A new type of recombinant collagen and its preparation method and use

Abstract

The present invention relates to a new type of recombinant collagen, and a preparation method and use thereof. Specifically, by designing a collagen core peptide segment with spaced inverse repeats, and then utilizing the flexibility of the sequence itself or adding a suitable linker, a self-folding recombinant collagen capable of forming a triple helix or a quasi-triple helix inside a single chain is produced. The produced recombinant collagen has enhanced and improved biological activity, stability and expression level.

Description

[0001] This application claims priority to Chinese invention patent application CN202211654764.3 filed on December 22, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The invention belongs to the field of biomedical materials, and specifically relates to a new type of recombinant collagen and a preparation method and application thereof. Background Art

[0003] Collagen is the most abundant and widely distributed macromolecular functional protein in animals. It is the main component of the extracellular matrix and is widely present in the skin, bones, tendons and other parts. It supports and protects the body and organs. Extracting collagen from animal tissues is an important source of collagen, but with the continuous improvement of safety and ethical requirements, as well as the current surge in demand for collagen applications, the production of recombinant collagen through microbial fermentation has become an inevitable trend. Recombinant collagen has the advantages of good safety, strong processability, and stable quality. It has been widely used in the fields of medical beauty and cosmetics to exert its biological effects such as anti-aging, whitening, and moisturizing.

[0004] Hosts for microbial expression of recombinant collagen include Escherichia coli, yeast, Bacillus, etc. According to the naming guidelines issued by the National Medical Products Administration in 2021, the expression products of microbial systems are mainly recombinant humanized collagen or recombinant collagen-like proteins. Like natural collagen, no matter what kind of recombinant collagen, its core sequence is composed of GXY units, where X and Y can be any amino acids except cysteine.

[0005] The biological activity of collagen is determined by its GXY core sequence, protein high-level structure and physicochemical properties. When selecting or designing a recombinant collagen sequence, some active sites are usually added according to demand, such as the cell adhesion site GER, the integrin binding site RGD, the discoidin binding site GVMGFO (O is hydroxyproline), etc. The recombinant collagen obtained by existing microbial expression technology is mostly a single α peptide chain, with no or very little triple helix structure of natural collagen, which not only affects the stability of the recombinant collagen, but also may cause it to lack some special biological activities. Compared with natural collagen with large molecular weight, poor solubility and poor absorption, recombinant collagen can express collagen peptides of specific molecular weight, so it has significant application advantages in the field of cosmetics. In theory, the smaller the molecular weight of collagen, the better the solubility, the easier it is to be absorbed by the skin, and the better the biological activity.

[0006] However, small molecular weight recombinant collagen is difficult to express in microbial systems, and the overall trend is that the smaller the molecular weight, the lower the expression, resulting in higher corresponding production costs. The current common method is to directly repeat the small molecule collagen sequence several times in series, and then detect the expression level of these strains and the biological activity of the products to screen better recombinant strains. The expression products formed by this method of forward sequence repetition are basically single α peptide chain proteins, which are not very stable and are prone to degradation during the fermentation process. This is also one of the important reasons for the low expression level. Moreover, the reported sequence forward repetition multiples range very wide, some are repeated 2 times, and some are even repeated 100 times. High repetition multiples will inevitably produce recombinant products with large relative molecular weights, which is inconsistent with the goal of expressing small molecule recombinant proteins. Summary of the invention

[0007] The present invention provides a new recombinant collagen protein, which forms a triple helix or triple helix-like self-folding structure by designing a collagen core peptide segment with spaced inverse repeats and utilizing the flexibility of the sequence itself or adding a suitable linker, thereby enhancing and improving the biological activity (such as cell proliferation activity), stability and expression level of the recombinant collagen protein.

[0008] A recombinant collagen protein, comprising at least three peptide segments in its structure, wherein the peptide segments are peptide segment A and reverse peptide segment A, and the peptide segment A and reverse peptide segment A are arranged alternately, and the peptide segment A comprises the following structure: (GXY) n , the reverse A peptide segment contains the following structure: (YXG) n , wherein G is glycine, X and Y are any natural or non-natural imino group or amino acid except cysteine ​​in each repeating unit independently, and n is selected from an integer greater than or equal to 3. In some embodiments, X and Y in each repeating unit are each independently selected from proline, hydroxyproline, alanine, lysine, glutamic acid, aspartic acid, serine, leucine, asparagine, arginine, phenylalanine, glutamine, threonine, isoleucine, valine, histidine, tryptophan, tyrosine, methionine, and glycine.

[0009] A recombinant collagen protein, comprising at least three peptide segments in its structure, wherein the peptide segments are peptide segment A and reverse peptide segment A, and the peptide segment A and reverse peptide segment A are arranged alternately, and the peptide segment A comprises the following structure: (GXY) n -B-(GXY) m , the reverse A peptide segment contains the following: (YXG) m -Reverse B-(YXG) n, wherein G is glycine, X and Y are any natural or non-natural imino group or amino acid other than cysteine ​​independently in each repeating unit, B is any peptide of any length, reverse B is the reverse sequence of B, and n and m are independently selected from integers greater than or equal to 3. In some embodiments, X and Y in each repeating unit are independently selected from proline, hydroxyproline, alanine, lysine, glutamic acid, aspartic acid, serine, leucine, asparagine, arginine, phenylalanine, glutamine, threonine, isoleucine, valine, histidine, tryptophan, tyrosine, methionine, and glycine. In some embodiments, the recombinant collagen comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 A peptide segments and reverse A peptide segments. In some embodiments, B is a fragment containing a GXY unit, and X and Y are independently selected from glycine, proline, hydroxyproline, alanine, lysine, glutamic acid, aspartic acid, serine, leucine, asparagine, arginine, phenylalanine, glutamine, threonine, isoleucine, valine, histidine, tryptophan, tyrosine, methionine, and glycine. In the present invention, the recombinant collagen contains 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 A peptide segments and reverse A peptide segments, which refers to the total number of A peptide segments and reverse A peptide segments included. For example, containing 3 A peptide segments and reverse A peptide segments means that the number of A peptide segments and reverse A peptide segments contained in the collagen is 3 in total, and the specific connection method is A peptide segment-reverse A peptide segment-A peptide segment.

[0010] In some embodiments, the A peptide segment in the recombinant collagen contains an amino acid motif that forms a folded or turned structure, and the motif is selected from GPNG, GPDG, GEDG, PPGK, PPGR, QPGR, and GPGA. In some embodiments, the A peptide segment in the recombinant collagen contains an amino acid motif that forms a folded or turned structure, and the motif is selected from GPNG, GPDG, GEDG, PPGK, PPGR, QPGR, and GPGA, and the amino acid motif that forms a folded or turned structure in the reversed A peptide segment is not reversed. In some embodiments, the A peptide segment in the recombinant collagen does not contain an amino acid motif that forms a folded or turned structure.

[0011] In some embodiments, in order to reduce the steric hindrance of the folding or turning structure, or to maintain the structural regularity of GXY, or to avoid the appearance of 4 or more consecutive identical amino acids and facilitate the synthesis of nucleic acid sequences, amino acid G is added to the end of the A peptide segment and / or the reverse A peptide segment.

[0012] In some embodiments, in the recombinant collagen, the A peptide segment and the reverse A peptide segment are arranged 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 30 times, 40 times, and 50 times.

[0013] In some embodiments, the A peptide segment and the reverse A peptide segment further comprise a linker, the linker is derived from a natural human collagen sequence, a natural human elastin sequence, a natural silk protein and other fibrous protein sequences, preferably derived from a natural human collagen sequence and a natural human elastin sequence, more preferably derived from the flexible sequence site of natural human collagen and natural human elastin, more preferably derived from the amino acid motifs of the flexible sequence site of natural human collagen and natural human elastin that are easy to form folded or corner structures. In some embodiments, the linker is selected from the amino acid motifs of GPNG, PPGK, GPGA, GVGG, VPGG, GLVPG, GVAPG, GVGGL, VPGVG, GVAPGV, GVGVAP, LGTIPG, PGAIPG, VAPGVG, and VGVAPG. In some embodiments, the A peptide segment and the reverse A peptide segment do not comprise a linker. In some embodiments, when the A peptide segment itself contains an amino acid motif that is easy to form a folded or turned structure, such as GPNG, GPDG, GEDG, PPGK, PPGR, QPGR, GPGA, etc., the A peptide segment and the reverse A peptide segment may not contain a linker.

[0014] In some embodiments, the A peptide segment is derived from a natural human collagen sequence, a natural bovine collagen sequence, a natural porcine collagen sequence, or a natural marine organism collagen sequence, preferably derived from a natural human collagen sequence, more preferably derived from type I, type II, type III, type IV, type VII, and type XVII collagen sequences of natural human collagen, more preferably derived from the triple helical region of type I, type II, type III, type IV, type VII, and type XVII collagen of natural human collagen, more preferably a peptide segment with a length of greater than or equal to 9 amino acids containing a GER, RGD, or GVMGFO active site derived from the triple helical region of type I, type II, type III, type IV, type VII, and type XVII collagen of natural human collagen.

[0015] In some embodiments, the A peptide segment is selected from natural human type I, type II, type III, type IV, type VII, and type XVII collagen fragment sequences.

[0016] In some embodiments, the peptide A segment is selected from any one of SEQ ID NO: 1 to SEQ ID NO: 17.

[0017] In some embodiments, the sequence of the A peptide segment in the recombinant collagen is selected from SEQ ID NO: 1 to SEQ ID NO: 17, and a linker is added between the A peptide segment and the reverse A peptide segment, and the linker is selected from GVGG, VPGG, GLVPG, GVAPG, GVGGL, LGGVG, PGGKP, VPGVG, GVAPGV, GVGVAP, LGTIPG, PGAIPG, PGVPGF, VAPGVG, and VGVAPG. In some embodiments, the sequence of the A peptide segment in the recombinant collagen is selected from SEQ ID NO: 1 to SEQ ID NO: 17, and a linker is added between the A peptide segment and the reverse A peptide segment, and the linker is VPGG.

[0018] In some embodiments, the sequence of the recombinant collagen is selected from SEQ ID NO: 19-23, 25, 26, 28, 29, 31, 32, 34, 35, 37, 38, 40, 41, 43, 44, 46, 47, 49, 50, 52, 53, 55, 56, 58, 59, 61, 62, 64, 65, 67, 68, 70, 71.

[0019] In some embodiments, the sequence of the recombinant collagen is selected from SEQ ID NO: 21, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71.

