Method for producing concentrated adhesive phase of silk fusion protein

Abstract

The present invention relates to a method for producing a concentrated phase of a silk fusion protein, the method comprising the following steps: preparing a solution of a silk fusion protein in an aqueous medium, and concentrating the silk fusion protein in the aqueous medium, wherein the fusion protein is isolated from a recombinant production host and comprises a filamentous protein sequence and two separate non-silk terminal module sequences flanking the filamentous protein sequence, such as a cellulose binding module, a SpyCatcher domain, the tenth type III module of fibronectin, and γ-crystallin D; wherein the method is performed so that the silk fusion protein is not precipitated and subsequently dissolved in the aqueous medium. The present invention also relates to the use of such a fusion protein as an adhesive.

Description

Field of the Invention

[0001] The present invention relates to the production of recombinant spider silk proteins and the use of the proteins in the fields of materials, biotechnology and medicine. The present invention particularly relates to such applications, which involve the preparation of composite materials, textiles, filters, biomedical coatings, wound dressings, drug release materials, cell scaffolds, adhesives, etc. Background of the Invention

[0003] Silk and silk-like proteins are a large class of proteins found in many different organisms, where they play a variety of different roles. A very famous example is the domesticated silk moth, Bombyx mori, which is used on an industrial scale to produce silk fibers. When making webs 1 Related silk proteins are produced by, for example, spiders. In addition, other organisms, such as mussels, produce silk proteins in their shells as part of pearl structures. 2 . Because the structural properties of silk proteins in natural materials are highly desirable, there is great industrial interest in the preparation of silk proteins. One envisions that industrially produced silk proteins could replace current synthetic or other natural polymers in specific applications or on a large scale. Although silk from the Bombyx mori family is widely used, there are limitations to its use and industrial scale-up. For scalability and process considerations, it is desirable to use recombinant organisms or cells that can be grown in bioreactors to produce silk proteins. It is also desirable to produce silk proteins in forms not found in nature to prepare new types of silk polymer materials. 3 .

[0004] The problem of forming fibers from recombinantly produced silk proteins is well documented in the literature. The present invention relates to the general problem of processing recombinantly produced silk proteins into a state from which fibers can be made, for example, and which is in the state found in naturally occurring silk proteins, such as spider webs. In a first step, bacteria or eukaryotic cells are used to produce the silk proteins. To this end, standard methods of recombinant DNA technology are used. 4 A typical method of proceeding is to collect the recombinant protein produced by the organism in an aqueous solution, which may be a medium in which the organism has been grown. The silk protein is then typically precipitated from the solution. After precipitation, the silk protein is collected and often freeze-dried. 5Then, some solubilization chemical (such as 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)) is used to 6-10 、Lithium bromide (LiBr) 11 or guanidinium thiocyanate (GdmSCN) to dissolve the collected and precipitated proteins 12 In the next step, excess dissolved chemicals are removed by, for example, dialysis or other forms of buffer exchange. The silk protein solution is typically concentrated or desalted and then extruded into a coagulation bath containing an antisolvent such as methanol or isopropanol. 10,12,13 Sometimes, this process is performed using microfluidic devices. 14,15 Alternative methods are also described. For example, the silk protein solution can also be processed by electrospinning to produce fibers. 16,17 .

[0005] Sometimes, proteins are purified using chromatography techniques rather than precipitation. In these cases, a peptide tag is genetically fused to the silk protein. Examples of these tags are histidine residues (His tag), thioredoxin (Trx) 18 or SUMO tag 19 When the tag (such as Trx or SUMO) is removed, the solubility of the protein decreases and fibril formation occurs. In these cases, fibrils form in solution or at the air-water interface and the fibers can be pulled out of the aqueous solution. 6 .

[0006] The present invention solves the problem of how silk proteins can be produced and processed for assembly into fibers or other structures, such as films or coatings, in a manner that does not require precipitation of the silk proteins or subsequent solubilization of the proteins. The present invention does not require the use of fusion tags, which must be removed by treatment with proteases or other means of cleaving the fusion protein tag. 20 The present invention allows the production of fibers of recombinant silk proteins without the use of a coagulation bath, such as isopropanol or methanol. The present invention has significant benefits in the preparation of fibers because the steps of solubilization, fusion protein removal, and the use of a coagulation bath are not required.

[0007] Dissolution of precipitated silk proteins requires the use of harsh chemicals, especially HFIP, lithium salts and GdmSCN, which are toxic or add substantial cost to the process. Dissolution can also cause physical damage to the silk proteins. Using proteases to cleave protein domains before assembly requires expensive proteases and adds processing steps, such as chromatography, which limit scalability.

[0008] The present invention discloses a manner in which recombinant silk proteins can enter a concentrated phase, which can then be used to form fibers, adhesives, coatings, films or other material structures. SUMMARY OF THE INVENTION

[0010] The object of the present invention is to provide a method for producing materials using recombinant silk protein by easy (facile) processing. The present invention reduces the processing steps required for silk protein purification, because only a concentration step is required for forming a concentrated silk protein solution. The concentrated silk solution is formed by a phase separation event, and does not involve the precipitation of protein or the subsequent dissolution of protein. The present invention can also omit other alternative processing methods, such as chromatographic separation and the possible removal of label proteins by peptide bond cleavage. The present invention has such an advantage that it enables the direct formation of fibers by stretching (stretching) or elongating (elongating) protein concentrated solutions in air or gaseous environments. Fibers can be pulled directly from the concentrate without squeezing out the silk protein in a liquid bath (liquid bath) to coagulate the protein (coagulate). Therefore, this processing step can be omitted. Advantages also include the possibility of preparing very long single fiber filaments (single fiber filament). In addition, the present invention provides the use of concentrated silk solutions as water-based adhesives for gluing various materials and surfaces.

[0011] Therefore, in one aspect, the present invention provides a method for producing a concentrated phase of a silk fusion protein, the method comprising the steps of:

[0012] a) preparing a solution of a silk fusion protein in an aqueous medium, wherein the fusion protein is isolated from a recombinant production host and comprises a silk protein sequence and two separate non-silk terminal module sequences flanking the silk protein sequence;

[0013] b) concentrating the fusion protein in the aqueous medium until liquid phase separation occurs;

[0014] c) collecting the protein-rich phase of the aqueous medium obtained in step b)

[0015] d) repeating steps b) and c) with the protein-rich phase obtained in step c) until a protein concentration of about 20-45% w / v is reached;

[0016] e) optionally separating aggregates of the fusion protein from the soluble fusion protein obtained in step d) and removing the aggregates from the solution;

[0017] f) concentrating the solution obtained in step d) or e) to a final protein concentration of about 60-80% w / v,

[0018] Steps a) to f) are performed so that the silk fusion protein is not precipitated and then dissolved in the aqueous medium.

[0019] In another aspect, the present invention provides a concentrate of spider silk fusion protein produced by the above method.

[0020] In another aspect, the present invention provides a recombinant spider silk fusion protein comprising a silk polymer repeat and two cellulose binding modules (CBMs) from Clostridium thermocellum flanking the silk polymer repeat, wherein the repeat sequence consists of a repeated A sequence of SEQ ID NO: 1 combined with a repeated Q sequence of SEQ ID NO: 2, and the cellulose binding module consists of a sequence of SEQ ID NO: 7, each module being connected to the repeat via a C-terminal linker of SEQ ID NO: 6 or an N-terminal linker of SEQ ID NO: 5.

[0021] On the other hand, the present invention provides a recombinant spider silk fusion protein, which comprises the spider silk protein repeat sequence Araneus diadematus ADF3 of SEQ ID NO:3, or the black widow spider (Latrodectus hesperus) AcSp1 sequence of SEQ ID NO:4; and two cellulose binding modules (CBM) from Clostridium thermocellum located on the flanks of the silk protein repeats, the cellulose binding modules consisting of the sequence of SEQ ID NO:7, and each module is connected to the repeat sequence ADF3 or the black widow spider AcSp1 sequence via a C-terminal linker of SEQ ID NO:6 or an N-terminal linker of SEQ ID NO:5.

[0022] On the other hand, the present invention provides a recombinant spider silk fusion protein, which comprises the spider silk protein repeat sequence ADF3 of SEQ ID NO: 3, and two SpyCatcher domains engineered from the fibronectin binding protein FbaB of Streptococcus pyogenes located on the flanks of the silk protein repeat sequence, wherein the SpyCatcher domain consists of the sequence of SEQ ID NO: 14, and each domain is connected to the repeat sequence ADF3 via a C-terminal linker of SEQ ID NO: 6 or an N-terminal linker of SEQ ID NO: 17.

[0023] On the other hand, the present invention provides a recombinant spider silk fusion protein, which comprises the spider silk protein repeat sequence ADF3 of SEQ ID NO: 3, and the tenth type III module of fibronectin from Homo sapiens located on the flank of the silk protein repeat sequence, wherein the tenth type III module of fibronectin consists of the sequence of SEQ ID NO: 18, and each domain is connected to the repeat sequence ADF3 via a C-terminal linker of SEQ ID NO: 6 or an N-terminal linker of SEQ ID NO: 17.

[0024] On the other hand, the present invention provides a recombinant spider silk fusion protein, which comprises the spider silk protein repeat sequence ADF3 of SEQ ID NO: 3, and two γ-crystallin D domains from Homo sapiens located on the flanks of the silk protein repeat sequence, wherein the γ-crystallin D domain consists of the sequence of SEQ ID NO: 20, and each domain is connected to the repeat sequence ADF3 via a C-terminal linker of SEQ ID NO: 6 or an N-terminal linker of SEQ ID NO: 17.

[0025] In another aspect, the present invention provides a recombinant nucleic acid expressing a recombinant spider silk fusion protein as defined above.

[0026] In another aspect, the present invention provides a host cell expressing a recombinant nucleic acid expressing a recombinant spider silk fusion protein as defined above.

[0027] In another aspect, the present invention provides an adhesive comprising a silk fusion protein as an active ingredient, wherein the silk fusion protein comprises a filamentous protein sequence and two separate non-silk terminal module sequences flanking the filamentous protein sequence.

[0028] In another aspect, the present invention relates to the use of a silk fusion protein as an adhesive, wherein the silk fusion protein comprises a filamentous protein sequence and two separate non-silk terminal module sequences flanking the filamentous protein sequence.

