thermocellum genome (SEQ ID NO. 224). The resulting gene was spliced to the Cel48S dockerin gene obtained in Example 3 and to a gene having aspartic acid substituted for the No. 18 and No. 50 asparagines of the Cel48S dockerin, and pXU-Cel8A-Cel48Sdoc and pXU-Cel8A-Cel48S-N-D-doc vectors were prepared each having a HXT3 homologous region, HOR7 promoter and His-tag upstream and a Tdh3 terminator, Ura3 marker and HXT3 homologous region downstream from the respective gene (FIG. 9). The resulting vectors were each introduced into the CtCBDcoh yeast obtained in Example 2 to obtain CtCBDcohCel8A48Sdoc and CtCBDcohCel8A48SdocN-D, each displaying a cohesin from C. thermocellum on the cell surface and simultaneously producing a dockerin-type cellulase or amino acid-substituted dockerin-type cellulase.
Example 8
The two yeasts CtCBDcohCel8A48Sdoc and CtCBDcohCel8A48SdocN-D obtained in Example 7 were cultured for 24 hours at 30° C. in YP+2% glucose medium, and the equivalent of OD 600=0.5, 62.5 μl was collected, washed once with PBS solution, mixed with PBS+1 mg/ml BSA+anti-His-FITC solution, reacted for 30 minutes at 4° C., and washed twice with PBS solution, and the displayed amount of CelA on the yeast cell surface was evaluated by flow cytometry. An increase in the displayed amount of CelA was confirmed due to amino acid substitution (FIG. 10).
Example 9
The two yeasts CtCBDcohCel8A48Sdoc and CtCBDcohCel8A48SdocN-D obtained in Example 7 were cultured for 24 hours at 30° C. in YP+2% glucose medium, and the equivalent of OD 600=1, 1 ml was collected, washed with 50 mM acetic acid buffer pH 6.0 solution, mixed with 1% CMC, 20 mM acetic acid buffer pH 6.0 solution, and reacted for 2 hours at 40° C. to decompose the CMC. CMC decomposition activity was increased by amino acid substitution (FIG. 11), indicating improved saccharification ability of the yeast.
[Sequence Table Free Text]
SEQ ID NOS. 217, 218, 219, 220, 222, 223: Primers
[Sequence Tables]

## Claims

1. An isolated protein comprising a dockerin domain that includes an amino acid sequence associated with cohesin-dockerin binding wherein the amino acid sequence comprises at least one sequence selected from the group consisting of SEQ ID NOS: 3-5, 8, 12, 13, 15-18, 20, 21, 23, 25, 27, 28, 30-32, 35, 38, 42, 46, 47, 50, 51, 53, 54, 61-63, 74-84, 86, 88-90, 94-97, 99-108, 110-137, 139, 140, 142-149, 152-155, 157, 158, 160-166, 169-171, 173, 175, 177, 179, 183-186, 188-194, 196-199, 201-205, 207-210, 213, and 214 where said sequence is modified by substitution of an aspartic acid for an asparagine in an intrinsic predicted N-type sugar chain modification site, thereby eliminating in vivo sugar chain modification of said sequence when produced in an eukaryotic microorganism.

2. The isolated protein according to claim 1, wherein the amino acid sequence comprises at least one sequence selected from the group consisting of SEQ ID NOS: 74-84, 86, 88-90, 94-97, 99-108, 110-137, 139, 140, 142-149, 152-155, 157, 158, 160-166, 169-171, 173, 175, 177, 179, 183-185, 188-194, 196-199, 201-205, 207-210, 213, and 214 where said sequence is modified by substitution of an aspartic acid for an asparagine in an intrinsic predicted N-type sugar chain modification site, thereby eliminating in vivo sugar chain modification of said sequence when produced in an eukaryotic microorganism.

3. The isolated protein according to claim 1, wherein the amino acid sequence comprises at least one sequence selected from the group consisting of SEQ ID NOS: 75-80, 82, 83, 88, 89, 94-97, 100-107, 111-137, 139, 140, 143-148, 153, 154, 161-166, 169, 170, 183-186, 189-194, 197, 198, 201-204, and 207-210 where said sequence is modified by substitution of an aspartic acid for an asparagine in an intrinsic predicted N-type sugar chain modification site, thereby eliminating in vivo sugar chain modification of said sequence when produced in an eukaryotic microorganism.

4. The isolated protein according to claim 1, wherein the amino acid sequence comprises at least one sequence selected from the group consisting of SEQ ID NOS: 3-5, 8, 12, 13, 15-18, 20, 21, 23, 25, 27, 28, 30-32, 35, 38, 42, 46, 47, 50, 51, 53, 54, and 61-63 where said sequence is modified by substitution of an aspartic acid for an asparagine in an intrinsic predicted N-type sugar chain modification site, thereby eliminating in vivo sugar chain modification of said sequence when produced in an eukaryotic microorganism.

5. The isolated protein according to claim 1, which has cellulolysis-promoting activity.

6. The isolated protein according to claim 5, wherein the cellulolysis-promoting activity is cellulase activity.

7. The isolated protein according to claim 5, wherein the protein further comprises an amino acid sequence from Clostridium thermocellum, which confers the cellulolysis-promoting activity.