[0020] The present invention also provides a polynucleotide encoding the recombinant collagen of the present invention. In some embodiments, the polynucleotide encodes a recombinant collagen selected from SEQ ID NO: 19-23, 25, 26, 28, 29, 31, 32, 34, 35, 37, 38, 40, 41, 43, 44, 46, 47, 49, 50, 52, 53, 55, 56, 58, 59, 61, 62, 64, 65, 67, 68, 70, 71. In some embodiments, the polynucleotide is selected from SEQ ID NO:73-77, 79, 80, 82, 83, 85, 86, 88, 89, 91, 92, 94, 95, 97, 98, 100, 101, 103, 104, 106, 107, 109, 110, 112, 113, 115, 116, 118, 119, 121, 122, 124, 125.

[0021] The present invention also provides an expression vector, which is incorporated with the polynucleotide of the present invention. In some embodiments, the expression vector is incorporated with a polynucleotide selected from SEQ ID NO: 73-77, 79, 80, 82, 83, 85, 86, 88, 89, 91, 92, 94, 95, 97, 98, 100, 101, 103, 104, 106, 107, 109, 110, 112, 113, 115, 116, 118, 119, 121, 122, 124, 125.

[0022] The present invention also provides a host cell transformed with the polynucleotide of the present invention and expressing the polynucleotide. In some embodiments, the host cell is transformed with a polynucleotide selected from SEQ ID NO: 73-77, 79, 80, 82, 83, 85, 86, 88, 89, 91, 92, 94, 95, 97, 98, 100, 101, 103, 104, 106, 107, 109, 110, 112, 113, 115, 116, 118, 119, 121, 122, 124, 125 and expressing the polynucleotide. The present invention also provides a host cell transformed with the expression vector of the present invention. In some embodiments, the host cell is transformed with an expression vector incorporating a polynucleotide selected from SEQ ID NO: 73-77, 79, 80, 82, 83, 85, 86, 88, 89, 91, 92, 94, 95, 97, 98, 100, 101, 103, 104, 106, 107, 109, 110, 112, 113, 115, 116, 118, 119, 121, 122, 124, 125. In some embodiments, the host cell is selected from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Bacillus. In some embodiments, the expression vector is selected from pET22a, pET28a, pET32a or an expression vector transformed by the aforementioned vector, and the host cell is Escherichia coli BL21 (DE3). In some embodiments, the expression vector is selected from pPIC9, pPIC9K, pPICZαA or an expression vector modified by the aforementioned vectors, and the host cell is Pichia pastoris X33 or GS115 or SMD1168.

[0023] The present invention also provides a method for producing the recombinant collagen of the present invention in a host cell, the method comprising culturing the host cell of the present invention under culture conditions that produce the recombinant collagen of the present invention. In some embodiments, the method for producing the recombinant collagen of the present invention further comprises recovering the recombinant collagen from the culture and / or host cell. In some embodiments, the method for producing the recombinant collagen of the present invention further comprises a step of purifying the recombinant collagen.

[0024] The present invention also provides a composition, which comprises the recombinant collagen of the present invention.

[0025] The present invention also provides application of the recombinant collagen of the present invention in biomedical materials.

[0026] Beneficial effects:

[0027] The present invention enhances activity (e.g., cell proliferation activity), stability, or expression by designing an interval reverse repeat core peptide segment in recombinant collagen. The GXY characteristic sequence of collagen is selected as the core sequence, the first peptide segment and the third peptide segment are kept in the forward direction, and the second peptide segment is designed in the reverse direction; when the peptide segment is repeated more than 3 times, it is connected in reverse order at intervals. The flexible structure of the peptide segment sequence itself is utilized to form or dislocate the peptide segment to form a self-folding structure, preferably a triple helix or a triple helix-like folding structure; if necessary, a suitable linker is added between two adjacent peptide segments, and the linker is used to form or dislocate the peptide segment to form a self-folding structure, preferably a triple helix or a triple helix-like folding structure. Thereby enhancing and improving the biological activity, stability, and expression of the recombinant collagen. Compared with the prior art sequence forward repeat tandem expression, the recombinant collagen of the present invention obtains high expression and high biological activity, and the molecular weight of the expression product, i.e., the recombinant collagen, is closer to the concept of small molecule collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : The first sequence design method of the present invention

[0029] Figure 2 : The second sequence design method of the present invention

[0030] Figure 3 :SDS-PAGE gel image of recombinant collagen related to core unit sequence Seq 1 to Seq 17

[0031] Figure 4 :Comparison of cell proliferation activity of recombinant collagen with different expression strategies of core unit sequence Seq 1

[0032] Figure 5 :Size exclusion chromatograms of recombinant collagen with three expression strategies for core unit sequence Seq 1

[0033] Figure 6 :Circular dichroism spectra of recombinant collagen with three expression strategies of core unit sequence Seq 1

[0034] Attached Figure 1 and attached Figure 2 This is only to express the design concept of the present invention, and does not limit the specific repetition mode of the core sequence of the present invention to a strict inverted repetition. For example, when Seq 1 is reversely repeated three times, the flanking amino acid motif "GPNG" that is easy to form a folded or corner structure is not reversed. For another example, in order to maintain the structural regularity of GXY during the repetition design of Seq 2, "G" is added to the end of each repetition. Collagen with this type of design also belongs to the scope of the present invention.

[0035] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent manufacturers unless otherwise specified.

[0036] Example 1: Screening of GXY core sequence units

[0037] The most preferred GXY core sequence unit of the present invention is derived from a peptide with a length of 9 amino acids or more with active sites such as GER and RGD in the triple helical region of type I, type II, type III, type IV, type VII, and type XVII collagen of natural human collagen. The present invention lists but is not limited to the following 17 GXY core sequence units that meet the above description (Table 1: Seq 1 to Seq 17), which respectively or simultaneously contain potential active sites such as GER and RGD and / or potential flanking amino acid motifs such as GPNG, GPDG, GEDG, PPGK, PPGR, QPGR, and GPGA that are prone to forming folding or turning structures (such as GPNG in Seq 1, PPGK in Seq 2, GPGA in Seq 4, and GPGA in Seq 6 in Table 1. When there are flanking amino acid motifs that are prone to forming folding or turning structures in the core sequence, these special amino acid motifs in the inverted repeat sequence are not reversed).

[0038] Table 1. 17 GXY core sequence units

[0039]

[0040]

[0041] Example 2: Screening for possible linker sequences to be added

[0042] The atomic distances and adjacent dihedral angles of protein polypeptide chains are the main factors that determine the secondary structure of proteins. Based on these two characteristics, protein secondary structures can be divided into α-helix, β-fold, β-turn, random coil, polyproline type II helix, etc., among which β-fold or β-turn can cause significant bending of the polypeptide chain. Various types of β-fold or β-turn structures have been identified in natural human collagen and natural human elastin. Therefore, in theory, these structures can be used to make the polypeptide chain form possible bends when designing recombinant collagen. Moreover, the amino acid sequences corresponding to these structures are derived from natural human proteins themselves and will not cause any safety risks to recombinant collagen.

[0043] The most preferred linker sequences that may need to be added in the present invention are derived from amino acid motifs that are easy to form folded or corner structures in the flexible sequence sites of natural human collagen and natural human elastin, including but not limited to GPNG, GPDG, GEDG, PPGK, PPGR, QPGR, GPGA and other motifs, as well as GVGG, VPGG, GLVPG, GVAPG, GVGGL, LGGVG, PGGKP, VPGVG, GVAPGV, GVGVAP, LGTIPG, PGAIPG, PGVPGF, VAPGVG, VGVAPG and other motifs, and any combination of these motifs. The present invention lists but is not limited to 4 types of linkers that may need to be added in the embodiments according to the above description, which are named linker 1 (GVGG), linker 2 (VPGG), linker 3 (PGAIPG), and linker 4 (PGGKP) respectively.

[0044] Example 3: Preparation of recombinant collagen

[0045] Construction of recombinant collagen expression strain

[0046] The present invention is based on the screened natural human type I, type II, type III, type IV, type VII, and type XVII recombinant collagen sequences, such as but not limited to the 17 GXY core unit sequences mentioned above, by designing the interval reverse repeat to be greater than or equal to a positive integer multiple of 3, and adding the above-described screened linker between two adjacent repeated peptide segments (one is a forward sequence and the other is a reverse sequence) when necessary, and then optimizing the nucleic acid sequence according to the codon preference of the expression host such as Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Bacillus, etc., connecting the codon-optimized nucleic acid sequence to a suitable vector by gene synthesis or long primer splicing, and then transforming the corresponding host strain, coating the resistance screening plate, selecting the positive clone for PCR verification and gene sequencing, and obtaining the recombinant collagen expression strain. The expression strain with the same multiple of forward repeats of the GXY core unit sequence is obtained by the same or similar construction method as a control.

[0047] The amino acid sequences of the recombinant collagen constructed based on the core unit sequence mentioned in the present invention are listed as follows. Seq 1 contains an amino acid motif GPNG that is easy to form a folded or turned structure. When designing an inverted repeat sequence, this motif can be regarded as a self-contained linker that is not reversed. Similar situations are shown in the corresponding construction sequences of Seq 2, Seq 4 and Seq 6 in Table 1. In some embodiments, such as the corresponding construction sequences of Seq 2, Seq 3, Seq 5 to 17, in order to reduce the steric hindrance of the folded or turned structure, or to maintain the structural regularity of GXY, or to avoid the appearance of 4 or more consecutive identical amino acids and facilitate the synthesis of nucleic acid sequences, amino acids G are also added to the ends of the core unit sequence.