[0029] In another aspect, the present invention relates to a method for preparing a composite, comprising the steps of: a) providing a first substrate, b) applying an adhesive according to any one of claims 15 to 28 to said substrate, and c) contacting the substrate obtained from step b) with a second substrate or surface to form a composite, wherein the adhesion of said first substrate to said second substrate or surface is enhanced by said adhesive. Sequence Listing <110> Aalto University Foundation sr. <120> Method for producing concentrated adhesive phase of silk fusion protein <130> AALTO18PCT <150> FI 20175544 <151> 2017-06-13 <150> FI 20176165 <151> 2017-12-22 <160> twenty three <170> BiSSAP 1.3 <210> 1 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> Block A <400> 1 Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly 1 5 10 15 Tyr Gly Pro Gly Ser Gly Gln Gln 20 <210> 2 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Block Q <400> 2 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly 1 5 10 15 Pro Gly Gln Gln 20 <210> 3 <211> 499 <212> PRT <213> Araneus diadematus <220> <223> ADF3 <400> 3 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly 1 5 10 15 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gly Tyr 20 25 30 Gly Pro Gly Ser Gly Gln Gln Gly Pro Ser Gln Gln Gly Pro Gly Gln 35 40 45 Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala 50 55 60 Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro 65 70 75 80 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala 85 90 95 Ala Gly Gly Asn Gly Pro Gly Ser Gly Gln Gln Gly Ala Gly Gln Gln 100 105 110 Gly Pro Gly Gln Gln Gly Pro Gly Gly Ser Ala Ala Ala Ala Ala Ala 115 120 125 Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly 130 135 140 Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala 145 150 155 160 Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gly Pro Gly Gln Gln 165 170 175 Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala 180 185 190 Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly 195 200 205 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 210 215 220 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly 225 230 235 240 Tyr Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 245 250 255 Tyr Gly Pro Gly Ala Ser Ala Ala Ser Ala Ala Ser Gly Gly Tyr Gly 260 265 270 Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln 275 280 285 Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly 290 295 300 Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 305 310 315 320 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 325 330 335 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly 340 345 350 Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 355 360 365 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 370 375 380 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly 385 390 395 400 Gln Gly Ala Tyr Gly Pro Gly Ala Ser Ala Ala Ala Gly Ala Ala Gly 405 410 415 Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 420 425 430 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 435 440 445 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly 450 455 460 Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly 465 470 475 480 Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 485 490 495 Gly Gly Gln <210> 4 <211> 396 <212> PRT <213> Black widow spider (Latrodectus hesperus) <220> <223> AcSp1 <400> 4 Phe Gly Gly Pro Ser Ala Gly Gly Asp Val Ala Ala Lys Leu Ala Arg 1 5 10 15 Ser Leu Ala Ser Thr Leu Ala Ser Ser Gly Val Phe Arg Ala Ala Phe 20 25 30 Asn Ser Arg Val Ser Thr Pro Val Ala Val Gln Leu Thr Asp Ala Leu 35 40 45 Val Gln Lys Ile Ala Ser Asn Leu Gly Leu Asp Tyr Ala Thr Ala Ser 50 55 60 Lys Leu Arg Lys Ala Ser Gln Ala Val Ser Lys Val Arg Met Gly Ser 65 70 75 80 Asp Thr Asn Ala Tyr Ala Leu Ala Ile Ser Ser Ala Leu Ala Glu Val 85 90 95 Leu Ser Ser Ser Gly Lys Val Ala Asp Ala Asn Ile Asn Gln Ile Ala 100 105 110 Pro Gln Leu Ala Ser Gly Ile Val Leu Gly Val Ser Thr Thr Ala Pro 115 120 125 Gln Phe Gly Val Asp Leu Ser Ser Ile Asn Val Asn Leu Asp Ile Ser 130 135 140 Asn Val Ala Arg Asn Met Gln Ala Ser Ile Gln Gly Gly Pro Ala Pro 145 150 155 160 Ile Thr Ala Glu Gly Pro Asp Phe Gly Ala Gly Tyr Pro Gly Gly Ala 165 170 175 Pro Thr Asp Leu Ser Gly Leu Asp Met Gly Ala Pro Ser Asp Gly Ser 180 185 190 Arg Gly Gly Asp Ala Thr Ala Lys Leu Leu Gln Ala Leu Val Pro Ala 195 200 205 Leu Leu Lys Ser Asp Val Phe Arg Ala Ile Tyr Lys Arg Gly Thr Arg 210 215 220 Lys Gln Val Val Gln Tyr Val Thr Asn Ser Ala Leu Gln Gln Ala Ala 225 230 235 240 Ser Ser Leu Gly Leu Asp Ala Ser Thr Ile Ser Gln Leu Gln Thr Lys 245 250 255 Ala Thr Gln Ala Leu Ser Ser Val Ser Ala Asp Ser Asp Ser Thr Ala 260 265 270 Tyr Ala Lys Ala Phe Gly Leu Ala Ile Ala Gln Val Leu Gly Thr Ser 275 280 285 Gly Gln Val Asn Asp Ala Asn Val Asn Gln Ile Gly Ala Lys Leu Ala 290 295 300 Thr Gly Ile Leu Arg Gly Ser Ser Ala Val Ala Pro Arg Leu Gly Ile 305 310 315 320 Asp Leu Ser Gly Ile Asn Val Asp Ser Asp Ile Gly Ser Val Thr Ser 325 330 335 Leu Ile Leu Ser Gly Ser Thr Leu Gln Met Thr Ile Pro Ala Gly Gly 340 345 350 Asp Asp Leu Ser Gly Gly Tyr Pro Gly Gly Phe Pro Ala Gly Ala Gln 355 360 365 Pro Ser Gly Gly Ala Pro Val Asp Ser Asn Ile Gly Leu Val Gly Thr 370 375 380 Gln Asp Val Ala Ile Gly Val Ser Gln Pro Val Asp 385 390 395 <210> 5 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> Connector N <400> 5 Pro Ser Ala Ser Ala Ser Ala Ser Ala Gly Ala Ser Ala Ala Ala Ser 1 5 10 15 Ala Gly Ala Gly Ala Gly Ala 20 <210> 6 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Connector C <400> 6 Ala Ser Ala Ser Ala Ser Ala Ala Ala Ser Ala Ala Ser Thr Val Ala 1 5 10 15 Asn Ser Ser Ser 20 <210> 7 <211> 154 <212> PRT <213> Clostridium thermocellum <220> <223> CBM <400> 7 Asn Leu Lys Val Glu Phe Tyr Asn Ser Asn Pro Ser Asp Thr Thr Asn 1 5 10 15 Ser Ile Asn Pro Gln Phe Lys Val Thr Asn Thr Gly Ser Ser Ala Ile 20 25 30 Asp Leu Ser Lys Leu Thr Leu Arg Tyr Tyr Tyr Thr Val Asp Gly Gln 35 40 45 Lys Asp Gln Thr Phe Trp Cys Asp His Ala Ala Ile Ile Gly Ser Asn 50 55 60 Gly Ser Tyr Asn Gly Ile Thr Ser Asn Val Lys Gly Thr Phe Val Lys 65 70 75 80 Met Ser Ser Ser Thr Asn Asn Ala Asp Thr Tyr Leu Glu Ile Ser Phe 85 90 95 Thr Gly Gly Thr Leu Glu Pro Gly Ala His Val Gln Ile Gln Gly Arg 100 105 110 Phe Ala Lys Asn Asp Trp Ser Asn Tyr Thr Gln Ser Asn Asp Tyr Ser 115 120 125 Phe Lys Ser Ala Ser Gln Phe Val Glu Trp Asp Gln Val Thr Ala Tyr 130 135 140 Leu Asn Gly Val Leu Val Trp Gly Lys Glu 145 150 <210> 8 <211> 860 <212> PRT <213> Artificial Sequence <220> <223> CBM-L_N-AQ12-L_C-CBM-H6 Fusion Protein <400> 8 Met Gly Asn Leu Lys Val Glu Phe Tyr Asn Ser Asn Pro Ser Asp Thr 1 5 10 15 Thr Asn Ser Ile Asn Pro Gln Phe Lys Val Thr Asn Thr Gly Ser Ser 20 25 30 Ala Ile Asp Leu Ser Lys Leu Thr Leu Arg Tyr Tyr Tyr Thr Val Asp 35 40 45 Gly Gln Lys Asp Gln Thr Phe Trp Cys Asp His Ala Ala Ile Ile Gly 50 55 60 Ser Asn Gly Ser Tyr Asn Gly Ile Thr Ser Asn Val Lys Gly Thr Phe 65 70 75 80 Val Lys Met Ser Ser Ser Thr Asn Asn Ala Asp Thr Tyr Leu Glu Ile 85 90 95 Ser Phe Thr Gly Gly Thr Leu Glu Pro Gly Ala His Val Gln Ile Gln 100 105 110 Gly Arg Phe Ala Lys Asn Asp Trp Ser Asn Tyr Thr Gln Ser Asn Asp 115 120 125 Tyr Ser Phe Lys Ser Ala Ser Gln Phe Val Glu Trp Asp Gln Val Thr 130 135 140 Ala Tyr Leu Asn Gly Val Leu Val Trp Gly Lys Glu Pro Ser Ala Ser 145 150 155 160 Ala Ser Ala Ser Ala Gly Ala Ser Ala Ala Ala Ser Ala Gly Ala Gly 165 170 175 Ala Gly Ala Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 180 185 190 Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala 195 200 205 Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Ser Gln Gln Gly 210 215 220 Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala 225 230 235 240 Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln 245 250 255 Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala 260 265 270 Ala Ala Ala Ala Gly Gly Asn Gly Pro Gly Ser Gly Gln Gln Gly Ala 275 280 285 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Ser Ala Ala Ala 290 295 300 Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly 305 310 315 320 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala 325 330 335 Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gly Pro 340 345 350 Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser 355 360 365 Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln 370 375 380 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly 385 390 395 400 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr 405 410 415 Gly Pro Gly Tyr Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly 420 425 430 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ser Ala Ala Ser Gly 435 440 445 Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 450 455 460 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala 465 470 475 480 Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln 485 490 495 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly 500 505 510 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr 515 520 525 Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln 530 535 540 Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln 545 550 555 560 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly 565 570 575 Pro Gly Gly Gln Gly Ala Tyr Gly Pro Gly Ala Ser Ala Ala Ala Gly 580 585 590 Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln 595 600 605 Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln 610 615 620 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly 625 630 635 640 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr 645 650 655 Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln 660 665 670 Gln Gly Pro Gly Gly Gln Ala Ser Ala Ser Ala Ser Ala Ala Ala Ser 675 680 685 Ala Ala Ser Thr Val Ala Asn Ser Ser Ser Asn Leu Lys Val Glu Phe 690 695 700 Tyr Asn Ser Asn Pro Ser Asp Thr Thr Asn Ser Ile Asn Pro Gln Phe 705 710 715 720 Lys Val Thr Asn Thr Gly Ser Ser Ala Ile Asp Leu Ser Lys Leu Thr 725 730 735 Leu Arg Tyr Tyr Tyr Thr Val Asp Gly Gln Lys Asp Gln Thr Phe Trp 740 745 750 Cys Asp His Ala Ala Ile Ile Gly Ser Asn Gly Ser Tyr Asn Gly Ile 755 760 765 Thr Ser Asn Val Lys Gly Thr Phe Val Lys Met Ser Ser Ser Thr Asn 770 775 780 Asn Ala Asp Thr Tyr Leu Glu Ile Ser Phe Thr Gly Gly Thr Leu Glu 785 790 795 800 Pro Gly Ala His Val Gln Ile Gln Gly Arg Phe Ala Lys Asn Asp Trp 805 810 815 Ser Asn Tyr Thr Gln Ser Asn Asp Tyr Ser Phe Lys Ser Ala Ser Gln 820 825 830 Phe Val Glu Trp Asp Gln Val Thr Ala Tyr Leu Asn Gly Val Leu Val 835 840 845 Trp Gly Lys Glu Leu Glu His His His His His His 850 855 860 <210> 9 <211> 860 <212> PRT <213> Artificial Sequence <220> <223> CBM-L_N-Nat.AQ12-L_C-CBM-H6 Fusion Protein <400> 9 Met Gly Asn Leu Lys Val Glu Phe Tyr Asn Ser Asn Pro Ser Asp Thr 1 5 10 15 Thr Asn Ser Ile Asn Pro Gln Phe Lys Val Thr Asn Thr Gly Ser Ser 20 25 30 Ala Ile Asp Leu Ser Lys Leu Thr Leu Arg Tyr Tyr Tyr Thr Val Asp 35 40 45 Gly Gln Lys Asp Gln Thr Phe Trp Cys Asp His Ala Ala Ile Ile Gly 50 55 60 Ser Asn Gly Ser Tyr Asn Gly Ile Thr Ser Asn Val Lys Gly Thr Phe 65 70 75 80 Val Lys Met Ser Ser Ser Thr Asn Asn Ala Asp Thr Tyr Leu Glu Ile 85 90 95 Ser Phe Thr Gly Gly Thr Leu Glu Pro Gly Ala His Val Gln Ile Gln 100 105 110 Gly Arg Phe Ala Lys Asn Asp Trp Ser Asn Tyr Thr Gln Ser Asn Asp 115 120 125 Tyr Ser Phe Lys Ser Ala Ser Gln Phe Val Glu Trp Asp Gln Val Thr 130 135 140 Ala Tyr Leu Asn Gly Val Leu Val Trp Gly Lys Glu Pro Ser Ala Ser 145 150 155 160 Ala Ser Ala Ser Ala Gly Ala Ser Ala Ala Ala Ser Ala Gly Ala Gly 165 170 175 Ala Gly Ala Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 180 185 190 Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala 195 200 205 Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Ser Gln Gln Gly 210 215 220 Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala 225 230 235 240 Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln 245 250 255 Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala 260 265 270 Ala Ala Ala Ala Gly Gly Asn Gly Pro Gly Ser Gly Gln Gln Gly Ala 275 280 285 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Ser Ala Ala Ala 290 295 300 Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly 305 310 315 320 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala 325 330 335 Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gly Pro 340 345 350 Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser 355 360 365 Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln 370 375 380 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly 385 390 395 400 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr 405 410 415 Gly Pro Gly Tyr Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly 420 425 430 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ser Ala Ala Ser Gly 435 440 445 Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 450 455 460 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala 465 470 475 480 Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln 485 490 495 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly 500 505 510 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr 515 520 525 Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln 530 535 540 Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln 545 550 555 560 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly 565 570 575 Pro Gly Gly Gln Gly Ala Tyr Gly Pro Gly Ala Ser Ala Ala Ala Gly 580 585 590 Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln 595 600 605 Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln 610 615 620 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly 625 630 635 640 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr 645 650 655 Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln 660 665 670 Gln Gly Pro Gly Gly Gln Ala Ser Ala Ser Ala Ser Ala Ala Ala Ser 675 680 685 Ala Ala Ser Thr Val Ala Asn Ser Ser Ser Asn Leu Lys Val Glu Phe 690 695 700 Tyr Asn Ser Asn Pro Ser Asp Thr Thr Asn Ser Ile Asn Pro Gln Phe 705 710 715 720 Lys Val Thr Asn Thr Gly Ser Ser Ala Ile Asp Leu Ser Lys Leu Thr 725 730 735 Leu Arg Tyr Tyr Tyr Thr Val Asp Gly Gln Lys Asp Gln Thr Phe Trp 740 745 750 Cys Asp His Ala Ala Ile Ile Gly Ser Asn Gly Ser Tyr Asn Gly Ile 755 760 765 Thr Ser Asn Val Lys Gly Thr Phe Val Lys Met Ser Ser Ser Thr Asn 770 775 780 Asn Ala Asp Thr Tyr Leu Glu Ile Ser Phe Thr Gly Gly Thr Leu Glu 785 790 795 800 Pro Gly Ala His Val Gln Ile Gln Gly Arg Phe Ala Lys Asn Asp Trp 805 810 815 Ser Asn Tyr Thr Gln Ser Asn Asp Tyr Ser Phe Lys Ser Ala Ser Gln 820 825 830 Phe Val Glu Trp Asp Gln Val Thr Ala Tyr Leu Asn Gly Val Leu Val 835 840 845 Trp Gly Lys Glu Leu Glu His His His His His His 850 855 860 <210> 10 <211> 757 <212> PRT <213> Artificial Sequence <220> <223> CBM-L_N-L.hes.AcSp1-L_C-CBM-H6 Fusion Protein <400> 10 Met Gly Asn Leu Lys Val Glu Phe Tyr Asn Ser Asn Pro Ser Asp Thr 1 5 10 15 Thr Asn Ser Ile Asn Pro Gln Phe Lys Val Thr Asn Thr Gly Ser Ser 20 25 30 Ala Ile Asp Leu Ser Lys Leu Thr Leu Arg Tyr Tyr Tyr Thr Val Asp 35 40 45 Gly Gln Lys Asp Gln Thr Phe Trp Cys Asp His Ala Ala Ile Ile Gly 50 55 60 Ser Asn Gly Ser Tyr Asn Gly Ile Thr Ser Asn Val Lys Gly Thr Phe 65 70 75 80 Val Lys Met Ser Ser Ser Thr Asn Asn Ala Asp Thr Tyr Leu Glu Ile 85 90 95 Ser Phe Thr Gly Gly Thr Leu Glu Pro Gly Ala His Val Gln Ile Gln 100 105 110 Gly Arg Phe Ala Lys Asn Asp Trp Ser Asn Tyr Thr Gln Ser Asn Asp 115 120 125 Tyr Ser Phe Lys Ser Ala Ser Gln Phe Val Glu Trp Asp Gln Val Thr 130 135 140 Ala Tyr Leu Asn Gly Val Leu Val Trp Gly Lys Glu Pro Ser Ala Ser 145 150 155 160 Ala Ser Ala Ser Ala Gly Ala Ser Ala Ala Ala Ser Ala Gly Ala Gly 165 170 175 Ala Gly Ala Phe Gly Gly Pro Ser Ala Gly Gly Asp Val Ala Ala Lys 180 185 190 Leu Ala Arg Ser Leu Ala Ser Thr Leu Ala Ser Ser Gly Val Phe Arg 195 200 205 Ala Ala Phe Asn Ser Arg Val Ser Thr Pro Val Ala Val Gln Leu Thr 210 215 220 Asp Ala Leu Val Gln Lys Ile Ala Ser Asn Leu Gly Leu Asp Tyr Ala 225 230 235 240 Thr Ala Ser Lys Leu Arg Lys Ala Ser Gln Ala Val Ser Lys Val Arg 245 250 255 Met Gly Ser Asp Thr Asn Ala Tyr Ala Leu Ala Ile Ser Ser Ala Leu 260 265 270 Ala Glu Val Leu Ser Ser Ser Gly Lys Val Ala Asp Ala Asn Ile Asn 275 280 285 Gln Ile Ala Pro Gln Leu Ala Ser Gly Ile Val Leu Gly Val Ser Thr 290 295 300 Thr Ala Pro Gln Phe Gly Val Asp Leu Ser Ser Ile Asn Val Asn Leu 305 310 315 320 Asp Ile Ser Asn Val Ala Arg Asn Met Gln Ala Ser Ile Gln Gly Gly 325 330 335 Pro Ala Pro Ile Thr Ala Glu Gly Pro Asp Phe Gly Ala Gly Tyr Pro 340 345 350 Gly Gly Ala Pro Thr Asp Leu Ser Gly Leu Asp Met Gly Ala Pro Ser 355 360 365 Asp Gly Ser Arg Gly Gly Asp Ala Thr Ala Lys Leu Leu Gln Ala Leu 370 375 380 Val Pro Ala Leu Leu Lys Ser Asp Val Phe Arg Ala Ile Tyr Lys Arg 385 390 395 400 Gly Thr Arg Lys Gln Val Val Gln Tyr Val Thr Asn Ser Ala Leu Gln 405 410 415 Gln Ala Ala Ser Ser Leu Gly Leu Asp Ala Ser Thr Ile Ser Gln Leu 420 425 430 Gln Thr Lys Ala Thr Gln Ala Leu Ser Ser Val Ser Ala Asp Ser Asp 435 440 445 Ser Thr Ala Tyr Ala Lys Ala Phe Gly Leu Ala Ile Ala Gln Val Leu 450 455 460 Gly Thr Ser Gly Gln Val Asn Asp Ala Asn Val Asn Gln Ile Gly Ala 465 470 475 480 Lys Leu Ala Thr Gly Ile Leu Arg Gly Ser Ser Ala Val Ala Pro Arg 485 490 495 Leu Gly Ile Asp Leu Ser Gly Ile Asn Val Asp Ser Asp Ile Gly Ser 500 505 510 Val Thr Ser Leu Ile Leu Ser Gly Ser Thr Leu Gln Met Thr Ile Pro 515 520 525 Ala Gly Gly Asp Asp Leu Ser Gly Gly Tyr Pro Gly Gly Phe Pro Ala 530 535 540 Gly Ala Gln Pro Ser Gly Gly Ala Pro Val Asp Ser Asn Ile Gly Leu 545 550 555 560 Val Gly Thr Gln Asp Val Ala Ile Gly Val Ser Gln Pro Val Asp Ala 565 570 575 Ser Ala Ser Ala Ser Ala Ala Ala Ser Ala Ala Ser Thr Val Ala Asn 580 585 590 Ser Ser Ser Asn Leu Lys Val Glu Phe Tyr Asn Ser Asn Pro Ser Asp 595 600 605 Thr Thr Asn Ser Ile Asn Pro Gln Phe Lys Val Thr Asn Thr Gly Ser 610 615 620 Ser Ala Ile Asp Leu Ser Lys Leu Thr Leu Arg Tyr Tyr Tyr Thr Val 625 630 635 640 Asp Gly Gln Lys Asp Gln Thr Phe Trp Cys Asp His Ala Ala Ile Ile 645 650 655 Gly Ser Asn Gly Ser Tyr Asn Gly Ile Thr Ser Asn Val Lys Gly Thr 660 665 670 Phe Val Lys Met Ser Ser Ser Thr Asn Asn Ala Asp Thr Tyr Leu Glu 675 680 685 Ile Ser Phe Thr Gly Gly Thr Leu Glu Pro Gly Ala His Val Gln Ile 690 695 700 Gln Gly Arg Phe Ala Lys Asn Asp Trp Ser Asn Tyr Thr Gln Ser Asn 705 710 715 720 Asp Tyr Ser Phe Lys Ser Ala Ser Gln Phe Val Glu Trp Asp Gln Val 725 730 735 Thr Ala Tyr Leu Asn Gly Val Leu Val Trp Gly Lys Glu Leu Glu His 740 745 750 His His His His His 755 <210> 11 <211> 2580 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding fusion protein CBM-L_N-AQ12-L_C-CBM-H6 <400> 11 atgggcaatc tgaaagtgga gttctataat agcaatccga gcgataccac caatagcatt 60 aatccgcagt ttaaagttac caataccggt agcagcgcaa ttgatctgag caaactgacc 120 ctgcgttatt actataccgt tgatggtcag aaagatcaga cattttggtg tgatcatgca 180 gccattattg gtagcaatgg tagctataat ggcattacca gcaatgtgaa aggcaccttt 240 gttaaaatga gcagcagcac caataatgcc gatacctatc tggaaattag ctttaccggt 300 ggcaccctgg aaccgggtgc acatgttcag attcagggtc gttttgcaaa aaatgattgg 360 agcaattata cccagagcaa cgactatagc tttaaaagcg caagccagtt tgttgaatgg 420 gatcaggtta ccgcatatct gaatggtgtt ctggtttggg gtaaagaacc gagcgctagc 480 gcaagcgcat cagccggtgc aagtgctgca gcaagtgccg gtgctggtgc gggtgcaggt 540 cctggtcagc agggtccggg tcaacaagga cctggacagc aaggaccgta tggtccaggt 600 gcatcagctg cagccgcagc agcgggtggt tatggtccgg gaagcggtca gcaaggccct 660 tcacaacagg gaccaggcca acagggtcct ggcggtcaag gtccttatgg acctggtgct 720 tctgctgcgg cagcggctgc cggtggctat ggccctggta gtggccagca agggcctggt 780 ggccagggtc catatggccc aggttctagt gccgcagctg ctgctgcagg cggtaatgga 840 ccgggttcag gacaacaagg tgcagggcag caaggtcccg gacaacaggg tccaggtggt 900 agtgcagcag cggcagcagc tggcggatat ggaccaggta gtgggcaaca aggcccaggt 960 caacaagggc cagggggtca aggcccatac ggtccgggtg cttccgcagc cgcagctgca 1020 gcaggcggtt acggtcctgg tagtggtcaa ggtccaggcc agcaaggacc aggtggacaa 1080 gggccttacg gaccaggcgc atctgcggca gcagcagccg cagggggata tggtcctggt 1140 tcaggcagc agggaccagg tcagcaaggt ccaggtcagc agggacctgg gggtcaggga 1200 ccttacggtc ctggcgcaag tgcagctgca gcggcagcgg gtggctacgg accgggttat 1260 ggccagcagg gaccgggaca gcagggaccc ggtggacagg gtccgtatgg accgggtgca 1320 agtgcagcat cagcagcaag tggtggttac ggacctggct caggacagca aggccctggc 1380 caacaaggcc ctggcggaca gggaccctat gggccaggtg ccagcgctgc agcagccgca 1440 gccggtggat acggtccagg ctctggtcaa caaggtcctg ggcaacaagg tcctggccag 1500 cagggtccag gacagcaagg gcctggcggt caaggaccgt acggaccggg tgccagcgca 1560 gcgggctcag cggcaggcgg ttatggtcca ggatcaggcc agcaaggtcc gggtcagcaa 1620 ggcccagggc agcaaggacc gggtcaacag ggaccgggtc agcagggtcc tgggcaacag 1680 ggtccgggac aacagggacc aggtcaacaa ggaccgggtc aacaaggtcc aggtggtcag 1740 ggtgcatatg gtcctggcgc ttcagcagca gcaggggctg caggggtta tggcccaggt 1800 agcggtcagc agggacccgg acaacaaggc cctggacaac agggtcccgg tcagcaaggg 1860 ccaggccaac aaggtccagg acaacaagga ccagggcagc agggtccagg ccaacaaggc 1920 ccttatggtc cgggtgccag tgctgcggca gcggcagctg ggggttatgg tccaggctct 1980 ggacagcagg gacctggcca gcaaggacct gggcagcaag ggccaggcgg tcaggcaagt 2040 gcatcagcaa gcgcagcagc gagtgcagca agcaccgttg cgaattctag ctccaacctg 2100 aaagttgagt tttataactc caatccgtca gatacgacga acagtattaa ccctcagttc 2160 aaagtgacaa acaccggttc aagcgccatc gatctgtcaa aactgacatt acgttactat 2220 tatacagtgg acggccagaa agaccaaacc ttctggtgcg accatgccgc aattatcggt 2280 tcaaatggtt catataacgg tatcacgagc aacgtcaaag gtacattcgt gaaaatgagt 2340 agctcaacca acaacgcaga tacctactta gaaatttcat ttacgggtgg tacgttagaa 2400 ccaggtgcgc acgtgcagat ccaaggtcgc ttcgccaaaa acgactggtc aaattataca 2460 cagtcgaatg actacagctt caaatcagca tcacagttcg tagagtggga tcaagtgaca 2520 gcctacctga acggcgtgct ggtgtggggc aaagaactcg agcaccacca ccaccaccac 2580 <210> 12 <211> 2580 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding fusion protein CBM-L_N-Nat.AQ12-L_C-CBM-H6 <400> 12 atgggcaatc tgaaagtgga gttctataat agcaatccga gcgataccac caatagcatt 60 aatccgcagt ttaaagttac caataccggt agcagcgcaa ttgatctgag caaactgacc 120 ctgcgttatt actataccgt tgatggtcag aaagatcaga cattttggtg tgatcatgca 180 gccattattg gtagcaatgg tagctataat ggcattacca gcaatgtgaa aggcaccttt 240 gttaaaatga gcagcagcac caataatgcc gatacctatc tggaaattag ctttaccggt 300 ggcaccctgg aaccgggtgc acatgttcag attcagggtc gttttgcaaa aaatgattgg 360 agcaattata cccagagcaa cgactatagc tttaaaagcg caagccagtt tgttgaatgg 420 gatcaggtta ccgcatatct gaatggtgtt ctggtttggg gtaaagaacc gagcgctagc 480 gcaagcgcat cagccggtgc aagtgctgca gcaagtgccg gtgctggtgc gggtgcaggt 540 cctggtcagc agggtccggg tcaacaagga cctggacagc aaggaccgta tggtccaggt 600 gcatcagctg cagccgcagc agcgggtggt tatggtccgg gaagcggtca gcaaggccct 660 tcacaacagg gaccaggcca acagggtcct ggcggtcaag gtccttatgg acctggtgct 720 tctgctgcgg cagcggctgc cggtggctat ggccctggta gtggccagca agggcctggt 780 ggccagggtc catatggccc aggttctagt gccgcagctg ctgctgcagg cggtaatgga 840 ccgggttcag gacaacaagg tgcagggcag caaggtcccg gacaacaggg tccaggtggt 900 agtgcagcag cggcagcagc tggcggatat ggaccaggta gtgggcaaca aggcccaggt 960 caacaagggc cagggggtca aggcccatac ggtccgggtg cttccgcagc cgcagctgca 1020 gcaggcggtt acggtcctgg tagtggtcaa ggtccaggcc agcaaggacc aggtggacaa 1080 gggccttacg gaccaggcgc atctgcggca gcagcagccg cagggggata tggtcctggt 1140 tcagggcagc agggaccagg tcagcaaggt ccaggtcagc agggacctgg gggtcaggga 1200 ccttacggtc ctggcgcaag tgcagctgca gcggcagcgg gtggctacgg accgggttat 1260 ggccagcagg gaccgggaca gcagggaccc ggtggacagg gtccgtatgg accgggtgca 1320 agtgcagcat cagcagcaag tggtggttac ggacctggct caggacagca aggccctggc 1380 caacaaggcc ctggcggaca gggaccctat gggccaggtg ccagcgctgc agcagccgca 1440 gccggtggat acggtccagg ctctggtcaa caaggtcctg ggcaacaagg tcctggccag 1500 cagggtccag gacagcaagg gcctggcggt caaggaccgt acggaccggg tgccagcgca 1560 gcgggctcag cggcaggcgg ttatggtcca ggatcaggcc agcaaggtcc gggtcagcaa 1620 ggcccagggc agcaaggacc gggtcaacag ggaccgggtc agcagggtcc tgggcaacag 1680 ggtccgggac aacagggacc aggtcaacaa ggaccgggtc aacaaggtcc aggtggtcag 1740 ggtgcatatg gtcctggcgc ttcagcagca gcaggggctg caggggtta tggcccaggt 1800 agcggtcagc agggacccgg acaacaaggc cctggacaac agggtcccgg tcagcaaggg 1860 ccaggccaac aaggtccagg acaacaagga ccagggcagc agggtccagg ccaacaaggc 1920 ccttatggtc cgggtgccag tgctgcggca gcggcagctg ggggttatgg tccaggctct 1980 ggcagcagg gacctggcca gcaaggacct gggcagcaag ggccaggcgg tcaggcaagt 2040 gcatcagcaa gcgcagcagc gagtgcagca agcaccgttg cgaattctag ctccaacctg 2100 aaagttgagt tttataactc caatccgtca gatacgacga acagtattaa ccctcagttc 2160 aaagtgacaa acaccggttc aagcgccatc gatctgtcaa aactgacatt acgttactat 2220 tatacagtgg acggccagaa agaccaaacc ttctggtgcg accatgccgc aattatcggt 2280 tcaaatggtt catataacgg tatcacgagc aacgtcaaag gtacattcgt gaaaatgagt 2340 agctcaacca acaacgcaga tacctactta gaaatttcat ttacgggtgg tacgttagaa 2400 ccaggtgcgc acgtgcagat ccaaggtcgc ttcgccaaaa acgactggtc aaattataca 2460 cagtcgaatg actacagctt caaatcagca tcacagttcg tagagtggga tcaagtgaca 2520 gcctacctga acggcgtgct ggtgtggggc aaagaactcg agcaccacca ccaccaccac 2580 <210> 13 <211> 2271 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence encoding the fusion protein CBM-L_N-L.hes.AcSp1-L_C-CBM-H6 <400> 13 atgggcaatc tgaaagtcga attttacaac agcaatccga gcgataccac gaacagcatc 60 aatccgcagt ttaaggttac gaacacgggc