[0048] Seq 18 (Seq 1 forward repeated 3 times):

[0049] MGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNG

[0050] Seq 19 (Seq 1 with spaced inverted repeats 3 times):

[0051] MGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGAPGRFGPAGREGAAGPLGNAGPEGPSGDKGDEGKAGGPNGGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNG

[0052] Seq 20 (Seq 1 spaced inverted repeat 3 times + adapter 1):

[0053] MGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGGVGGAPGRFGPAGREGAAGPLGNAGPEGPSGDKGDEGKAGGPNGGVGGGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNG

[0054] Seq 21 (Seq 1 spaced inverted repeat 3 times + adapter 2):

[0055] MGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGVPGGAPGRFGPAGREGAAGPLGNAGPEGPSGDKGDEGKAGGPNGVPGGGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNG

[0056] Seq 22 (Seq 1 spaced inverted repeat 3 times + adapter 3):

[0057] MGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGPGAIPGAPGRFGPAGREGAAGPLGNAGPEGPSGDKGDEGKAGGPNGPGAIPGGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNG

[0058] Seq 23 (Seq 1 spaced inverted repeat 3 times + adapter 4):

[0059] MGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGPGGKPAPGRFGPAGREGAAGPLGNAGPEGPSGDKGDEGKAGGPNGPGGKPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNG

[0060] Seq 24 (Seq 2 forward repeated 3 times):

[0061] MGERGGPGGPGPQGPPGKGGERGGPGGPGPQGPPGKGGERGGPGGPGPQGPPGKG

[0062] Seq 25 (Seq 2 spaced inverted repeat 3 times):

[0063] MGERGGPGGPGPQGPPGKGGQPGPGGPGGREGPPGKGGERGGPGGPGPQGPPGKG

[0064] Seq 26 (Seq 2 spaced inverted repeat 3 times + adapter 2):

[0065] MGERGGPGGPGPQGPPGKGVPGGGQPGPGGPGGREGPPGKGVPGGGERGGPGGPGPQGPPGKG

[0066] Seq 27 (Seq 3 forward repeated 3 times):

[0067] MGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPP

[0068] Seq 28 (Seq 3 interspaced inverted repeats 3 times):

[0069] MGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGPPGPPGAAGKGGEPGPPGAGGRLGPAGALGPPGGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPP

[0070] Seq 29 (Seq 3 spacer inverted repeat 3x + linker 2):

[0071] MGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGVPGGPPGPPGAAGKGGEPGPPGAGGRLGPAGALGPPGVPGGGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPP

[0072] Seq 30 (Seq 4 direct repeat 3x):

[0073] MGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGA

[0074] Seq 31 (Seq 4 spacer inverted repeat 3x):

[0075] MGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGAGPEGKDGKPGPSGLGGREGPMGQLGPTGAAGPPGPPGGPGAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGA

[0076] Seq 32 (Seq 4 spacer inverted repeat 3x + linker 2):

[0077] MGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGAVPGGGPEGKDGKPGPSGLGGREGPMGQLGPTGAAGPPGPPGGPGAVPGGGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGA

[0078] Seq 33 (Seq 5 direct repeat 3x):

[0079] MGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPP

[0080] Seq 34 (Seq 5 Spacer Inverted Repeat x3):

[0081] MGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGPPGGEGKEGPAGREGKGGPEGNQGPAGPFGAPGPPGTEGREGGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPP

[0082] Seq 35 (Seq 5 Spacer Inverted Repeat x3 + Linker 2):

[0083] MGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPVPGGGPPGGEGKEGPAGREGKGGPEGNQGPAGPFGAPGPPGTEGREGVPGGGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPP

[0084] Seq 36 (Seq 6 Direct Repeat x3):

[0085] MGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAGGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAGGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAG

[0086] Seq 37 (Seq 6 Spacer Inverted Repeat x3):

[0087] MGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAGGPSGREGKVGQPGPPGAPGSGGPPGAVGPPGGEGKEGPAGREGGPGAGGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAG

[0088] Seq 38 (Seq 6 Spacer Inverted Repeat 3X + Linker 2):

[0089] MGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAGVPGGPSGREGKVGQPGPPGAPGSGGPPGAVGPPGGEGKEGPAGREGGPGAGVPGGGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAGG

[0090] Seq 39 (Seq 7 Direct Repeat 3X):

[0091] MGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGEN

[0092] Seq 40 (Seq 7 Spacer Inverted Repeat 3X):

[0093] MGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGNEGRDGKGGPSGDRGPLGDSGPNGDRGPEGATGALGPLGQPGPPGREGSLGNAGPKGSEGKVGQPGPSGRPGPMGGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGEN

[0094] Seq 41 (Seq 7 Spacer Inverted Repeat 3X + Linker 2):

[0095] MGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENVPGGGNEGRDGKGGPSGDRGPLGDSGPNGDRGPEGATGALGPLGQPGPPGREGSLGNAGPKGSEGKVGQPGPSGRPGPMGVPGGGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGEN

[0096] Seq 42 (3 - fold forward repeat of Seq 8):

[0097] MGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPGKDGTSGHPGPIGPPGPRGNRGERGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPGKDGTSGHPGPIGPPGPRGNRGERGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPGKDGTSGHPGPIGPPGPRGNRGER

[0098] Seq 43 (3 - fold inverted repeat with spacer of Seq 8):

[0099] MGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPGKDGTSGHPGPIGPPGPRGNRGERGREGRNGRPGPPGIPGPHGSTGDKGPGSPGVPGRPGAPGPSGIAGQQGAPGPSGPAGPNGPFGRHGKIGAAGREGTEGKDGRPGGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPGKDGTSGHPGPIGPPGPRGNRGER

[0100] Seq 44 (3 - fold inverted repeat with spacer of Seq 8 + linker 2):

[0101] MGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPGKDGTSGHPGPIGPPGPRGNRGERGVPGGREGRNGRPGPPGIPGPHGSTGDKGPGSPGVPGRPGAPGPSGIAGQQGAPGPSGPAGPNGPFGRHGKIGAAGREGTEGKDGRPGVPGGGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPGKDGTSGHPGPIGPPGPRGNRGER

[0102] Seq 45 (3 - fold direct repeat of Seq 9):

[0103] MGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGP

[0104] Seq 46 (3 - fold inverted repeat of Seq 9 with spacer):

[0105] MGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPGGPGGREGNKGPAGDNGKPGPFGMVGPQGRPGSPGPPGPRGSEGQSGPPGPKGDSGPGGPSGPMGRMGPGGPVGDRGPEGAAGRPGAPGPAGREGAPGKEGPIGNPGAPGRFGPAGREGGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGP

[0106] Seq 47 (Seq 9 Spacer Inverted Repeat 3x + Linker 2):

[0107] MGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGVPGGPGGPGGREGNKGPAGDNGKPGPFGMVGPQGRPGSPGPPGPRGSEGQSGPPGPKGDSGPGGPSGPMGRMGPGGPVGDRGPEGAAGRPGAPGPAGREGAPGKEGPIGNPGAPGRFGPAGREGVPGGGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGP

[0108] Seq 48 (Seq 10 Direct Repeat 3x):

[0109] MGERGFPGERGVQGPPGPAGERGFPGERGVQGPPGPAGERGFPGERGVQGPPGPA

[0110] Seq 49 (Seq 10 Spacer Inverted Repeat 3x):

[0111] MGERGFPGERGVQGPPGPAGAPGPPGQVGREGPFGREGGERGFPGERGVQGPPGPA

[0112] Seq 50 (Seq 10 spaced inverted repeat 3 times + adapter 2):

[0113] MGERGFPGERGVQGPPGPAGVPGGAPGPPGQVGREGPFGREGVPGGGERGFPGERGVQGPPGPA

[0114] Seq 51 (Seq 11 forward repeated 3 times):

[0115] MGERGGPGSRGFPGADGVAGPKGPAGERGERGGPGSRGFPGADGVAGPKGPAGERGERGGPGSRGFPGADGVAGPKGPAGER

[0116] Seq 52 (Seq 11 spaced inverted repeat 3 times):

[0117] MGERGGPGSRGFPGADGVAGPKGPAGERGREGAPGKPGAVGDAGPFGRSGPGGREGGERGGPGSRGFPGADGVAGPKGPAGER

[0118] Seq 53 (Seq 11 spaced inverted repeat 3 times + adapter 2):

[0119] MGERGGPGSRGFPGADGVAGPKGPAGERGVPGGREGAPGKPGAVGDAGPFGRSGPGGGREGVPGGGERGGPGSRGFPGADGVAGPKGPAGER

[0120] Seq 54 (Seq 12 forward repeated 3 times):

[0121] MGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKD

[0122] Seq 55 (Seq 12 spaced inverted repeat 3 times):

[0123] MGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGDKGPSGDAGKPGADGRDGKPGPLGAAGREGPMGGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKD

[0124] Seq 56 (Seq 12 Spacer Reverse Repeat 3x + Linker 2):

[0125] MGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVPGGDKGPSGDAGKPGADGRDGKPGPLGAAGREGPMGVPGGGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKD

[0126] Seq 57 (Seq 13 Forward Repeat 3x):

[0127] MGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPP

[0128] Seq 58 (Seq 13 Spacer Reverse Repeat 3x):

[0129] MGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPPGPPGREGSAGSPGQKGPEGSPGPLGPFGREGRQGPLGVVGRQGAIGQPGGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPP

[0130] Seq 59 (Seq 13 Spacer Reverse Repeat 3x + Linker 2):

[0131] MGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPPGVPGGPPGREGSAGSPGQKGPEGSPGPLGPFGREGRQGPLGVVGRQGAIGQPGVPGGGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPP

[0132] Seq 60 (3 - fold forward repeat of Seq 14):

[0133] MGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGERGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGERGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGER

[0134] Seq 61 (3 - fold spaced reverse repeat of Seq 14):

[0135] MGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGERGREGAKGPEGAAGKPGPFGMVGAQGRAGPPGPPGPRGDQGAPGPPGTKGDPGGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGER

[0136] Seq 62 (3 - fold spaced reverse repeat of Seq 14 + linker 2):

[0137] MGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGERGVPGGREGAKGPEGAAGKPGPFGMVGAQGRAGPPGPPGPRGDQGAPGPPGTKGDPGVPGGGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGER

[0138] Seq 63 (3 - fold forward repeat of Seq 15):

[0139] MGERGEPGLVGDPGPRGEPGPKGAPGLPGQPGPPGLPGVPGQAGAPGERGEPGLVGDPGPRGEPGPKGAPGLPGQPGPPGLPGVPGQAGAPGERGEPGLVGDPGPRGEPGPKGAPGLPGQPGPPGLPGVPGQAGAP

[0140] Seq 64 (3 - fold spaced reverse repeat of Seq 15):

[0141] MGERGEPGLVGDPGPRGEPGPKGAPGLPGQPGPPGLPGVPGQAGAPGPAGAQGPVGPLGPPGPQGPLGPAGKPGPEGRPGPDGVLGPEGREGGERGEPGLVGDPGPRGEPGPKGAPGLPGQPGPPGLPGVPGQAGAP

[0142] Seq 65 (Seq 15 spaced inverted repeat 3 times + adapter 2):

[0143] MGERGEPGLVGDPGPRGEPGPKGAPGLPGQPGPPGLPGVPGQAGAPGVPGGPAGAQGPVGPLGPPGPQGPLGPAGKPGPEGRPGPDGVLGPEGREGVPGGGERGEPGLVGDPGPRGEPGPKGAPGLPGQPGPPGLPGVPGQAGAP

[0144] Seq 66 (Seq 16 forward repeated 3 times):

[0145] MGERGPPGPAGERGLPGVAGRPGAQGPEGPPGPTGRQGERGEPGPRGDPGERGPPGPAGERGLPGVAGRPGAQGPEGPPGPTGRQGERGEPGPRGDPGERGPPGPAGERGLPGVAGRPGAQGPEGPPGPTGRQGERGEPGPRGDP

[0146] Seq 67 (Seq 16 spaced inverted repeat 3 times):

[0147] MGERGPPGPAGERGLPGVAGRPGAQGPEGPPGPTGRQGERGEPGPRGDPGPDGRPGPEGREGQRGTPGPPGEPGQAGPRGAVGPLGREGAPGPPGREGGERGPPGPAGERGLPGVAGRPGAQGPEGPPGPTGRQGERGEPGPRGDP

[0148] Seq 68 (Seq 16 spaced inverted repeat 3 times + adapter 2):

[0149] MGERGPPGPAGERGLPGVAGRPGAQGPEGPPGPTGRQGERGEPGPRGDPGVPGGPDGRPGPEGREGQRGTPGPPGEPGQAGPRGAVGPLGREGAPGPPGREGVPGGGERGPPGPAGERGLPGVAGRPGAQGPEGPPGPTGRQGERGEPGPRGDP

[0150] Seq 69 (Seq 17 forward repeated 3 times):

[0151] MGPQGEVGDPGAEGPMGQRGREGPRGEAGPPGDRGERGPQGEVGDPGAEGPMGQRGREGPRGEAGPPGDRGERGPQGEVGDPGAEGPMGQRGREGPRGEAGPPGDRGER

[0152] Seq 70 (Seq 17 spaced inverted repeat 3 times):

[0153] MGPQGEVGDPGAEGPMGQRGREGPRGEAGPPGDRGERGREGRDGPPGAEGRPGERGRQGMPGEAGPDGVEGQPGGPQGEVGDPGAEGPMGQRGREGPRGEAGPPGDRGER

[0154] Seq 71 (Seq 17 spaced inverted repeat 3 times + adapter 2):

[0155] MGPQGEVGDPGAEGPMGQRGREGPRGEAGPPGDRGERGVPGGREGRDGPPGAEGRPGERGRQGMPGEAGPDGVEGQPGVPGGGPQGEVGDPGAEGPMGQRGREGPRGEAGPPGDRGER

[0156] A portion of the sequence of the present invention is expressed in Escherichia coli, the vector is derived from pET22a, pET28a, pET32a, etc. or is appropriately modified on the basis of these vectors, and the host is Escherichia coli BL21 (DE3); a portion of the sequence is expressed in Pichia pastoris, the vector is derived from pPIC9, pPIC9K, pPICZαA, etc. or is appropriately modified on the basis of these vectors, and the host is Pichia pastoris X33 or GS115 or SMD1168; a portion of the sequence is expressed in both Escherichia coli and Pichia pastoris, and the vector and host are the same as described above.

[0157] The nucleic acid sequences of the recombinant collagen constructed based on the core unit sequence mentioned in the present invention are listed as follows:

[0158] Seq 72 (Seq 1 forward repeat 3 times (expressed in E. coli)):

[0159] ATGGGTGCTAAAGGTGAAGACGGTAAAGACGGTTCTCCGGGTGAACCGGGTGCTAACGGTCTGCCGGGTGCTGCTGGTGAACGTGGTGCTCCGGGTTTCCGTGGTCCGGCTGGTCCGAACGGTGGTGCTAAAGGTGAAGACGGTAAAGACGGTTCTCCGGGTGAACCGGGTGCTAACGGTCTGCCGGGTGCTGCTGGTGAACGTGGTGCTCCGGGTTTCCGTGGTCCGGCTGGTCCGAACGGTGGTGCTAAAGGTGAAGACGGTAAAGACGGTTCTCCGGGTGAACCGGGTGCTAACGGTCTGCCGGGTGCTGCTGGTGAACGTGGTGCTCCGGGTTTCCGTGGTCCGGCTGGTCCGAACGGTTAA

[0160] Seq 73 (Seq 1 spacer reverse repeat 3 times (expressed in E. coli)):

[0161] ATGGGTGCTAAAGGTGAAGACGGTAAAGACGGTTCTCCGGGTGAACCGGGTGCTAACGGTCTGCCGGGTGCTGCTGGTGAACGTGGTGCTCCGGGTTTCCGTGGTCCGGCTGGTCCGAACGGTGCTCCGGGTCGTTTCGGTCCGGCTGGTCGTGAAGGTGCTGCTGGTCCGCTGGGTAACGCTGGTCCGGAAGGTCCGTCTGGTGACAAAGGTGACGAAGGTAAAGCTGGTGGTCCGAACGGTGGTGCTAAAGGTGAAGACGGTAAAGACGGTTCTCCGGGTGAACCGGGTGCTAACGGTCTGCCGGGTGCTGCTGGTGAACGTGGTGCTCCGGGTTTCCGTGGTCCGGCTGGTCCGAACGGTTAA

[0162] Seq 74 (Seq 1 spacer reverse repeat 3 times + linker 1 (expressed in E. coli)):

[0163] ATGGGTGCTAAAGGTGAAGACGGTAAAGACGGTTCTCCGGGTGAACCGGGTGCTAACGGTCTGCCGGGTGCTGCTGGTGAACGTGGTGCTCCGGGTTTCCGTGGTCCGGCTGGTCCGAACGGTGGTGTTGGTGGTGCTCCGGGTCGTTTCGGTCCGGCTGGTCGTGAAGGTGCTGCTGGTCCGCTGGGTAACGCTGGTCCGGAAGGTCCGTCTGGTGACAAAGGTGACGAAGGTAAAGCTGGTGGTCCGAACGGTGGTGTTGGTGGTGGTGCTAAAGGTGAAGACGGTAAAGACGGTTCTCCGGGTGAACCGGGTGCTAACGGTCTGCCGGGTGCTGCTGGTGAACGTGGTGCTCCGGGTTTCCGTGGTCCGGCTGGTCCGAACGGTTAA

[0164] Seq 75 (Seq 1 Spacer Inverted Repeat 3x + Linker 2 (expressed in E. coli)):

[0165] ATGGGTGCTAAAGGTGAAGACGGTAAAGACGGTTCTCCGGGTGAACCGGGTGCTAACGGTCTGCCGGGTGCTGCTGGTGAACGTGGTGCTCCGGGTTTCCGTGGTCCGGCTGGTCCGAACGGTGTTCCGGGTGGTGCTCCGGGTCGTTTCGGTCCGGCTGGTCGTGAAGGTGCTGCTGGTCCGCTGGGTAACGCTGGTCCGGAAGGTCCGTCTGGTGACAAAGGTGACGAAGGTAAAGCTGGTGGTCCGAACGGTGTTCCGGGTGGTGGTGCTAAAGGTGAAGACGGTAAAGACGGTTCTCCGGGTGAACCGGGTGCTAACGGTCTGCCGGGTGCTGCTGGTGAACGTGGTGCTCCGGGTTTCCGTGGTCCGGCTGGTCCGAACGGTTAA

[0166] Seq 76 (Seq 1 Spacer Inverted Repeat 3x + Linker 3 (expressed in E. coli)):

[0167] ATGGGTGCTAAAGGTGAAGACGGTAAAGACGGTTCTCCGGGTGAACCGGGTGCTAACGGTCTGCCGGGTGCTGCTGGTGAACGTGGTGCTCCGGGTTTCCGTGGTCCGGCTGGTCCGAACGGTCCGGGTGCTATCCCGGGTGCTCCGGGTCGTTTCGGTCCGGCTGGTCGTGAAGGTGCTGCTGGTCCGCTGGGTAACGCTGGTCCGGAAGGTCCGTCTGGTGACAAAGGTGACGAAGGTAAAGCTGGTGGTCCGAACGGTCCGGGTGCTATCCCGGGTGGTGCTAAAGGTGAAGACGGTAAAGACGGTTCTCCGGGTGAACCGGGTGCTAACGGTCTGCCGGGTGCTGCTGGTGAACGTGGTGCTCCGGGTTTCCGTGGTCCGGCTGGTCCGAACGGTTAA

[0168] Seq 77 (Seq 1 spacer inverted repeat 3x + linker 4 (expressed in E. coli)):

[0169] ATGGGTGCTAAAGGTGAAGACGGTAAAGACGGTTCTCCGGGTGAACCGGGTGCTAACGGTCTGCCGGGTGCTGCTGGTGAACGTGGTGCTCCGGGTTTCCGTGGTCCGGCTGGTCCGAACGGTCCGGGTGGTAAACCGGCTCCGGGTCGTTTCGGTCCGGCTGGTCGTGAAGGTGCTGCTGGTCCGCTGGGTAACGCTGGTCCGGAAGGTCCGTCTGGTGACAAAGGTGACGAAGGTAAAGCTGGTGGTCCGAACGGTCCGGGTGGTAAACCGGGTGCTAAAGGTGAAGACGGTAAAGACGGTTCTCCGGGTGAACCGGGTGCTAACGGTCTGCCGGGTGCTGCTGGTGAACGTGGTGCTCCGGGTTTCCGTGGTCCGGCTGGTCCGAACGGTTAA

[0170] Seq 78 (Seq 2 direct repeat 3x (expressed in E. coli)):

[0171] ATGGGTGAACGTGGTGGTCCGGGTGGTCCGGGTCCGCAGGGTCCGCCGGGTAAAGGTGGTGAACGTGGTGGTCCGGGTGGTCCGGGTCCGCAGGGTCCGCCGGGTAAAGGTGGTGAACGTGGTGGTCCGGGTGGTCCGGGTCCGCAGGGTCCGCCGGGTAAAGGTTAA

[0172] Seq 79 (Seq 2 spacer inverted repeat 3 times (expressed in E. coli)):

[0173] ATGGGTGAACGTGGTGGTCCGGGTGGTCCGGGTCCGCAGGGTCCGCCGGGTAAAGGTGGTCAGCCGGGTCCGGGTGGTCCGGGTGGTCGTGAAGGTCCGCCGGGTAAAGGTGGTGAACGTGGTGGTCCGGGTGGTCCGGGTCCGCAGGGTCCGCCGGGTAAAGGTTAA

[0174] Seq 80 (Seq 2 spacer inverted repeat 3 times + linker 2 (expressed in E. coli)):

[0175] ATGGGTGAACGTGGTGGTCCGGGTGGTCCGGGTCCGCAGGGTCCGCCGGGTAAAGGTGTTCCGGGTGGTGGTCAGCCGGGTCCGGGTGGTCCGGGTGGTCGTGAAGGTCCGCCGGGTAAAGGTGTTCCGGGTGGTGGTGAACGTGGTGGTCCGGGTGGTCCGGGTCCGCAGGGTCCGCCGGGTAAAGGTTAA

[0176] Seq 81 (Seq 3 direct repeat 3 times (expressed in E. coli)):