agctccgcaa ttgatctgtc aaaactgacc 120 ctgcgttatt actatacggt ggatggccag aaggaccaaa ccttttggtg cgaccatgcg 180 gccattatcg gctcgaacgg tagctacaat ggcatcacct cgaacgtcaa aggcacgttc 240 gtgaagatga gctctagtac caacaatgcc gatacgtatc tggaaattag ttttaccggc 300 ggtacgctgg aaccgggtgc acacgttcag atccaaggtc gcttcgctaa aaacgattgg 360 tctaattaca cccagtctaa cgactatagt tttaagtccg cgtcacagtt cgttgaatgg 420 gatcaagtca cggcctacct gaatggcgtg ctggtttggg gtaaagaacc gagcgcgagc 480 gcaagcgcaa gtgccggtgc aagcgcagca gcatcagccg gtgctggtgc aggcgccttt 540 ggtggtccga gtgccggtgg tgatgttgca gcaaaactgg cacgtagcct ggcaagcacc 600 ctggcaagca gcggtgtttt tcgtgcagca tttaatagcc gtgttagcac accggttgca 660 gttcagctga ccgatgcact ggttcagaaa attgcaagca atctgggtct ggattatgca 720 accgcaagca aactgcgtaa agcaagccag gcagttagca aagttcgtat gggtagcgat 780 accaatgcat atgcactggc aattagcagc gcactggcag aagttctgag cagcagcggt 840 aaagttgcag atgcaaacat taatcagatt gcaccgcagc tggcaagcgg tattgttctg 900 ggtgttagca ccaccgcacc gcagtttggt gttgatctga gcagcattaa tgtgaacctg 960 gatattagca atgtggcacg taatatgcag gcaagcattc agggtggtcc ggcaccgatt 1020 acagccgaag gtccggattt tggtgcaggt tatccgggtg gtgcaccgac cgatctgagc 1080 ggtctggata tgggtgcacc gagtgatggt agccgtggtg gtgatgcaac cgccaaactg 1140 ctgcaggcac tggttccggc actgctgaaa agtgatgtgt ttcgtgcaat ctataaacgt 1200 ggcacccgta aacaggttgt tcagtatgtt accaatagcg cactgcagca ggcagcaagc 1260 agcctgggcc tggatgcaag caccattagc cagctgcaga ccaaagcaac ccaggcactg 1320 agcagcgtta gcgcagatag cgatagcacc gcatatgcaa aagcatttgg tctggcaatt 1380 gcacaggttc tgggcaccag cggtcaggtt aatgatgcaa atgtgaatca gattggtgca 1440 aaactggcca ccggtattct gcgtggtagc agcgcagttg caccgcgtct gggtattgat 1500 ctgagtggta ttaatgtgga tagcgatatt ggtagcgtta ccagcctgat tctgagcggt 1560 agcaccctgc agatgaccat tccggcaggc ggtgatgatc tgtcaggtgg ctatccgggt 1620 ggctttcctg ccggtgcaca gccgagtggt ggcgcaccgg ttgatagcaa tattggtctg 1680 gttggcaccc aggatgttgc aattggtgtt agccagccgg ttgatgccag cgcaagcgca 1740 agtgcagcag caagcgcagc cagcaccgtt gcaatagct ctagcaatct gaaagtcgaa 1800 ttttacaaca gcaatccgag cgataccacg aacagcatca atccgcagtt taaggttacg 1860 aacacgggca gctccgcaat tgatctgtca aaactgaccc tgcgttatta ctatacggtg 1920 gatggccaga aggaccaaac cttttggtgc gaccatgcgg ccattatcgg ctcgaacggt 1980 agctacaatg gcatcacctc gaacgtcaaa ggcacgttcg tgaagatgag ctctagtacc 2040 aacaatgccg atacgtatct ggaaattagt tttaccggcg gtacgctgga accgggtgca 2100 cacgttcaga tccaaggtcg cttcgctaaa aacgattggt ctaattacac ccagtctaac 2160 gactatagtt ttaagtccgc gtcacagttc gttgaatggg atcaagtcac ggcctacctg 2220 aatggcgtgc tggtttgggg taaagaactc gagcaccacc accaccacca c 2271 <210> 14 <211> 116 <212> PRT <213> Streptococcus pyogenes <220> <223> SpyCatcher <400> 14 Gly Ala Met Val Asp Thr Leu Ser Gly Leu Ser Ser Glu Gln Gly Gln 1 5 10 15 Ser Gly Asp Met Thr Ile Glu Glu Asp Ser Ala Thr His Ile Lys Phe 20 25 30 Ser Lys Arg Asp Glu Asp Gly Lys Glu Leu Ala Gly Ala Thr Met Lys 35 40 45 Leu Arg Asp Ser Ser Gly Lys Thr Ile Ser Thr Trp Ile Ser Asp Gly 50 55 60 Gln Val Lys Asp Phe Tyr Leu Tyr Pro Gly Lys Tyr Thr Phe Val Glu 65 70 75 80 Thr Ala Ala Pro Asp Gly Tyr Glu Val Ala Thr Ala Ile Thr Phe Thr 85 90 95 Val Asn Glu Gln Gly Gln Val Thr Val Asn Gly Lys Ala Thr Lys Gly 100 105 110 Asp Ala His Ile 115 <210> 15 <211> 781 <212> PRT <213> Artificial sequence <220> <223> SpyCatcher-L_ N-Nat.AQ12-L_C- SpyCatcher-H6 <400> 15 Met Gly Ala Met Val Asp Thr Leu Ser Gly Leu Ser Ser Glu Gln Gly 1 5 10 15 Gln Ser Gly Asp Met Thr Ile Glu Glu Asp Ser Ala Thr His Ile Lys 20 25 30 Phe Ser Lys Arg Asp Glu Asp Gly Lys Glu Leu Ala Gly Ala Thr Met 35 40 45 Lys Leu Arg Asp Ser Ser Gly Lys Thr Ile Ser Thr Trp Ile Ser Asp 50 55 60 Gly Gln Val Lys Asp Phe Tyr Leu Tyr Pro Gly Lys Tyr Thr Phe Val 65 70 75 80 Glu Thr Ala Ala Pro Asp Gly Tyr Glu Val Ala Thr Ala Ile Thr Phe 85 90 95 Thr Val Asn Glu Gln Gly Gln Val Thr Val Asn Gly Lys Ala Thr Lys 100 105 110 Gly Asp Ala His Ile Ser Ala Ser Ala Ser Ala Ser Ala Gly Ala Ser 115 120 125 Ala Ala Ala Ser Ala Gly Ala Gly Ala Gly Ala Gly Pro Gly Gln Gln 130 135 140 Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly 145 150 155 160 Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly 165 170 175 Gln Gln Gly Pro Ser Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly 180 185 190 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly 195 200 205 Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 210 215 220 Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Gly Gly Asn Gly 225 230 235 240 Pro Gly Ser Gly Gln Gln Gly Ala Gly Gln Gln Gly Pro Gly Gln Gln 245 250 255 Gly Pro Gly Gly Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro 260 265 270 Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly 275 280 285 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr 290 295 300 Gly Pro Gly Ser Gly Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln 305 310 315 320 Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly 325 330 335 Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 340 345 350 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala 355 360 365 Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Tyr Gly Gln Gln Gly 370 375 380 Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala 385 390 395 400 Ser Ala Ala Ser Ala Ala Ser Gly Gly Tyr Gly Pro Gly Ser Gly Gln 405 410 415 Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro 420 425 430 Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser 435 440 445 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 450 455 460 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala 465 470 475 480 Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly 485 490 495 Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 500 505 510 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 515 520 525 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Ala Tyr Gly 530 535 540 Pro Gly Ala Ser Ala Ala Ala Gly Ala Ala Gly Gly Tyr Gly Pro Gly 545 550 555 560 Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 565 570 575 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 580 585 590 Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala 595 600 605 Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly 610 615 620 Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Ala Ser 625 630 635 640 Ala Ser Ala Ser Ala Ala Ala Ser Ala Ala Ser Thr Val Ala Asn Ser 645 650 655 Ser Ser Ala Met Val Asp Thr Leu Ser Gly Leu Ser Ser Glu Gln Gly 660 665 670 Gln Ser Gly Asp Met Thr Ile Glu Glu Asp Ser Ala Thr His Ile Lys 675 680 685 Phe Ser Lys Arg Asp Glu Asp Gly Lys Glu Leu Ala Gly Ala Thr Met 690 695 700 Lys Leu Arg Asp Ser Ser Gly Lys Thr Ile Ser Thr Trp Ile Ser Asp 705 710 715 720 Gly Gln Val Lys Asp Phe Tyr Leu Tyr Pro Gly Lys Tyr Thr Phe Val 725 730 735 Glu Thr Ala Ala Pro Asp Gly Tyr Glu Val Ala Thr Ala Ile Thr Phe 740 745 750 Thr Val Asn Glu Gln Gly Gln Val Thr Val Asn Gly Lys Ala Thr Lys 755 760 765 Gly Asp Ala His Ile Leu Glu His His His His His His 770 775 780 <210> 16 <211> 2346 <212> DNA <213> Artificial sequence <220> <223> SpyCatcher-L_N-Nat.AQ12-L_C-SpyCatcher-H6 <400> 16 atgggcgcaa tggttgatac cctgagcggt ctgagcagcg aacagggtca gagcggtgat 60 atgaccattg aagaagatag cgcaacccac atcaaattca gcaaacgtga tgaagatggt 120 aaagaactgg caggcgcaac catgaaactg cgtgatagca gcggtaaaac cattagcacc 180 tggattagtg atggtcaggt gaaagatttt tatctgtacc ctggcaaata cacctttgtt 240 gaaaccgcag caccggatgg ttatgaagtt gcaaccgcaa ttacctttac cgttaatgaa 300 cagggccagg ttaccgtgaa tggtaaagca accaaaggtg atgcacatat tagtgctagc 360 gcaagcgcat cagccggtgc aagtgctgca gcaagtgccg gtgctggtgc gggtgcaggt 420 cctggtcagc agggtccggg tcaacaagga cctggacagc aaggaccgta tggtccaggt 480 gcatcagctg cagccgcagc agcgggtggt tatggtccgg gaagcggtca gcaaggccct 540 tcacaacagg gaccaggcca acagggtcct ggcggtcaag gtccttatgg acctggtgct 600 tctgctgcgg cagcggctgc cggtggctat ggccctggta gtggccagca agggcctggt 660 ggccagggtc catatggccc aggttctagt gccgcagctg ctgctgcagg cggtaatgga 720 ccgggttcag gacaacaagg tgcagggcag caaggtcccg gacaacaggg tccaggtggt 780 agtgcagcag cggcagcagc tggcggatat ggaccaggta gtgggcaaca aggcccaggt 840 caacaagggc cagggggtca aggcccatac ggtccgggtg cttccgcagc cgcagctgca 900 gcaggcggtt acggtcctgg tagtggtcaa ggtccaggcc agcaaggacc aggtggacaa 960 gggccttacg gaccaggcgc atctgcggca gcagcagccg cagggggata tggtcctggt 1020 tcagggcagc agggaccagg tcagcaaggt ccaggtcagc agggacctgg gggtcaggga 1080 ccttacggtc ctggcgcaag tgcagctgca gcggcagcgg gtggctacgg accgggttat 1140 ggccagcagg gaccgggaca gcagggaccc ggtggacagg gtccgtatgg accgggtgca 1200 agtgcagcat cagcagcaag tggtggttac ggacctggct caggacagca aggccctggc 1260 caacaaggcc ctggcggaca gggaccctat gggccaggtg ccagcgctgc agcagccgca 1320 gccggtggat acggtccagg ctctggtcaa caaggtcctg ggcaacaagg tcctggccag 1380 cagggtccag gacagcaagg gcctggcggt caaggaccgt acggaccggg tgccagcgca 1440 gcgggctcag cggcaggcgg ttatggtcca ggatcaggcc agcaaggtcc gggtcagcaa 1500 ggcccagggc agcaaggacc gggtcaacag ggaccgggtc agcagggtcc tgggcaacag 1560 ggtccgggac aacagggacc aggtcaacaa ggaccgggtc aacaaggtcc aggtggtcag 1620 ggtgcatatg gtcctggcgc ttcagcagca gcaggggctg caggggtta tggcccaggt 1680 agcggtcagc agggacccgg acaacaaggc cctggacaac agggtcccgg tcagcaaggg 1740 ccaggccaac aaggtccagg acaacaagga ccagggcagc agggtccagg ccaacaaggc 1800 ccttatggtc cgggtgccag tgctgcggca gcggcagctg ggggttatgg tccaggctct 1860 ggacagcagg gacctggcca gcaaggacct gggcagcaag ggccaggcgg tcaggcaagt 1920 gcatcagcaa gcgcagcagc gagtgcagca agcaccgttg cgaattctag ctccgcaatg 1980 gttgataccc tgagcggtct gagcagcgaa cagggtcaga gcggtgatat gaccattgaa 2040 gaagatagcg caacccacat caaattcagc aaacgtgatg aagatggtaa agaactggca 2100 ggcgcaacca tgaaactgcg tgatagcagc ggtaaaacca ttagcacctg gattagtgat 2160 ggtcaggtga aagattttta tctgtaccct ggcaaataca cctttgttga aaccgcagca 2220 ccggatggtt atgaagttgc aaccgcaatt acctttaccg ttaatgaaca gggccaggtt 2280 accgtgaatg gtaaagcaac caaaggtgat gcacatattc tcgagcacca ccaccaccac 2340 cactga 2346 <210> 17 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Linker N <400> 17 Ser Ala Ser Ala Ser Ala Ser Ala Gly Ala Ser Ala Ala Ala Ser Ala 1 5 10 15 Gly Ala Gly Ala Gly Ala 20 <210> 18 <211> 100 <212> PRT <213> Homo sapiens <220> <223> The tenth type III module of fibronectin <400> 18 Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg 65 70 75 80 Gly Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95 Glu Ile Pro Ser 100 <210> 19 <211> 744 <212> PRT <213> Artificial sequence <220> <223> FB10-L_N-ADF3-L_N-FB10-H6 fusion protein <400> 19 Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg 65 70 75 80 Gly Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95 Glu Ile Pro Ser Ala Ser Ala Ser Ala Ser Ala Gly Ala Ser Ala Ala 100 105 110 Ala Ser Ala Gly Ala Gly Ala Gly Ala Gly Pro Gly Gln Gln Gly Pro 115 120 125 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser 130 135 140 Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln 145 150 155 160 Gly Pro Ser Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly 165 170 175 Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr 180 185 190 Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 195 200 205 Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Gly Gly Asn Gly Pro Gly 210 215 220 Ser Gly Gln Gln Gly Ala Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 225 230 235 240 Gly Gly Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser 245 250 255 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 260 265 270 Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro 275 280 285 Gly Ser Gly Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 290 295 300 Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly 305 310 315 320 Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln 325 330 335 Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala 340 345 350 Ala Ala Ala Gly Gly Tyr Gly Pro Gly Tyr Gly Gln Gln Gly Pro Gly 355 360 365 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala 370 375 380 Ala Ser Ala Ala Ser Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly 385 390 395 400 Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala 405 410 415 Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln 420 425 430 Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln 435 440 445 Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala 450 455 460 Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly 465 470 475 480 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln 485 490 495 Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln 500 505 510 Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Ala Tyr Gly Pro