[0177] ATGGGTCCACCAGGTCTGGCTGGTGCACCAGGTCTGCGTGGTGGTGCTGGTCCTCCAGGTCCAGAAGGTGGTAAAGGTGCAGCTGGTCCTCCTGGTCCACCTGGTCCACCAGGTCTGGCTGGTGCTCCAGGTCTGCGTGGTGGTGCTGGTCCTCCAGGTCCTGAAGGTGGTAAAGGTGCAGCAGGTCCACCAGGTCCTCCTGGTCCTCCTGGTCTGGCTGGTGCTCCAGGTCTGCGTGGTGGTGCTGGTCCACCAGGTCCGGAAGGTGGTAAAGGTGCAGCGGGTCCACCAGGCCCTCCTTAA

[0178] Seq 82 (Seq 3 spacer inverted repeat 3x (expressed in E. coli)):

[0179] ATGGGTCCTCCAGGTCTGGCAGGTGCACCAGGTCTGCGTGGTGGTGCTGGTCCACCAGGTCCAGAAGGTGGTAAAGGTGCAGCAGGTCCACCAGGTCCACCTGGTCCTCCAGGTCCACCAGGTGCTGCTGGTAAAGGTGGCGAACCAGGTCCTCCAGGTGCTGGTGGTCGTCTGGGTCCTGCTGGTGCTCTGGGTCCTCCTGGTGGTCCTCCAGGTCTGGCTGGTGCTCCTGGTCTGCGTGGTGGTGCAGGTCCACCTGGTCCTGAAGGTGGTAAAGGTGCTGCTGGTCCGCCTGGTCCGCCATAA

[0180] Seq 83 (Seq 3 spacer inverted repeat 3x + linker 2 (expressed in E. coli)):

[0181] ATGGGTCCACCAGGTCTGGCTGGTGCACCAGGTCTGCGTGGTGGTGCAGGTCCACCAGGTCCAGAAGGTGGCAAAGGTGCTGCAGGTCCACCTGGTCCTCCAGGTGTTCCAGGTGGTCCACCAGGTCCTCCTGGTGCAGCTGGTAAAGGTGGTGAACCTGGTCCACCAGGTGCTGGTGGTCGTCTGGGTCCAGCTGGTGCTCTGGGTCCTCCTGGTGTACCAGGTGGTGGTCCACCAGGTCTGGCAGGTGCTCCTGGTCTGCGTGGTGGTGCTGGTCCACCAGGTCCTGAAGGTGGTAAAGGTGCAGCAGGCCCTCCTGGTCCGCCATAA

[0182] Seq 84 (Seq 4 forward repeat 3 times (expressed in E. coli)):

[0183] ATGGGTCCTCCTGGTCCTCCAGGTGCAGCAGGTACTCCGGGTCTGCAAGGTATGCCGGGTGAACGTGGTGGCCTGGGTTCTCCTGGTCCAAAAGGTGATAAAGGTGAACCTGGTGGTCCTGGTGCAGGTCCTCCAGGTCCACCAGGTGCTGCAGGTACTCCAGGTCTGCAGGGTATGCCTGGTGAACGTGGTGGTCTGGGTTCTCCAGGTCCTAAAGGTGACAAAGGCGAACCAGGTGGTCCAGGTGCTGGTCCACCAGGTCCACCGGGTGCTGCTGGTACTCCAGGTCTGCAAGGTATGCCAGGTGAACGTGGTGGTCTGGGCTCTCCAGGTCCGAAAGGCGACAAAGGTGAACCGGGTGGTCCTGGTGCGTAA

[0184] Seq 85 (Seq 4 spacer reverse repeat 3 times (expressed in E. coli)):

[0185] ATGGGTCCACCAGGTCCACCTGGTGCTGCTGGTACTCCAGGTCTGCAAGGTATGCCTGGTGAACGTGGTGGTCTGGGCTCTCCTGGTCCGAAAGGTGACAAAGGTGAACCTGGTGGTCCGGGTGCTGGTCCTGAAGGTAAAGACGGTAAACCGGGTCCTTCCGGTCTGGGTGGTCGTGAAGGTCCGATGGGTCAGCTGGGTCCAACTGGTGCTGCAGGTCCTCCAGGTCCTCCTGGTGGTCCAGGTGCAGGTCCACCTGGTCCTCCAGGTGCAGCAGGTACTCCTGGTCTGCAGGGCATGCCAGGCGAACGTGGTGGTCTGGGTAGCCCTGGTCCGAAAGGCGACAAAGGTGAACCAGGTGGTCCAGGTGCTTAA

[0186] Seq 86 (Seq 4 spacer inverted repeat 3 times + linker 2 (expressed in E. coli)):

[0187] ATGGGTCCTCCAGGTCCTCCAGGTGCAGCAGGTACCCCAGGTCTGCAAGGTATGCCAGGCGAACGTGGTGGTCTGGGTTCTCCGGGTCCGAAAGGTGACAAAGGTGAACCTGGTGGTCCAGGTGCTGTTCCTGGTGGTGGTCCGGAAGGCAAAGATGGCAAACCAGGCCCGTCTGGTCTGGGTGGTCGTGAAGGTCCGATGGGTCAACTGGGTCCTACCGGTGCTGCTGGTCCACCTGGTCCACCAGGTGGTCCTGGTGCAGTTCCAGGTGGTGGTCCACCAGGTCCACCTGGTGCTGCAGGTACTCCTGGTCTGCAGGGTATGCCGGGTGAACGTGGTGGTCTGGGCTCTCCTGGCCCTAAAGGTGATAAAGGTGAACCGGGTGGTCCGGGTGCATAA

[0188] Seq 87 (Seq 5 direct repeat 3 times (expressed in E. coli)):

[0189] ATGGGTGAACGCGGTGAAACCGGTCCACCAGGTCCAGCTGGTTTCCCAGGTGCTCCTGGTCAGAACGGTGAACCAGGTGGTAAAGGCGAACGTGGCGCTCCAGGTGAAAAGGGCGAAGGTGGTCCGCCTGGTGAACGTGGTGAAACTGGTCCACCGGGTCCGGCAGGTTTTCCAGGTGCTCCAGGTCAGAACGGTGAACCGGGCGGTAAAGGTGAACGTGGTGCACCGGGCGAAAAAGGTGAAGGTGGTCCTCCGGGCGAACGTGGTGAAACTGGCCCACCAGGTCCAGCAGGTTTTCCAGGTGCACCTGGTCAAAACGGCGAACCGGGTGGTAAAGGTGAACGTGGCGCTCCGGGTGAAAAAGGCGAAGGTGGTCCGCCGTAA

[0190] Seq 88 (Seq 5 spacer inverted repeat 3 - fold (expressed in E. coli)):

[0191] ATGGGTGAACGTGGTGAAACTGGTCCTCCTGGTCCGGCTGGTTTCCCTGGTGCTCCAGGTCAGAATGGCGAACCAGGTGGTAAAGGTGAGCGTGGCGCACCGGGTGAAAAAGGTGAAGGTGGTCCGCCAGGTCCTCCAGGTGGTGAAGGTAAAGAAGGTCCGGCCGGTCGTGAAGGTAAAGGTGGTCCTGAAGGTAACCAAGGTCCGGCAGGTCCTTTTGGTGCACCGGGTCCTCCTGGTACCGAAGGTCGTGAAGGTGGTGAACGTGGTGAAACTGGTCCGCCGGGTCCAGCAGGTTTTCCAGGTGCACCAGGTCAGAATGGTGAACCGGGTGGCAAAGGCGAACGTGGTGCTCCTGGTGAGAAAGGCGAAGGTGGTCCTCCGTAA

[0192] Seq 89 (Seq 5 spacer inverted repeat 3 - fold + linker 2 (expressed in E. coli)):

[0193] ATGGGCGAACGTGGTGAAACCGGTCCACCTGGTCCAGCTGGTTTCCCAGGTGCACCAGGTCAAAACGGTGAACCAGGTGGTAAAGGTGAACGTGGTGCTCCGGGTGAAAAAGGTGAAGGTGGTCCACCTGTGCCAGGTGGTGGTCCACCTGGTGGTGAAGGTAAAGAAGGCCCGGCGGGTCGTGAAGGTAAAGGTGGTCCAGAAGGTAACCAGGGTCCGGCTGGTCCATTTGGTGCACCAGGTCCGCCTGGTACTGAAGGTCGTGAAGGTGTTCCAGGTGGTGGCGAACGTGGTGAAACGGGTCCACCTGGTCCGGCTGGTTTTCCAGGTGCTCCTGGTCAGAATGGTGAACCGGGTGGCAAAGGTGAACGTGGCGCTCCTGGTGAAAAAGGTGAAGGCGGTCCGCCGTAA

[0194] Seq 90 (3 - fold direct repeat of Seq 6 (expressed in E. coli)):

[0195] ATGGGTGAACGTGGTGCTCCGGGTGAAAAAGGTGAAGGTGGTCCGCCGGGTGTTGCTGGTCCGCCGGGTGGTTCTGGTCCGGCTGGTCCGCCGGGTCCGCAGGGTGTTAAAGGTGAACGTGGTTCTCCGGGTGGTCCGGGTGCTGGTGGTGAACGTGGTGCTCCGGGTGAAAAAGGTGAAGGTGGTCCGCCGGGTGTTGCTGGTCCGCCGGGTGGTTCTGGTCCGGCTGGTCCGCCGGGTCCGCAGGGTGTTAAAGGTGAACGTGGTTCTCCGGGTGGTCCGGGTGCTGGTGGTGAACGTGGTGCTCCGGGTGAAAAAGGTGAAGGTGGTCCGCCGGGTGTTGCTGGTCCGCCGGGTGGTTCTGGTCCGGCTGGTCCGCCGGGTCCGCAGGGTGTTAAAGGTGAACGTGGTTCTCCGGGTGGTCCGGGTGCTGGTTAA

[0196] Seq 91 (3 - fold interspersed inverted repeat of Seq 6 (expressed in E. coli)):

[0197] ATGGGTGAACGTGGTGCTCCGGGTGAAAAAGGTGAAGGTGGTCCGCCGGGTGTTGCTGGTCCGCCGGGTGGTTCTGGTCCGGCTGGTCCGCCGGGTCCGCAGGGTGTTAAAGGTGAACGTGGTTCTCCGGGTGGTCCGGGTGCTGGTGGTCCGTCTGGTCGTGAAGGTAAAGTTGGTCAGCCGGGTCCGCCGGGTGCTCCGGGTTCTGGTGGTCCGCCGGGTGCTGTTGGTCCGCCGGGTGGTGAAGGTAAAGAAGGTCCGGCTGGTCGTGAAGGTGGTCCGGGTGCTGGTGGTGAACGTGGTGCTCCGGGTGAAAAAGGTGAAGGTGGTCCGCCGGGTGTTGCTGGTCCGCCGGGTGGTTCTGGTCCGGCTGGTCCGCCGGGTCCGCAGGGTGTTAAAGGTGAACGTGGTTCTCCGGGTGGTCCGGGTGCTGGTTAA

[0198] Seq 92 (Seq 6 spacer inverted repeat 3x + linker 2 (expressed in E. coli)):

[0199] ATGGGTGAACGTGGTGCTCCGGGTGAAAAAGGTGAAGGTGGTCCGCCGGGTGTTGCTGGTCCGCCGGGTGGTTCTGGTCCGGCTGGTCCGCCGGGTCCGCAGGGTGTTAAAGGTGAACGTGGTTCTCCGGGTGGTCCGGGTGCTGGTGTTCCGGGTGGTCCGTCTGGTCGTGAAGGTAAAGTTGGTCAGCCGGGTCCGCCGGGTGCTCCGGGTTCTGGTGGTCCGCCGGGTGCTGTTGGTCCGCCGGGTGGTGAAGGTAAAGAAGGTCCGGCTGGTCGTGAAGGTGGTCCGGGTGCTGGTGTTCCGGGTGGTGGTGAACGTGGTGCTCCGGGTGAAAAAGGTGAAGGTGGTCCGCCGGGTGTTGCTGGTCCGCCGGGTGGTTCTGGTCCGGCTGGTCCGCCGGGTCCGCAGGGTGTTAAAGGTGAACGTGGTTCTCCGGGTGGTCCGGGTGCTGGTGGTTAA

[0200] Seq 93 (3 - fold direct repeat of Seq 7 (expressed in E. coli)):

[0201] ATGGGTATGCCGGGTCCGCGTGGTTCTCCGGGTCCGCAGGGTGTTAAAGGTGAATCTGGTAAACCGGGTGCTAACGGTCTGTCTGGTGAACGTGGTCCGCCGGGTCCGCAGGGTCTGCCGGGTCTGGCTGGTACCGCTGGTGAACCGGGTCGTGACGGTAACCCGGGTTCTGACGGTCTGCCGGGTCGTGACGGTTCTCCGGGTGGTAAAGGTGACCGTGGTGAAAACGGTATGCCGGGTCCGCGTGGTTCTCCGGGTCCGCAGGGTGTTAAAGGTGAATCTGGTAAACCGGGTGCTAACGGTCTGTCTGGTGAACGTGGTCCGCCGGGTCCGCAGGGTCTGCCGGGTCTGGCTGGTACCGCTGGTGAACCGGGTCGTGACGGTAACCCGGGTTCTGACGGTCTGCCGGGTCGTGACGGTTCTCCGGGTGGTAAAGGTGACCGTGGTGAAAACGGTATGCCGGGTCCGCGTGGTTCTCCGGGTCCGCAGGGTGTTAAAGGTGAATCTGGTAAACCGGGTGCTAACGGTCTGTCTGGTGAACGTGGTCCGCCGGGTCCGCAGGGTCTGCCGGGTCTGGCTGGTACCGCTGGTGAACCGGGTCGTGACGGTAACCCGGGTTCTGACGGTCTGCCGGGTCGTGACGGTTCTCCGGGTGGTAAAGGTGACCGTGGTGAAAACTAA

[0202] Seq 94 (3 - fold inverted repeat of the spacer of Seq 7 (expressed in E. coli)):

[0203] ATGGGTATGCCGGGTCCGCGTGGTTCTCCGGGTCCGCAGGGTGTTAAAGGTGAATCTGGTAAACCGGGTGCTAACGGTCTGTCTGGTGAACGTGGTCCGCCGGGTCCGCAGGGTCTGCCGGGTCTGGCTGGTACCGCTGGTGAACCGGGTCGTGACGGTAACCCGGGTTCTGACGGTCTGCCGGGTCGTGACGGTTCTCCGGGTGGTAAAGGTGACCGTGGTGAAAACGGTAACGAAGGTCGTGACGGTAAAGGTGGTCCGTCTGGTGACCGTGGTCCGCTGGGTGACTCTGGTCCGAACGGTGACCGTGGTCCGGAAGGTGCTACCGGTGCTCTGGGTCCGCTGGGTCAGCCGGGTCCGCCGGGTCGTGAAGGTTCTCTGGGTAACGCTGGTCCGAAAGGTTCTGAAGGTAAAGTTGGTCAGCCGGGTCCGTCTGGTCGTCCGGGTCCGATGGGTGGTATGCCGGGTCCGCGTGGTTCTCCGGGTCCGCAGGGTGTTAAAGGTGAATCTGGTAAACCGGGTGCTAACGGTCTGTCTGGTGAACGTGGTCCGCCGGGTCCGCAGGGTCTGCCGGGTCTGGCTGGTACCGCTGGTGAACCGGGTCGTGACGGTAACCCGGGTTCTGACGGTCTGCCGGGTCGTGACGGTTCTCCGGGTGGTAAAGGTGACCGTGGTGAAAACTAA

[0204] Seq 95 (Seq 7 spacer inverted repeat 3x + linker 2 (expressed in E. coli)):

[0205] ATGGGTATGCCTGGTCCACGTGGTTCCCCAGGTCCGCAAGGTGTTAAAGGCGAAAGCGGTAAACCTGGTGCTAATGGTCTGAGCGGTGAACGTGGTCCACCAGGTCCACAGGGTCTGCCTGGTCTGGCAGGTACTGCGGGTGAGCCTGGTCGTGATGGTAATCCGGGTTCTGACGGTCTGCCAGGTCGTGATGGCTCTCCAGGTGGTAAAGGTGATCGTGGTGAGAATGTTCCGGGTGGCGGCAACGAAGGTCGTGATGGTAAAGGTGGTCCGTCTGGTGACCGTGGTCCACTGGGTGATTCTGGTCCTAACGGCGATCGTGGTCCGGAAGGTGCAACCGGTGCACTGGGTCCTCTGGGTCAACCAGGTCCTCCAGGTCGTGAAGGTTCTCTGGGTAACGCCGGTCCGAAAGGCAGCGAAGGTAAGGTTGGTCAGCCAGGTCCTTCTGGTCGTCCGGGTCCAATGGGTGTACCAGGTGGTGGTATGCCGGGTCCACGTGGTTCTCCAGGTCCGCAAGGTGTAAAAGGTGAATCTGGTAAACCGGGTGCAAACGGTCTGTCTGGCGAACGTGGCCCTCCAGGTCCACAAGGTCTGCCTGGTCTGGCGGGTACTGCTGGTGAACCGGGTCGTGATGGTAACCCGGGTTCTGATGGTCTGCCTGGTCGTGATGGTTCCCCGGGTGGTAAAGGTGATCGCGGTGAAAACTAA

[0206] Seq 96 (3 - fold forward repeat of Seq 8 (expressed in E. coli)):

[0207] ATGGGTCCTCGTGGCGATAAAGGTGAAACGGGTGAGCGTGGTGCAGCTGGTATTAAAGGCCATCGTGGTTTTCCAGGTAATCCAGGTGCTCCAGGTTCTCCTGGTCCGGCAGGTCAACAGGGTGCTATCGGTTCTCCTGGTCCAGCTGGTCCTCGCGGTCCTGTAGGTCCATCTGGTCCTGGTAAAGATGGTACCAGCGGTCATCCGGGTCCTATTGGTCCACCTGGTCCGCGTGGCAATCGTGGTGAACGTGGTCCGCGCGGTGACAAAGGTGAAACTGGTGAACGTGGTGCGGCTGGTATTAAAGGTCACCGTGGCTTTCCAGGTAACCCAGGTGCTCCTGGTTCTCCAGGCCCTGCTGGTCAACAAGGTGCCATTGGTTCTCCTGGTCCGGCTGGTCCACGTGGTCCTGTTGGTCCATCTGGTCCGGGTAAAGATGGTACCTCTGGTCATCCAGGTCCAATTGGTCCTCCGGGTCCGCGTGGTAATCGTGGTGAACGTGGCCCGCGTGGTGACAAAGGCGAAACTGGTGAACGCGGTGCAGCAGGCATCAAAGGTCATCGTGGTTTCCCGGGTAACCCAGGTGCACCTGGTTCTCCGGGTCCAGCAGGTCAACAGGGTGCAATTGGTTCTCCAGGTCCAGCAGGTCCACGTGGTCCTGTGGGTCCTAGCGGTCCAGGTAAAGATGGTACCTCCGGTCATCCTGGTCCGATTGGTCCGCCAGGTCCACGTGGTAACCGTGGTGAACGTTAA

[0208] Seq 97 (Seq 8 Spacer Inverted Repeat x3 (expressed in E. coli)):

[0209] ATGGGTCCGCGTGGTGATAAGGGCGAAACTGGTGAACGTGGCGCTGCGGGTATCAAAGGCCACCGTGGTTTTCCGGGTAACCCTGGTGCACCGGGTTCTCCTGGTCCTGCTGGTCAGCAGGGTGCAATTGGTTCCCCTGGTCCTGCTGGTCCACGTGGTCCAGTTGGTCCTTCTGGTCCGGGTAAAGACGGTACCTCCGGTCATCCAGGTCCAATTGGTCCACCTGGTCCACGTGGTAACCGTGGTGAACGCGGTCGTGAAGGTCGTAATGGTCGTCCAGGCCCACCTGGTATTCCGGGTCCACACGGTTCTACTGGCGACAAAGGTCCGGGTTCTCCAGGTGTTCCTGGTCGTCCAGGTGCTCCAGGTCCGTCTGGTATTGCTGGTCAACAAGGTGCTCCGGGTCCATCTGGTCCTGCAGGTCCTAATGGTCCTTTCGGTCGTCACGGTAAAATTGGCGCTGCAGGTCGTGAAGGTACTGAAGGTAAAGATGGTCGTCCTGGCGGTCCGCGTGGTGATAAAGGCGAAACTGGTGAACGTGGTGCAGCTGGCATCAAAGGTCACCGTGGCTTCCCAGGTAATCCAGGTGCACCTGGTTCTCCAGGTCCAGCAGGCCAACAAGGTGCTATTGGTTCTCCGGGTCCAGCAGGTCCTCGTGGTCCAGTTGGTCCGTCTGGTCCAGGTAAAGATGGCACTAGCGGTCATCCGGGTCCAATTGGTCCTCCTGGTCCTCGTGGTAACCGTGGCGAACGCTAA

[0210] Seq 98 (Seq 8 Spacer Inverted Repeat x3 + Adapter 2 (expressed in E. coli)):