Gly 515 520 525 Ala Ser Ala Ala Ala Gly Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly 530 535 540 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln 545 550 555 560 Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln 565 570 575 Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala 580 585 590 Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly 595 600 605 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Ala Ser Ala Ser 610 615 620 Ala Ser Ala Ala Ala Ser Ala Ala Ser Thr Val Ala Asn Ser Ser Ser 625 630 635 640 Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 645 650 655 Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr 660 665 670 Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 675 680 685 Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro 690 695 700 Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly Asp 705 710 715 720 Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile 725 730 735 Leu Glu His His His His His His 740 <210> 20 <211> 175 <212> PRT <213> Homo sapiens <220> <223> γ-Crystallin D <400> 20 Met Gly Lys Ile Thr Leu Tyr Glu Asp Arg Gly Phe Gln Gly Arg His 1 5 10 15 Tyr Glu Cys Ser Ser Asp His Pro Asn Leu Gln Pro Tyr Leu Ser Arg 20 25 30 Cys Asn Ser Ala Arg Val Asp Ser Gly Cys Trp Met Leu Tyr Glu Gln 35 40 45 Pro Asn Tyr Ser Gly Leu Gln Tyr Phe Leu Arg Arg Gly Asp Tyr Ala 50 55 60 Asp His Gln Gln Trp Met Gly Leu Ser Asp Ser Val Arg Ser Cys Arg 65 70 75 80 Leu Ile Pro His Ser Gly Ser His Arg Ile Arg Leu Tyr Glu Arg Glu 85 90 95 Asp Tyr Arg Gly Gln Met Ile Glu Phe Thr Glu Asp Cys Ser Cys Leu 100 105 110 Gln Asp Arg Phe Arg Phe Asn Glu Ile His Ser Leu Asn Val Leu Glu 115 120 125 Gly Ser Trp Val Leu Tyr Glu Leu Ser Asn Tyr Arg Gly Arg Gln Tyr 130 135 140 Leu Leu Met Pro Gly Asp Tyr Arg Arg Tyr Gln Asp Trp Gly Ala Thr 145 150 155 160 Asn Ala Arg Val Gly Ser Leu Arg Arg Val Ile Asp Phe Ser Pro 165 170 175 <210> 21 <211> 897 <212> PRT <213> Artificial sequence <220> <223> CRYS-L_N-ADF3-L_c-CRYS-H6 fusion protein <400> 21 Met Gly Lys Ile Thr Leu Tyr Glu Asp Arg Gly Phe Gln Gly Arg His 1 5 10 15 Tyr Glu Cys Ser Ser Asp His Pro Asn Leu Gln Pro Tyr Leu Ser Arg 20 25 30 Cys Asn Ser Ala Arg Val Asp Ser Gly Cys Trp Met Leu Tyr Glu Gln 35 40 45 Pro Asn Tyr Ser Gly Leu Gln Tyr Phe Leu Arg Arg Gly Asp Tyr Ala 50 55 60 Asp His Gln Gln Trp Met Gly Leu Ser Asp Ser Val Arg Ser Cys Arg 65 70 75 80 Leu Ile Pro His Ser Gly Ser His Arg Ile Arg Leu Tyr Glu Arg Glu 85 90 95 Asp Tyr Arg Gly Gln Met Ile Glu Phe Thr Glu Asp Cys Ser Cys Leu 100 105 110 Gln Asp Arg Phe Arg Phe Asn Glu Ile His Ser Leu Asn Val Leu Glu 115 120 125 Gly Ser Trp Val Leu Tyr Glu Leu Ser Asn Tyr Arg Gly Arg Gln Tyr 130 135 140 Leu Leu Met Pro Gly Asp Tyr Arg Arg Tyr Gln Asp Trp Gly Ala Thr 145 150 155 160 Asn Ala Arg Val Gly Ser Leu Arg Arg Val Ile Asp Phe Ser Pro Ser 165 170 175 Ala Ser Ala Ser Ala Ser Ala Gly Ala Ser Ala Ala Ala Ser Ala Gly 180 185 190 Ala Gly Ala Gly Ala Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly 195 200 205 Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala 210 215 220 Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Ser Gln 225 230 235 240 Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro 245 250 255 Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser 260 265 270 Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ser Ser 275 280 285 Ala Ala Ala Ala Ala Ala Gly Gly Asn Gly Pro Gly Ser Gly Gln Gln 290 295 300 Gly Ala Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Ser Ala 305 310 315 320 Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly 325 330 335 Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala 340 345 350 Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln 355 360 365 Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly 370 375 380 Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly 385 390 395 400 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly 405 410 415 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly 420 425 430 Gly Tyr Gly Pro Gly Tyr Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 435 440 445 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ser Ala Ala 450 455 460 Ser Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln 465 470 475 480 Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala 485 490 495 Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly 500 505 510 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly 515 520 525 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly 530 535 540 Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 545 550 555 560 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 565 570 575 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln 580 585 590 Gln Gly Pro Gly Gly Gln Gly Ala Tyr Gly Pro Gly Ala Ser Ala Ala 595 600 605 Ala Gly Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro 610 615 620 Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly 625 630 635 640 Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln 645 650 655 Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly 660 665 670 Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro 675 680 685 Gly Gln Gln Gly Pro Gly Gly Gln Ala Ser Ala Ser Ala Ser Ala Ala 690 695 700 Ala Ser Ala Ala Ser Thr Val Ala Asn Ser Ser Ser Gly Lys Ile Thr 705 710 715 720 Leu Tyr Glu Asp Arg Gly Phe Gln Gly Arg His Tyr Glu Cys Ser Ser 725 730 735 Asp His Pro Asn Leu Gln Pro Tyr Leu Ser Arg Cys Asn Ser Ala Arg 740 745 750 Val Asp Ser Gly Cys Trp Met Leu Tyr Glu Gln Pro Asn Tyr Ser Gly 755 760 765 Leu Gln Tyr Phe Leu Arg Arg Gly Asp Tyr Ala Asp His Gln Gln Trp 770 775 780 Met Gly Leu Ser Asp Ser Val Arg Ser Cys Arg Leu Ile Pro His Ser 785 790 795 800 Gly Ser His Arg Ile Arg Leu Tyr Glu Arg Glu Asp Tyr Arg Gly Gln 805 810 815 Met Ile Glu Phe Thr Glu Asp Cys Ser Cys Leu Gln Asp Arg Phe Arg 820 825 830 Phe Asn Glu Ile His Ser Leu Asn Val Leu Glu Gly Ser Trp Val Leu 835 840 845 Tyr Glu Leu Ser Asn Tyr Arg Gly Arg Gln Tyr Leu Leu Met Pro Gly 850 855 860 Asp Tyr Arg Arg Tyr Gln Asp Trp Gly Ala Thr Asn Ala Arg Val Gly 865 870 875 880 Ser Leu Arg Arg Val Ile Asp Phe Ser Leu Glu His His His His His 885 890 895 His <210> 22 <211> 2228 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding the fusion protein FB10-L_N-ADF3-L_N-FB10-H6 <400> 22 tgttagtgat gttccgcgtg atctggaagt tgttgcagca accccgacca gcctgctgat 60 tagctgggat gcaccggcag ttaccgttcg ttattatcgt attacctatg gtgaaaccgg 120 tggtaatagt ccggttcaag aatttaccgt tccgggtagc aaaagcaccg caaccattag 180 cggtctgaaa ccgggtgttg attacaccat taccgtttat gccgttaccg gtcgtggtga 240 ttcaccggca agcagcaaac cgattagcat taactatcgt accgaaattc cgagcgctag 300 cgcaagcgca agtgccggtg caagcgcagc agcatcagcc ggtgctggtg caggcgccgg 360 cccgggtcag caaggcccag gacagcaagg cccgggtcag cagggaccgt acggccctgg 420 tgcctcggcc gcagcagctg cagcaggagg atatggacct ggatctggtc agcagggccc 480 gtcgcagcag ggcccgggtc aacaaggccc aggcggccag ggcccgtatg gaccgggggc 540 gagtgccgct gccgctgccg ctggcggtta tgggcctggg tcaggtcagc aaggtcctgg 600 gggtcaggga ccatatgggc ctgggagttc ggccgcagcc gctgcggcgg gcggtaatgg 660 gcctggctcc gggcaacaag gcgctggtca acaaggacct gggcagcagg ggccgggcgg 720 tagcgctgcg gcagcagctg caggtgggta cggtccagga tcgggacaac aaggaccagg 780 tcaacagggg cctggcgggc aaggacctta tgggccggga gcctccgcgg cggcggctgc 840 agctggtggt tacggaccgg ggtcaggcca aggaccgggc cagcagggac cgggggggca 900 gggtccatac ggcccagggg cttcagcagc tgcagcagcg gcgggcggct atggtcctgg 960 ttcgggccag caagggccag ggcaacaagg tccagggcaa caagggcctg gcgggcaagg 1020 accttacggc ccaggagcgt cagcggcggc tgcggctgcc gggggctacg gcccgggcta 1080 tggtcagcag gggccgggtc agcagggacc gggtggtcag gggccatatg gtccaggggc 1140 ctctgccgca agcgcggcga gtgggggcta tgggccgggc tcgggacagc agggccctgg 1200 ccagcagggg ccagggggcc aaggaccgta tggaccaggc gcatcggcgg cagccgccgc 1260 ggcgggagga tatggtcctg ggagcggtca gcagggaccg ggccagcaag gacctggtca 1320 gcaagggcca ggtcaacaag ggccgggtgg gcaagggcca tacgggccag gagcctcagc 1380 agcggctgcc gcggctggtg ggtacggtcc gggttcgggc cagcaaggac ctggacaaca 1440 gggtccaggt cagcaaggcc caggccaaca gggtccgggg cagcaagggc caggtcagca 1500 aggtcctggt caacaaggtc cagggcagca gggaccgggc cagcaagggc caggcggtca 1560 aggtgcgtat ggaccggggg catctgccgc tgcaggggcc gccggtggtt atggcccggg 1620 ctccgggcag caagggcctg ggcaacaggg gcctggccaa caaggcccag gacagcaagg 1680 tccaggccaa caggggcctg ggcaacaagg gcctggccag caagggcctg gccaacaagg 1740 tccgtatggt cctggcgcaa gcgcggcagc ggcggcagcc ggtggatacg gcccaggaag 1800 cgggcaacaa ggtcctgggc agcaggggcc tggtcaacaa ggaccgggcg gtcaggccag 1860 cgcaagcgca agtgcagcag caagcgcagc cagcaccgtt gcgaattctt ctagcgttag 1920 tgatgttccg cgtgatctgg aagttgttgc agcaaccccg accagcctgc tgattagctg 1980 ggatgcaccg gcagttaccg ttcgttatta tcgtattacc tatggtgaaa ccggtggtaa 2040 tagtccggtt caagaattta ccgttccggg tagcaaaagc accgcaacca ttagcggtct 2100 gaaaccgggt gttgattaca ccattaccgt ttatgccgtt accggtcgtg gtgattcacc 2160 ggcaagcagc aaaccgatta gcattaacta tcgtaccgaa attctcgagc accaccacca 2220 ccaccact 2228 <210> 23 <211> 2689 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence encoding the fusion protein CRYS-L_N-ADF3-L_c-CRYS-H6 <400> 23 tgggtaaaat taccctgtat gaagatcgtg gttttcaggg tcgtcattat gaatgtagca 60 gcgatcatcc gaatctgcag ccgtatctga gccgttgtaa tagcgcacgt gttgatagcg 120 gttgttggat gctgtatgaa cagccgaatt atagtggtct gcagtatttt ctgcgtcgcg 180 gtgattatgc agatcatcag cagtggatgg gtctgagcga tagcgttcgt agctgtcgtc 240 tgattccgca tagcggtagc catcgtattc gtctgtatga gcgtgaagat tatcgtggtc 300 agatgattga atttacggaa gattgtagct gtctgcagga tcgttttcgc tttaatgaaa 360 ttcatagcct gaatgtgctg gaaggtagct gggttctgta cgaactgagc aattatcgcg 420 gtcgtcagta tctgctgatg cctggtgatt atcgtcgtta tcaggattgg ggtgcaacca 480 atgcgcgtgt tggtagcctg cgtcgtgtta ttgattttag cccgagcgct agcgcaagcg 540 catcagccgg tgcaagtgct gcagcaagtg ccggtgctgg tgcgggtgca ggtcctggtc 600 agcagggtcc gggtcaacaa ggacctggac agcaaggacc gtatggtcca ggtgcatcag 660 ctgcagccgc agcagcgggt ggttatggtc cgggaagcgg tcagcaaggc ccttcacaac 720 agggaccagg ccaacagggt cctggcggtc aaggtcctta tggacctggt gcttctgctg 780 cggcagcggc tgccggtggc tatggccctg gtagtggcca gcaagggcct ggtggccagg 840 gtccatatgg cccaggttct agtgccgcag ctgctgctgc aggcggtaat ggaccgggtt 900 caggacaaca aggtgcaggg cagcaaggtc ccggacaaca gggtccaggt ggtagtgcag 960 cagcggcagc agctggcgga tatggaccag gtagtgggca acaaggccca ggtcaacaag 1020 ggccaggggg tcaaggccca tacggtccgg gtgcttccgc agccgcagct gcagcaggcg 1080 gttacggtcc tggtagtggt caaggtccag gccagcaagg accaggtgga caagggcctt 1140 acggaccagg cgcatctgcg gcagcagcag ccgcaggggg atatggtcct ggttcagggc 1200 agcagggacc aggtcagcaa ggtccaggtc agcagggacc tgggggtcag ggaccttacg 1260 gtcctggcgc aagtgcagct gcagcggcag cgggtggcta cggaccgggt tatggccagc 1320 agggaccggg acagcaggga cccggtggac agggtccgta tggaccgggt gcaagtgcag 1380 catcagcagc aagtggtggt tacggacctg gctcaggaca gcaaggccct ggccaacaag 1440 gccctggcgg acagggaccc tatgggccag gtgccagcgc tgcagcagcc gcagccggtg 1500 gatacggtcc aggctctggt caacaaggtc ctgggcaaca aggtcctggc cagcagggtc 1560 caggacagca agggcctggc ggtcaaggac cgtacggacc gggtgccagc gcagcggctg 1620 cagcggcagg cggttatggt ccaggatcag gccagcaagg tccgggtcag caaggcccag 1680 ggcagcaagg accgggtcaa cagggaccgg gtcagcaggg tcctgggcaa cagggtccgg 1740 gacaacaggg accaggtcaa caaggaccgg gtcaacaagg tccaggtggt cagggtgcat 1800 atggtcctgg cgcttcagca gcagcagggg ctgcagggg ttatggccca ggtagcggtc 1860 agcagggacc cggacaacaa ggccctggac aacagggtcc cggtcagcaa gggccaggcc 1920 aacaaggtcc aggacaacaa ggaccagggc agcagggtcc aggccaacaa ggccccttatg 1980 gtccgggtgc cagtgctgcg gcagcggcag ctggggtta tggtccaggc tctggacagc 2040 agggacctgg ccagcaagga cctgggcagc aagggccagg cggtcaggca agtgcatcag 2100 caagcgcagc agcgagtgca gcaagcaccg ttgcgaattc tagctccggt aaaattaccc 2160 tgtatgaga tcgtggttt cagggtcgtc attatgaatg tagcagcgat catccgaatc 2220 tgcagccgta tctgagccgt tgtaatagcg cacgtgttga tagcggttgt tggatgctgt 2280 atgaacagcc gaattatagt ggtctgcagt attttctgcg tcgcggtgat tatgcagatc 2340 atcagcagtg gatgggtctg agcgatagcg ttcgtagctg tcgtctgatt ccgcatagcg 2400 gtagccatcg tattcgtctg tatgagcgtg aagattatcg tggtcagatg attgaattta 2460 cggaagattg tagctgtctg caggatcgtt ttcgctttaa tgaaattcat agcctgaatg 2520 tgctggaagg tagctgggtt ctgtacgaac tgagcaatta tcgcggtcgt cagtatctgc 2580 tgatgcctgg tgattatcgt cgttatcagg attggggtgc aaccaatgcg cgtgttggta 2640 gcctgcgtcg tgttattgat tttagcctcg agcaccacca ccaccacca 2689 BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The filamentous protein phase separates into concentrated phases. The darker liquid at the bottom is the filamentous protein phase formed by concentration-induced phase separation.