[0211] ATGGGTCCGCGCGGTGACAAGGGTGAAACTGGTGAACGTGGTGCTGCTGGTATCAAAGGTCACCGCGGTTTTCCAGGTAATCCTGGTGCTCCTGGTTCTCCAGGTCCGGCAGGTCAGCAGGGTGCAATTGGTTCTCCAGGTCCTGCTGGTCCTCGTGGTCCGGTTGGTCCATCTGGTCCGGGTAAAGATGGTACCTCTGGCCACCCAGGCCCTATTGGTCCTCCTGGTCCGCGTGGTAACCGTGGTGAACGTGGTGTCCCGGGTGGTCGTGAAGGTCGTAATGGTCGTCCGGGTCCACCTGGTATTCCTGGTCCGCACGGTAGCACCGGTGACAAAGGTCCAGGTTCTCCAGGTGTTCCGGGTCGTCCTGGTGCACCTGGTCCTTCTGGTATCGCTGGTCAGCAAGGTGCTCCAGGTCCATCTGGTCCAGCTGGTCCAAACGGTCCTTTCGGCCGTCATGGTAAAATCGGCGCTGCCGGCCGTGAAGGTACTGAAGGTAAAGATGGTCGTCCGGGTGTTCCAGGTGGTGGTCCTCGTGGTGACAAAGGCGAAACCGGTGAACGCGGTGCTGCAGGTATTAAAGGTCATCGTGGTTTTCCAGGTAACCCGGGTGCACCAGGTTCTCCTGGTCCTGCAGGCCAGCAAGGTGCTATTGGTTCTCCAGGTCCAGCTGGTCCACGTGGTCCAGTAGGTCCTTCTGGTCCGGGCAAAGATGGCACTTCCGGCCACCCAGGTCCAATTGGTCCGCCTGGTCCTCGTGGTAATCGTGGTGAACGTTAA

[0212] Seq 99 (3 - fold forward repeat of Seq 9 (expressed in Pichia pastoris)):

[0213]

[0214] Seq 100 (Seq 9 with 3-fold inverted repeats (expressed in Pichia pastoris)):

[0215]

[0216] Seq 101 (Seq 9 interspaced inverted repeat 3 times + adapter 2 (expressed in Pichia pastoris)):

[0217]

[0218] Seq 102 (Seq 10 forward repeated 3 times (expressed in E. coli)):

[0219] ATGGGCGAACGTGGCTTTCCGGGCGAACGTGGTGTTCAAGGCCCACCAGGTCCTGCAGGTGAACGTGGTTTCCCGGGTGAACGTGGTGTTCAAGGTCCGCCAGGTCCTGCTGGTGAACGTGGTTTTCCGGGCGAACGCGGTGTTCAAGGTCCACCTGGTCCGGCTTAA

[0220] Seq 103 (Seq 10 spaced inverted repeat 3 times (expressed in E. coli)):

[0221] ATGGGTGAACGTGGCTTCCCTGGTGAACGTGGCGTACAGGGTCCACCAGGTCCTGCTGGTGCACCTGGTCCACCTGGTCAGGTTGGTCGTGAAGGTCCGTTCGGCCGCGAAGGTGGTGAACGTGGTTTCCCGGGCGAACGTGGTGTACAAGGTCCGCCAGGTCCAGCTTAA

[0222] Seq 104 (Seq 10 spaced inverted repeat 3 times + adapter 2 (expressed in E. coli)):

[0223] ATGGGCGAACGCGGTTTCCAAGGTGAACGTGGTGTGCAGGGTCCTCCTGGTCCTGCAGGTGTTCCAGGTGGTGCTCCAGGTCCACCAGGTCAAGTAGGTCGTGAAGGTCCGTTTGGTCGTGAAGGTGTTCCAGGCGGTGGTGAACGTGGTTTTCCGGGCGAACGTGGTGTACAGGGTCCACCAGGTCCGGCATAA

[0224] Seq 105 (Seq 11 forward repeated 3 times (expressed in E. coli)):

[0225] ATGGGTGAACGTGGTGGCCCAGGTTCCCGTGGTTTTCCTGGTGCCGATGGCGTAGCAGGTCCAAAAGGTCCGGCCGGTGAACGTGGTGAACGTGGTGGTCCGGGTTCTCGTGGTTTCCCAGGTGCTGACGGTGTTGCTGGTCCGAAAGGTCCAGCTGGTGAACGTGGTGAGCGTGGTGGTCCGGGTTCTCGTGGTTTTCCGGGTGCGGATGGCGTTGCAGGTCCGAAAGGCCCTGCTGGTGAACGCTAA

[0226] Seq 106 (Seq 11 spacer inverted repeat 3 times (expressed in E. coli)):

[0227] ATGGGTGAACGTGGTGGTCCAGGTTCCCGTGGTTTTCCGGGTGCTGATGGTGTTGCCGGTCCGAAAGGTCCTGCGGGTGAACGTGGTCGTGAAGGTGCTCCGGGTAAACCGGGTGCAGTAGGTGATGCAGGTCCATTCGGTCGTTCTGGTCCGGGTGGTCGTGAAGGTGGTGAACGTGGTGGTCCGGGTTCTCGTGGTTTTCCGGGCGCTGATGGCGTAGCTGGCCCAAAAGGTCCTGCTGGCGAACGTTAA

[0228] Seq 107 (Seq 11 spacer inverted repeat 3 times + linker 2 (expressed in E. coli)):

[0229] ATGGGTGAACGTGGTGGTCCTGGTTCTCGTGGTTTTCCGGGCGCAGATGGTGTGGCTGGTCCGAAAGGTCCAGCAGGTGAACGTGGCGTACCTGGTGGTCGTGAAGGTGCCCCAGGTAAACCAGGTGCAGTAGGTGACGCTGGTCCTTTCGGTCGTTCTGGTCCGGGTGGTCGTGAAGGTGTTCCAGGTGGTGGTGAACGCGGCGGTCCGGGTTCTCGTGGTTTTCCAGGTGCAGATGGTGTAGCAGGTCCGAAAGGTCCGGCTGGTGAACGTTAA

[0230] Seq 108 (3 - fold direct repeat of Seq 12 (expressed in E. coli)):

[0231] ATGGGTATGCCAGGTGAACGTGGTGCAGCAGGCCTGCCAGGTCCAAAAGGTGATCGTGGCGACGCTGGTCCGAAAGGTGCTGACGGTTCTCCGGGCAAGGATGGTATGCCAGGTGAACGTGGTGCTGCTGGTCTGCCGGGTCCAAAAGGTGATCGTGGTGACGCAGGTCCGAAAGGTGCAGATGGTTCTCCGGGTAAAGACGGTATGCCAGGTGAACGTGGTGCGGCTGGTCTGCCAGGTCCTAAAGGTGACCGTGGTGATGCTGGCCCTAAAGGCGCAGATGGCTCTCCGGGTAAAGATTAA

[0232] Seq 109 (3 - fold inverted repeat with spacer of Seq 12 (expressed in E. coli)):

[0233] ATGGGTATGCCTGGTGAACGTGGCGCAGCTGGTCTGCCAGGTCCAAAAGGCGATCGTGGTGATGCAGGCCCGAAAGGTGCAGATGGTTCTCCGGGCAAAGATGGTGACAAAGGCCCGTCTGGTGATGCCGGTAAACCGGGTGCAGATGGTCGTGATGGTAAACCTGGTCCACTGGGTGCAGCAGGTCGTGAAGGTCCAATGGGTGGTATGCCGGGTGAACGTGGTGCTGCTGGTCTGCCTGGTCCGAAAGGTGATCGCGGTGATGCAGGCCCAAAAGGCGCTGATGGTTCTCCGGGTAAAGACTAA

[0234] Seq 110 (3 - fold inverted repeat with spacer of Seq 12 + linker 2 (expressed in E. coli)):

[0235] ATGGGTATGCCTGGTGAACGTGGTGCAGCTGGTCTGCCGGGTCCTAAAGGTGACCGTGGTGATGCAGGTCCGAAAGGTGCAGATGGTTCTCCGGGTAAAGATGGTGTTCCGGGCGGTGACAAAGGTCCGTCTGGTGATGCGGGCAAACCGGGTGCAGATGGTCGTGATGGTAAACCTGGTCCTCTGGGTGCTGCTGGTCGTGAAGGTCCGATGGGTGTTCCTGGCGGTGGTATGCCGGGTGAACGTGGTGCTGCTGGTCTGCCAGGTCCAAAGGGTGACCGTGGTGATGCTGGCCCAAAAGGTGCTGATGGTTCTCCGGGCAAAGACTAA

[0236] Seq 111 (Seq 13 forward repeat 3 times (expressed in E. coli)):

[0237] ATGGGTCCTCAGGGTATCGCTGGCCAACGTGGCGTTGTTGGTCTGCCAGGTCAACGTGGTGAACGTGGCTTTCCTGGTCTGCCAGGTCCAAGCGGTGAACCAGGCAAACAGGGTCCGTCTGGTGCATCTGGTGAACGTGGTCCGCCTGGTCCTCAGGGTATCGCAGGTCAACGTGGTGTCGTTGGCCTGCCAGGTCAGCGTGGTGAACGTGGTTTTCCTGGCCTGCCTGGTCCTTCTGGCGAACCAGGTAAACAGGGTCCATCTGGTGCGTCTGGTGAACGTGGTCCGCCAGGTCCACAAGGTATTGCAGGTCAGCGTGGTGTCGTTGGTCTGCCTGGTCAGCGTGGTGAACGCGGTTTCCCAGGTCTGCCGGGTCCATCTGGTGAACCGGGTAAACAGGGTCCGTCTGGCGCTTCTGGTGAACGTGGTCCGCCGTAA

[0238] Seq 112 (Seq 13 spacer reverse repeat 3 times (expressed in E. coli)):

[0239] ATGGGTCCGCAAGGCATCGCAGGTCAACGTGGTGTTGTTGGTCTGCCGGGTCAGCGTGGTGAACGTGGTTTTCCTGGTCTGCCGGGTCCTAGCGGTGAACCAGGTAAACAAGGCCCATCCGGTGCTTCTGGTGAACGTGGTCCACCAGGTCCTCCGGGTCGTGAAGGTTCTGCAGGTAGCCCTGGCCAGAAAGGTCCAGAAGGTTCTCCGGGTCCTCTGGGTCCGTTTGGTCGTGAAGGTCGTCAGGGTCCTCTGGGTGTCGTGGGTCGTCAAGGTGCAATTGGTCAGCCGGGTGGTCCGCAAGGTATTGCTGGTCAACGTGGTGTAGTTGGTCTGCCGGGTCAACGTGGTGAACGTGGTTTTCCGGGTCTGCCTGGTCCATCTGGTGAACCGGGTAAGCAGGGTCCGTCTGGTGCGTCTGGTGAACGTGGTCCGCCGTAA