[0032] Figure 2 .Samples collected from the phase-separated protein concentrate showed droplets of concentrated silk protein (optical microscopy).

[0033] Figure 3 .Cryo-electronmicrograph of phase-separated dense protein concentrate droplets.

[0034] Figure 4 .Fibers are pulled from phase separated dense protein concentrate droplets.

[0035] Figure 5 .Scanning electron micrograph of filaments pulled out and dried from phase-separated dense protein droplets.

[0036] Figure 6 .When the filamentous protein concentrate is deformed under tension, it forms a fibrous assembly.

[0037] Figure 7 . Schematic representation of plasmids (A) pPMA51, (B) pSA42, (C) pSA60, (D) pSA16 (E) pEEEt20, and (F) pSAEt210 containing the filamentous sequence.

[0038] Figure 8 .Samples collected from the phase-separated protein concentrate showed droplets of concentrated silk protein (optical microscopy).

[0039] Fig. 9 .Scanning electron micrograph of filaments pulled out and dried from phase-separated dense protein droplets.

[0040] Fig.10 .Filamentous protein concentrates form fibrous aggregates when deformed under tension.

[0041] Fig.11 .Scanning electron micrograph of molecular arrangement in a fibrous aggregate formed by filamentous molecules.

[0042] Fig.12 . Three-dimensional structure of the terminal domain. Left: Structure of the familial tricellulose binding module (CBM) from Clostridium thermocellum (PDB: 1NBC). Right: Structure of the SpyCatcher domain from Streptococcus pyogenes (PDB: 4MLI).

[0043] Fig.13 .Adhesion of different types of materials using protein concentrates.

[0044] Fig.14 . Lap-shear strength of protein concentrate adhesives for different types of materials.

[0045] Fig.15 The strength of this joint made with the protein concentrate adhesive was demonstrated by hanging a 4.5 kg weight from it.

[0046] Implementation Description

[0047] The present invention relates to forming a protein concentrate from a solution of filamentous protein after production of the filamentous protein in a recombinant production host. The concentrate is formed by concentrating the solution of the filamentous protein without requiring a purification step such as protein precipitation or chromatographic purification. The concentrate is characterized by a detectable and separate liquid phase that is different from the aqueous phase from which the concentrate is formed.

[0048] Therefore, the present invention provides a method for producing a concentrated phase of a silk fusion protein, the method comprising the following steps:

[0049] a) preparing a solution of a silk fusion protein in an aqueous medium, wherein the fusion protein is isolated from a recombinant production host and comprises a silk protein sequence and two separate non-silk terminal module sequences flanking the silk protein sequence;

[0050] b) concentrating the fusion protein in the aqueous medium until liquid phase separation occurs;

[0051] c) collecting the protein-rich phase of the aqueous medium obtained in step b)

[0052] d) repeating steps b) and c) with the protein-rich phase obtained in step c) until a protein concentration of about 20-45% w / v is reached;

[0053] e) optionally separating aggregates of the fusion protein from the soluble fusion protein obtained in step d) and removing the aggregates from the solution;

[0054] f) concentrating the solution obtained in step d) or e) to a final protein concentration of about 60-80% w / v,

[0055] Steps a) to f) are performed so that the silk fusion protein is not precipitated and then dissolved in the aqueous medium.

[0056] The expression "non-silk terminal module sequence" refers herein to two protein domains flanking the filamentous protein sequence, the two domains having identical or nearly identical (i.e., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or sequence identity) amino acid sequences. In an alternative embodiment, the non-silk terminal module sequences flanking the filamentous protein sequence have similar three-dimensional structures. The non-silk terminal module sequences do not exist in combination with the filamentous protein sequence in nature. Suitable terminal module sequences for the silk fusion protein of the present invention preferably have the following: Fig.12 The three-dimensional structure. The three-dimensional structure comprises continuous β chains forming β-folds, preferably at least four continuous β chains. The amino acid sequence length of the terminal module is preferably in the range of 90-250 amino acids, more preferably in the range of 98-239 amino acids (see Table 1). Preferably, the protein domain is selected from the following group: a carbohydrate binding module (CBM), such as a cellulose binding module from Clostridium thermocellum, which comprises the sequence of SEQ ID NO: 7. We also disclose a terminal module sequence based on the SpyCatcher domain engineered from the fibronectin binding protein FbaB of Streptococcus pyogenes, which comprises the sequence of SEQ ID NO: 14, γ-crystallin D from Homo sapiens, which comprises the sequence of SEQ ID NO: 20, and the tenth type III module of fibronectin from Homo sapiens, which comprises the sequence of SEQ ID NO: 18. For other preferred embodiments, suitable terminal module peptides for silk fusion proteins are listed in Table 1. In a preferred embodiment, a non-silk terminal module sequence can be paired with another non-silk terminal module sequence having a corresponding three-dimensional structure. For example, when one of the flanking terminal module sequences in the silk fusion protein is a CBM, the other can be a SpyCatcher domain.

[0057] The term "aqueous medium" herein refers to a liquid medium, which may preferably be distilled water or a buffer consisting of a mixture of water and a weak acid and its conjugate base.

[0058] The term "filamentous protein sequence" herein refers to an amino acid sequence comprising a repeating sequence capable of forming a fibrillar structure or a silk fiber. Preferably, the filamentous protein sequence is a spider silk protein sequence from a spider of the order Araneae or a variant thereof.

[0059] As used herein, the term "variant" refers to an amino acid or nucleic acid sequence with a high degree of homology to a parent sequence. Preferably, the variant has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology (i.e., sequence identity) sequence with the parent sequence. Two examples of algorithms suitable for determining percentage sequence identity and sequence similarity are BLASTp and BLASTn 2.0 algorithms, which are described in Altschul et al. (1997) Nucleic Acids Res 25 (17): 3389-3402.

[0060] Preferably, the above method further comprises the following steps:

[0061] g) preparing silk fusion protein filaments from the concentrate obtained in step f). More preferably, in step g), the filaments are prepared by pulling the filaments out of the concentrate using shear force. Figure 4 An example is shown in .

[0062] In a preferred embodiment, the method comprises a further initial step of isolating the recombinant fusion protein from a host cell expressing the fusion protein. In certain embodiments, the present invention requires the production of recombinant filamentous proteins (e.g., SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 15; SEQ ID NO: 19; SEQ ID NO: 21) in a form in which both the amino and carboxyl termini of the silk protein have additional non-silk terminal module sequences ( Figure 7). As discussed above, these non-filament terminal module sequences can be, for example, a cellulose binding module (CBM) (e.g., SEQ ID NO: 7), a SpyCatcher domain engineered from the fibronectin binding protein FbaB of Streptococcus pyogenes (e.g., SEQ ID NO: 14), γ-crystallin D from Homo sapiens (which comprises the sequence of SEQ ID NO: 20), and the tenth type III module of fibronectin from Homo sapiens (which comprises the sequence of SEQ ID NO: 18), variants thereof, or mixed pairs thereof (for other options, see Table 1). Additional protein sequences can also be incorporated between the non-filament terminal module sequence and the filament sequence to serve as an amino acid sequence of the linker (SEQ ID NO: 5; SEQ ID NO: 6). The filamentous portion can be a repeat of the A (SEQ ID NO: 1) and Q (SEQ ID NO: 2) sequences, or can be a sequence found in nature, such as SEQ ID NO: 3 or SEQ ID NO: 4. Preferably, the spider silk protein repeat sequence comprises 10-50 silk polymer repeats, wherein the repeat sequence consists of a repeat A sequence of SEQ ID NO: 1 or a variant thereof combined with a repeat Q sequence of SEQ ID NO: 2 or a variant thereof. More preferably, the spider silk protein repeat sequence is (AQ) 12. In another preferred embodiment, the spider silk protein repeat sequence is found in ADF3 from the spider Araneus cruciferus comprising SEQ ID NO: 3; the black widow spider AcSp1 sequence of SEQ ID NO: 4 or a variant thereof. The linker may preferably be selected from the group consisting of a C-terminal linker of SEQ ID NO: 6 and an N-terminal linker of SEQ ID NO: 5.

[0063] In a preferred embodiment, the concentration steps b) and f) of the method are performed by centrifugal force, dialysis such as ultrafiltration techniques, and / or evaporation. In the following examples, centrifugal force and evaporation are used at different stages of the method.

[0064] The present invention also provides a silky filament produced by the method of the present invention. Preferably, the silky filament is composed of a spider silk fusion protein as defined above. More preferably, the silky filament is composed of a spider silk fusion protein comprising 12 silk polymer repeats and two cellulose binding modules (CBM) from Clostridium thermocellum flanking the silk polymer repeats, wherein the repeat sequence consists of a repeated A sequence of SEQ ID NO: 1 combined with a repeated Q sequence of SEQ ID NO: 2, the cellulose binding module consists of a sequence of SEQ ID NO: 7, and each module is connected to the repeat via a C-terminal linker of SEQ ID NO: 6 or an N-terminal linker of SEQ ID NO: 5. In an alternative embodiment, the silk filaments are composed of the spider silk protein repeat sequence ADF3 of SEQ ID NO: 3, or the black widow spider AcSp1 sequence of SEQ ID NO: 4; and two cellulose binding modules (CBMs) from Clostridium thermocellum located on the sides of the silk protein repeats, the cellulose binding modules consisting of the sequence of SEQ ID NO: 7, and each module is connected to the repeat sequence ADF3 or the black widow spider AcSp1 sequence via a C-terminal linker of SEQ ID NO: 6 or an N-terminal linker of SEQ ID NO: 5.

[0065] The present invention also provides a concentrate of spider silk fusion proteins produced by steps a) to f) of the method defined above and in the claims.

[0066] The present invention further relates to a recombinant spider silk fusion protein comprising a silk polymer repeat and two cellulose binding modules (CBMs) from Clostridium thermocellum flanking the silk polymer repeat, wherein the repeat sequence consists of a repeating A sequence of SEQ ID NO: 1 combined with a repeating Q sequence of SEQ ID NO: 2, and the cellulose binding modules consist of a sequence of SEQ ID NO: 7, each module being linked to the repeat by a C-terminal linker of SEQ ID NO: 6 or an N-terminal linker of SEQ ID NO: 5. Preferably, the silk polymer repeat sequence is (AQ) 12. More preferably, the recombinant spider silk fusion protein comprises the sequence of SEQ ID NO: 8.

[0067] In an alternative embodiment, the recombinant spider silk fusion protein comprises the spider silk protein repeat sequence ADF3 of SEQ ID NO: 3, or the black widow spider AcSp1 sequence of SEQ ID NO: 4; and two cellulose binding modules (CBMs) from Clostridium thermocellum located on the sides of the silk protein repeats, the cellulose binding modules consisting of the sequence of SEQ ID NO: 7, and each module is connected to the repeat sequence ADF3 or the black widow spider AcSp1 sequence via a C-terminal linker of SEQ ID NO: 6 or an N-terminal linker of SEQ ID NO: 5.

[0068] In other alternative embodiments, the recombinant spider silk fusion protein comprises the spider silk protein repeat sequence ADF3 of SEQ ID NO: 3, and two SpyCatcher domains engineered from the fibronectin binding protein FbaB of Streptococcus pyogenes located on the flanks of the silk protein repeat sequence, wherein the SpyCatcher domain consists of the sequence of SEQ ID NO: 14, and each domain is connected to the repeat sequence ADF3 via a C-terminal linker of SEQ ID NO: 6 or an N-terminal linker of SEQ ID NO: 17.

[0069] In other alternative embodiments, the recombinant spider silk fusion protein comprises the spider silk protein repeat sequence ADF3 of SEQ ID NO: 3, and two tenth type III modules of fibronectin from Homo sapiens located on the sides of the silk protein repeat sequence, the tenth type III module of fibronectin consists of the sequence of SEQ ID NO: 18, and each domain is connected to the repeat sequence ADF3 via a C-terminal linker of SEQ ID NO: 6 or an N-terminal linker of SEQ ID NO: 17.

[0070] In other alternative embodiments, the recombinant spider silk fusion protein comprises the spider silk protein repeat sequence ADF3 of SEQ ID NO: 3, and two γ-crystallin D domains from Homo sapiens located on the sides of the silk protein repeat sequence, wherein the γ-crystallin D domain consists of the sequence of SEQ ID NO: 20, and each domain is connected to the repeat sequence ADF3 via a C-terminal linker of SEQ ID NO: 6 or an N-terminal linker of SEQ ID NO: 17.

[0071] Recombinant nucleic acids expressing fusion proteins as defined above and host cells comprising said nucleic acids are within the scope of the invention (eg, SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 16; SEQ ID NO: 22; SEQ ID NO: 23).

[0072] As defined above as optional step e): Prior to the formation of the concentrate, the solution may be clarified from cell debris and particulate matter by filtration or by centrifugal sedimentation. This step may be further enhanced by heating the solution to above 70 degrees Celsius in advance. A significant advantage of the present invention is that precipitation of the filamentous protein is not performed. The lack of a precipitation step also makes it unnecessary to involve a step of dissolving the precipitate. The use of dissolving solutions may result in damage to the filamentous protein, and the present invention is therefore advantageous in avoiding the use of these. Omitting precipitation and subsequent dissolution of the filamentous protein also facilitates processing and expansion of the method. The lack of a chromatographic purification step reduces costs and makes the method easier to scale up. The formation of the concentrate occurs as a phase separation event, wherein the concentrate of the filamentous protein clearly forms a protein-rich fluid phase (see Figure 1 The concentrate can be described as a phase-separated protein solution, a dope, or it can also be described as a protein coacervate. By imaging under a microscope, it is apparent that sometimes the protein concentrate is visible as droplets in the solution ( Figure 2 and 3 ). A significant advantage of the present invention is that, apart from the clarification and concentration steps, no further processing steps are required to form a high concentration of protein in the concentrated phase. The concentration of protein in the concentrate can be further increased, for example, by evaporation. The protein concentrate has some significant advantageous properties. From such a concentrate it is possible to prepare fibers of filamentous protein. The fibers are produced by applying an extensional force to the concentrate. Figure 4 As demonstrated, stretching of the concentrate results in the formation of filamentous protein fibers. Direct stretching of the protein concentrate to form fibers is advantageous because the fibers can be formed without the need for a coagulation bath. The fibers can be formed with the surrounding medium being air, so extrusion as a liquid is not necessary. Another distinct advantage of the protein concentrate is that it can be applied to a variety of surfaces and can be used as a water-based adhesive.

[0073] The adhesive of the present invention can be used by being applied to a substrate preferably selected from the group consisting of plastics, glass, metal, wood, paper, textiles and tissue substrates. That is, it can be used to adhere or fix substrates. The mode of use follows the general mode of adhesive use, and a typical mode is coating.

[0074] Examples of medical or biological applications of the adhesives of the present invention include, but are not limited to: (1) plastic surgery, such as treatment of bones, ligaments, tendons, menisci and muscles, and implantation of artificial materials; (2) treatment of punctures, lacerations and cuts, and ophthalmic attachments, such as corneal implants and artificial corneal implants; (3) dental attachments, such as fixing retainers, bridges or crowns in place, fixing loose teeth, repairing broken teeth, and fixing fillings in place; (4) surgical treatments, such as vascular attachment, cell tissue attachment, artificial material implants and wound closure; (5) plant attachment, such as bonding of transplanted parts and wound healing.

[0075] Industrial Applicability

[0076] Filamentous proteins are often used to form materials such as coatings, fibers, filters, composite materials, automotive parts, medical materials (wound dressings, drug delivery materials, coatings, cell growth matrices). Advantages are their biocompatibility, biodegradability, the possibility of designing and modifying properties, their mechanical properties, water-based processing, strength, toughness and elasticity. The materials can also be used in textile and clothing applications.

[0077] Adhesives have a very wide range of applications in industry, such as in the manufacture of composite materials or in fixing components of a material together. In composite materials, the adhesive is bonded to the other components, and the adhesive may also comprise a small portion of the composition. In composite materials, the adhesive acts as a matrix that holds the components of the material together. Other applications of adhesives are fixing components of a device together.

[0078] Gluing applications include biomedical or pharmaceutical industries. Adhesives can be used to glue tissue, skin, dental applications, wounds, bones, implants, sensors.

[0079] Having now generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the invention.

[0080] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. Example

[0081] Embodiment 1:

[0082] Protein production. The plasmid containing the filamentous protein sequence (SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; or SEQ ID NO: 16) (see Figure 7 ) was transformed into E. coli and grown in Luria Bertani (LB) medium (or other bacterial growth medium) until an optical density of about 0.5 AU was reached at 600 nm. Protein production was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) and the cells were allowed to grow for 12 hours. When an automatic induction medium such as MagicMediaTM (Thermo Fisher Scientific) was used, IPTG was not added. The cells were broken by ultrasound. Alternatively, a homogenizer such as Avestin Emulsiflex-C3 was used. Similarly, other cell lysis techniques can be used. Cell debris was removed by centrifugation.

[0083] Preparation of protein solution The clarified supernatant is heated to, for example, 75 degrees Celsius to precipitate material from the supernatant while the filamentous protein remains in solution.

[0084] In a subsequent step, the solution containing the protein is desalted using a desalting chromatography column or using ultrafiltration. The solvent for the protein can be changed during the desalting process to, for example, water or a 5-50 mM Tris buffer containing 0-100 mM NaCl. The concentration of the filamentous protein is determined by its absorbance at 280 nm.

[0085] Formation of concentrated protein phase . The fusion protein solution from the previous step was concentrated using a centrifugal concentrator at 25°C. During the increase in protein concentration, phase separation occurred and a concentrated protein-rich phase was formed. The protein-rich phase was collected and further processed. Phase separation occurred once the total concentration of the protein solution reached approximately 1% w / v. The protein was then further concentrated in a second concentration step. The protein-rich, phase-separated solution collected from the previous step was further gradually concentrated to approximately 10-15% w / v using a centrifugal concentrator at 25 degrees Celsius and 845-1500 relative centrifugal force. In order to determine the protein concentration at these concentrations, more accurate dry weight measurements were performed.

[0086] Increased concentration can also be achieved by different types of ultrafiltration techniques, dialysis or evaporation.

[0087] A third concentration step using the protein-rich phase The silk protein at a concentration of 10-15% w / v is further concentrated by evaporation to reach a concentration of about 20-45% w / v.

[0088] During the concentration step, a washing step may be necessary: ​​to obtain a homogenous and non-flocculating solution, aggregates and short filaments (usually nanometers in diameter and several micrometers in length, which usually form a network), as well as gel particles (about a few micrometers in size) are removed from the solution (by centrifugation). Removal of aggregates facilitates subsequent fiber pulling or other processing.

[0089] Storage steps : 20-45% w / v is the storage concentration of the dope solution. After the third concentration step, the samples were quickly frozen using liquid nitrogen.

[0090] Embodiment 2:

[0091] Fiber from protein concentrate A drop of the protein solution prepared in Example 1 (about 30%, or 20-45%) is further concentrated to 60-80% by evaporation and placed between two tip-shaped objects (such as the tips of tweezers).

[0092] When the protein content is about 60-80% w / v, filaments can be drawn from the protein solution. Figure 4 shown.

[0093] Embodiment 3:

[0094] Making adhesives from protein concentrates A drop of the protein solution prepared in Example 1 (about 30% or 20-50%) is placed between two materials (e.g. standard paper) and allowed to dry for 5 minutes until the protein concentrate solidifies and the papers adhere to each other. Fig.13 See examples of protein concentrates used as binders in .

[0095] Table 1. Other suitable peptides for use as non-silk terminal module sequences in the silk fusion proteins of the present invention.

[0096]

[0097]

[0098] References

[0099] 1. Ebrahimi, D. et al. Silk Its Mysteries, How It Is Made, and How It Is Used. ACS Biomater. Sci. Eng. 1, 864 876 (2015).

[0100] 2. Nudelman, F. et al. Forming nacreous layer of the shells of the bivalves Atrina rigida and Pinctada margaritifera: An environmental - and cryo - scanning electron microscopy study. J. Struct. Biol. 162, 290 - 300 (2008).

[0101] 3. Rising, A. & Johansson, J. Toward spinning artificial spidersilk. Nat. Chem. Biol. 11, 309–315 (2015).

[0102] 4. Green, M. & Sambrook, J. Molecular cloning. A laboratory manual. Zool. Res. (Cold Spring Harbor Laboratory Press, 2012). doi:10.3724 / SP.J.1141.2012.01075

[0103] 5. US7951908B2.

[0104] 6. Teule, F. et al. Combining flagelliform and dragline spider silk motifs to produce tunable synthetic biopolymer fibers. Biopolymers 97, 418 - 431 (2012).

[0105] 7. Adrianos, S. L. et al. Nephila clavipes flagelliform silk - like GGX motifs contribute to extensibility and spacer motifs contribute to strength in synthetic spider silk fibers. Biomacromolecules 14, 1751 - 1760 (2013).

[0106] 8. Albertson, A.E., Teule, F., Weber, W., Yarger, J.L. & Lewis, R.V. Effects of different post-spin stretching conditions on the mechanical properties of synthetic spider silk fibers. J. Mech. Behav. Biomed. Mater. 29, 225–234 (2014).

[0107] 9. Xia, X.-X. et al. Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber. Proc. Natl. Acad. Sci. U.S.A. 107, 14059–14063 (2010).

[0108] 10. Gnesa, E. et al. Conserved C-terminal domain of spider tubuliform spidroin 1 contributes to extensibility in synthetic fibers. Biomacromolecules 13, 304–312 (2012).

[0109] 11. Lin, S. et al. Predictive modelling-based design and experiments for synthesis and spinning of bioinspired silk fibres. Nat. Commun. 6, 1–12 (2015).

[0110] 12. Heidebrecht, A. et al. Biomimetic Fibers Made of Recombinant Spidroins with the Same Toughness as Natural Spider Silk. Adv. Mater. 27, 2189 2194 (2015).

[0112] 13. Teulé, F. et al. A protocol for the production of recombinant spidersilk-like proteins for artificial fiber spinning. Nat. Protoc. 4, 341 55 (2009).

[0113] 14. Rammensee, S., Slotta, U., Scheibel, T. & Bausch, A. R. Assembly mechanism of recombinant spider silk proteins. Proc. Natl. Acad. Sci. U.S.A. 105, 6590–6595 (2008).

[0114] 15. Kinahan, M. E. et al. Tunable Silk: Using Microfluidics to Fabricate Silk Fibers with Controllable Properties. Biomacromolecules 12, 1504 1511 (2011).

[0115] 16. Wong Po Foo, C. et al. Novel nanocomposites from spider silk-silica fusion (chimeric) proteins. Proc. Natl. Acad. Sci. U.S.A. 103, 9428 33 (2006).

[0116] 17. Lang, G., Jokisch, S. & Scheibel, T. Air filter devices including nonwoven meshes of electrospun recombinant spider silk proteins. J. Vis. Exp. 4, e50492 (2013).

[0117] 18. Hedhammar, M. et al. Structural Properties of Recombinant Nonrepetitive and Repetitive Parts of Major Ampullate Spidroin 1 from Euprosthenops australis: Implications for Fiber Formation Biochemistry 47, 3407–3417 (2008).

[0118] 19. Tremblay, M.-L. et al. Spider wrapping silk fibre architecture arising from its modular soluble protein precursor. Sci. Rep. 5, 11502 (2015).

[0119] 20. US8173772B2.

Claims

1. Use of a silk fusion protein as an adhesive, wherein the silk fusion protein comprises a silk-like protein sequence and two separate non-silk terminal module sequences flanking the silk-like protein sequence, wherein the non-silk terminal module sequences flanking the silk-like protein sequence comprise a cellulose binding module (CBM), or a SpyCatcher (SpyC) domain, wherein the silk-like protein sequence is a spider silk protein repeat sequence (AQ) 12, wherein the repeat sequence consists of a repeated A sequence of SEQ ID NO: 1 combined with a repeated Q sequence consisting of SEQ ID NO: 2, or wherein the silk-like protein sequence is a black widow spider (Latrodectus hesperus) AcSp1 sequence consisting of a sequence of SEQ ID NO: 4, wherein the cellulose binding module (CBM) is from Clostridium thermocellum and consists of a sequence of SEQ ID NO: 7, and wherein the SpyCatcher domain is a Streptococcus pyogenes (Streptococcus pyogenes) 12. The invention relates to an engineered variant of the fibronectin binding protein FbaB from Pseudomonas pyogenes, consisting of the sequence of SEQ ID NO:

14.

2. The use according to claim 1, wherein the silk fusion protein is in a composition with one or more selected from the group consisting of a surfactant, an oxidizing agent, a cross-linking agent and a filler.

3. An adhesive comprising a silk fusion protein as an active ingredient, wherein the silk fusion protein comprises a silk protein sequence and two separate non-silk terminal module sequences flanking the silk protein sequence, wherein the non-silk terminal module sequences flanking the silk protein sequence comprise a cellulose binding module (CBM), or a SpyCatcher domain, wherein the silk protein sequence is a spider silk protein repeat sequence (AQ) 12, wherein the repeat sequence consists of a repeated A sequence of SEQ ID NO: 1 combined with a repeated Q sequence consisting of SEQ ID NO: 2, or wherein the silk protein sequence is the black widow spider (Latrodectus hesperus) AcSp1 sequence consisting of the sequence of SEQ ID NO: 4, wherein the cellulose binding module (CBM) is from Clostridium thermocellum and consists of the sequence of SEQ ID NO: 7, and wherein the SpyCatcher domain is an engineered variant of the fibronectin binding protein FbaB of Streptococcus pyogenes and consists of the sequence of SEQ ID NO:

14.

4. An adhesive according to claim 3, wherein the cellulose binding module (CBM) or the SpyCatcher domain flanking the filamentous protein sequence is connected to the filamentous protein sequence via a linker sequence, wherein the linker sequence is selected from the group consisting of a C-terminal linker consisting of SEQ ID NO: 6 and an N-terminal linker consisting of SEQ ID NO:

5.

5. The adhesive according to claim 3 or 4, wherein the silk fusion protein is in a composition with one or more selected from the group consisting of a surfactant, an oxidizing agent, a cross-linking agent and a filler.

6. A method for producing a concentrated phase of a silk fusion protein, the method comprising the following steps: a) preparing a solution of silk fusion protein in an aqueous medium; b) concentrating the fusion protein in the aqueous medium until liquid phase separation occurs; c) collecting the protein-rich phase of the aqueous medium obtained in step b); d) repeating steps b) and c) with the protein-rich phase obtained in step c) until a protein concentration of 20-45% w / v is reached; e) optionally separating aggregates of the fusion protein from the soluble fusion protein obtained in step d) and removing the aggregates from the solution; f) concentrating the solution obtained in step d) or e) to a final protein concentration of 60-80% w / v, wherein steps a) to f) are performed so that the silk fusion protein is not precipitated and then dissolved in the aqueous medium, and wherein the silk fusion protein comprises a silk protein sequence and two separate non-silk terminal module sequences flanking the silk protein sequence, wherein the non-silk terminal module sequences flanking the silk protein sequence comprise a cellulose binding module (CBM), or a SpyCatcher domain, wherein the silk protein is a spider silk protein repeat sequence (AQ) 12, wherein the repeat sequence consists of a repeated A sequence of SEQ ID NO: 1 combined with a repeated Q sequence consisting of SEQ ID NO: 2, or wherein the silk protein is a black widow spider AcSp1 sequence consisting of the sequence of SEQ ID NO: 4, wherein the cellulose binding module (CBM) is from Clostridium thermocellum and consists of the sequence of SEQ ID NO: 7, and wherein the SpyCatcher domain is an engineered variant of the fibronectin binding protein FbaB of Streptococcus pyogenes and consists of the sequence of SEQ ID NO:

14.

7. The method according to claim 6, wherein the method comprises the further steps of: g) preparing silk fusion protein fibers from the concentrate obtained in step f).

8. The method according to claim 6, wherein steps b) and f) are performed by centrifugal force and / or evaporation.

9. The method according to claim 6, wherein the aqueous medium is water or an aqueous buffer.

10. The method according to claim 6, wherein the cellulose binding module or the SpyCatcher domain flanking the filamentous protein sequence is connected to the filamentous protein sequence via a linker sequence, wherein the linker sequence is selected from the group consisting of a C-terminal linker consisting of SEQ ID NO: 6 and an N-terminal linker consisting of SEQ ID NO:

5.

11. The method according to claim 7, wherein in step g) the fibers are prepared by pulling the fibers from a concentrate using a pulling force.

12. A method according to claim 6, comprising the further initial step of isolating the recombinant fusion protein from a host cell expressing said fusion protein.

13. A recombinant spider silk fusion protein comprising a silk polymer repeat and two cellulose binding modules (CBMs) from Clostridium thermocellum, wherein the repeat sequence consists of a repeated A sequence of SEQ ID NO: 1 combined with a repeated Q sequence of SEQ ID NO: 2, and the cellulose binding module consists of a sequence of SEQ ID NO: 7, each module being connected to the repeat via a C-terminal linker of SEQ ID NO: 6 or an N-terminal linker of SEQ ID NO: 5, wherein the silk polymer repeat sequence is (AQ)12.

14. The recombinant spider silk fusion protein according to claim 13, which consists of SEQ ID NO:

8.

15. A recombinant spider silk fusion protein comprising the black widow spider AcSp1 sequence of SEQ ID NO: 4; and two cellulose binding modules (CBMs) from Clostridium thermocellum, wherein the cellulose binding modules consist of the sequence of SEQ ID NO: 7, and each module is connected to the black widow spider AcSp1 sequence via a C-terminal linker of SEQ ID NO: 6 or an N-terminal linker of SEQ ID NO: 5.

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