[0240] Seq 113 (Seq 13 spacer inverted repeat 3 - fold + linker 2 (expressed in E. coli)):

[0241] ATGGGTCCGCAAGGTATCGCTGGCCAGCGTGGTGTTGTTGGTCTGCCAGGTCAACGTGGTGAACGTGGCTTCCCGGGTCTGCCAGGTCCTTCTGGTGAACCGGGTAAACAGGGTCCGTCTGGTGCTTCTGGCGAACGTGGTCCTCCTGGTGTTCCGGGTGGTCCTCCTGGTCGTGAAGGTTCTGCTGGTTCTCCGGGTCAGAAAGGTCCGGAAGGTTCTCCAGGTCCGCTGGGTCCATTCGGTCGTGAAGGTCGTCAGGGTCCGCTGGGTGTAGTGGGTCGTCAGGGTGCAATTGGTCAACCAGGTGTTCCTGGTGGTGGTCCGCAGGGTATCGCAGGTCAACGCGGTGTTGTAGGTCTGCCTGGTCAGCGTGGCGAACGTGGTTTTCCGGGTCTGCCAGGTCCATCCGGTGAACCTGGTAAACAGGGTCCTTCTGGTGCTAGCGGCGAACGTGGTCCGCCATAA

[0242] Seq 114 (3 - fold direct repeat of Seq 14 (expressed in E. coli)):

[0243] ATGGGTCCAGACGGTAAAACTGGTCCACCGGGTCCAGCAGGTCAGGATGGTCGTCCAGGTCCTCCAGGTCCACCAGGTGCACGTGGTCAGGCTGGTGTGATGGGTTTCCCGGGTCCAAAAGGTGCAGCTGGCGAACCGGGTAAAGCAGGTGAGCGTGGTCCGGATGGCAAAACTGGTCCGCCTGGTCCTGCTGGTCAAGATGGTCGTCCTGGTCCTCCTGGTCCACCTGGTGCACGTGGTCAGGCTGGTGTTATGGGTTTTCCGGGTCCGAAGGGTGCTGCAGGTGAACCTGGTAAAGCAGGTGAGCGTGGTCCAGACGGTAAAACTGGTCCTCCAGGTCCGGCTGGTCAAGATGGTCGTCCAGGTCCTCCTGGTCCACCTGGTGCTCGTGGTCAGGCTGGTGTGATGGGCTTTCCGGGTCCGAAAGGTGCTGCAGGCGAACCTGGTAAAGCAGGTGAACGTTAASeq 115 (3 - fold inverted repeat of the spacer of Seq 14 (expressed in E. coli)):

[0244] ATGGGCCCGGATGGTAAAACTGGTCCTCCTGGTCCGGCAGGTCAAGATGGTCGTCCTGGTCCACCAGGTCCTCCAGGTGCTCGTGGTCAGGCAGGTGTTATGGGTTTCCCGGGCCCAAAAGGTGCTGCGGGTGAACCTGGCAAAGCTGGTGAGCGTGGTCGTGAAGGTGCTAAAGGTCCAGAAGGTGCAGCTGGTAAACCGGGTCCGTTTGGCATGGTTGGTGCTCAAGGTCGTGCAGGTCCACCAGGTCCACCAGGTCCGCGTGGTGATCAGGGTGCTCCGGGTCCGCCGGGTACTAAAGGTGACCCAGGTGGTCCAGATGGCAAAACTGGTCCTCCTGGTCCTGCTGGTCAGGATGGTCGTCCTGGTCCTCCAGGTCCTCCGGGTGCACGCGGTCAAGCAGGTGTGATGGGTTTCCCGGGCCCTAAAGGTGCGGCCGGTGAACCGGGCAAAGCAGGTGAACGCTAA

[0245] Seq 116 (Seq 14 Spacer Inverted Repeat 3x + Linker 2 (expressed in E. coli)):

[0246] ATGGGTCCAGATGGTAAAACTGGTCCGCCAGGTCCGGCTGGTCAAGATGGTCGTCCAGGTCCTCCAGGTCCTCCAGGTGCTCGTGGTCAAGCTGGCGTAATGGGCTTTCCGGGTCCGAAAGGTGCTGCGGGTGAACCAGGTAAAGCTGGTGAACGTGGTGTACCGGGTGGTCGCGAAGGTGCAAAGGGTCCTGAAGGTGCGGCAGGTAAACCGGGTCCGTTCGGTATGGTTGGTGCACAAGGTCGTGCTGGTCCTCCAGGTCCTCCTGGTCCACGTGGTGATCAGGGTGCGCCGGGTCCACCTGGTACCAAAGGTGATCCTGGTGTACCGGGTGGTGGCCCAGACGGTAAAACCGGTCCACCGGGTCCTGCCGGTCAGGATGGTCGTCCTGGTCCACCGGGTCCACCTGGTGCCCGTGGCCAGGCAGGTGTCATGGGTTTTCCGGGTCCGAAAGGCGCCGCAGGCGAGCCGGGTAAAGCAGGCGAACGCTAA

[0247] Seq 117 (3 - fold forward repeat of Seq 15 (expressed in E. coli)):

[0248] ATGGGTGAACGCGGTGAGCCAGGTCTGGTGGGTGATCCTGGTCCACGTGGTGAACCGGGCCCTAAAGGTGCACCAGGTCTGCCAGGTCAGCCAGGTCCACCAGGTCTGCCAGGTGTACCGGGTCAAGCTGGTGCTCCTGGTGAACGTGGTGAACCAGGTCTGGTAGGTGATCCAGGTCCGCGTGGTGAACCGGGTCCAAAAGGTGCACCGGGTCTGCCAGGTCAACCTGGTCCTCCAGGTCTGCCTGGTGTTCCAGGTCAAGCTGGTGCTCCGGGTGAACGTGGTGAACCGGGTCTGGTTGGCGATCCAGGTCCTCGTGGTGAACCGGGTCCGAAAGGTGCTCCAGGTCTGCCTGGTCAACCTGGTCCACCGGGTCTGCCAGGTGTTCCTGGTCAGGCTGGCGCACCATAA

[0249] Seq 118 (3 - fold tandem inverted repeat of Seq 15 spacer (expressed in E. coli)):

[0250] ATGGGTGAGCGTGGTGAACCGGGTCTGGTTGGTGATCCAGGCCCTCGTGGTGAACCGGGTCCAAAAGGTGCGCCTGGTCTGCCAGGTCAACCAGGTCCACCAGGTCTGCCAGGTGTTCCAGGTCAAGCAGGTGCTCCAGGTCCTGCGGGTGCTCAAGGTCCAGTAGGTCCACTGGGTCCTCCAGGTCCTCAAGGTCCACTGGGTCCGGCAGGTAAACCTGGTCCGGAAGGTCGTCCGGGTCCAGATGGTGTACTGGGTCCTGAAGGTCGTGAAGGTGGTGAACGTGGCGAACCAGGTCTGGTTGGTGACCCAGGTCCACGTGGTGAACCGGGTCCTAAAGGTGCACCAGGTCTGCCAGGTCAGCCAGGTCCACCTGGTCTGCCTGGTGTTCCGGGTCAGGCTGGTGCACCATAA

[0251] Seq 119 (3 - fold tandem inverted repeat of Seq 15 spacer + linker 2 (expressed in E. coli)):

[0252] ATGGGCGAACGTGGCGAACCAGGTCTGGTAGGTGATCCGGGTCCTCGTGGTGAACCGGGTCCAAAAGGTGCTCCAGGTCTGCCAGGTCAACCGGGTCCACCAGGTCTGCCAGGTGTACCTGGTCAGGCAGGTGCGCCAGGTGTTCCTGGTGGTCCAGCAGGTGCGCAAGGTCCAGTTGGTCCACTGGGTCCACCTGGTCCTCAGGGTCCACTGGGTCCAGCTGGTAAACCGGGTCCTGAAGGTCGTCCTGGCCCAGATGGTGTTCTGGGTCCGGAAGGTCGTGAAGGTGTGCCTGGCGGTGGTGAACGTGGTGAACCGGGTCTGGTTGGTGATCCAGGTCCTCGTGGTGAACCAGGTCCAAAAGGTGCTCCGGGTCTGCCTGGTCAACCAGGTCCTCCAGGCCTGCCTGGTGTTCCAGGTCAGGCTGGTGCTCCTTAA

[0253] Seq 120 (3 - fold forward repeat of Seq 16 (expressed in E. coli)):

[0254] ATGGGTGAACGTGGTCCTCCAGGTCCAGCAGGTGAACGTGGTCTGCCGGGTGTTGCTGGTCGTCCTGGTGCTCAAGGTCCGGAAGGTCCGCCAGGTCCAACTGGTCGTCAAGGTGAACGTGGTGAACCGGGTCCGCGTGGTGATCCTGGTGAACGTGGTCCGCCAGGTCCTGCAGGTGAACGTGGTCTGCCTGGTGTAGCAGGTCGTCCAGGTGCTCAAGGTCCGGAAGGTCCTCCAGGTCCTACGGGTCGTCAGGGTGAACGTGGTGAACCTGGTCCGCGTGGTGATCCAGGTGAACGTGGTCCGCCTGGTCCAGCAGGTGAACGTGGTCTGCCAGGCGTTGCAGGTCGTCCAGGTGCTCAAGGTCCTGAAGGTCCACCAGGTCCAACTGGCCGTCAGGGTGAACGTGGCGAACCAGGTCCGCGTGGTGATCCATAA

[0255] Seq 121 (Seq 16 Spacer Inverted Repeat Triplication (expressed in E. coli)):

[0256] ATGGGTGAACGTGGTCCACCAGGTCCGGCAGGTGAACGTGGTCTGCCAGGTGTCGCTGGTCGTCCTGGTGCTCAGGGTCCTGAAGGTCCACCAGGTCCTACTGGTCGTCAGGGTGAACGTGGCGAACCAGGTCCACGTGGCGATCCTGGTCCAGATGGTCGTCCGGGTCCGGAAGGTCGTGAAGGTCAGCGTGGTACTCCAGGTCCACCGGGTGAACCAGGTCAAGCTGGTCCGCGTGGTGCAGTAGGTCCACTGGGTCGTGAAGGTGCTCCAGGTCCGCCAGGTCGTGAAGGTGGTGAACGTGGTCCTCCAGGTCCTGCTGGTGAACGCGGTCTGCCTGGTGTTGCTGGTCGTCCAGGTGCTCAAGGTCCAGAAGGTCCACCAGGCCCGACTGGTCGTCAAGGTGAACGCGGTGAACCTGGTCCTCGTGGTGATCCGTAA

[0257] Seq 122 (Seq 16 Spacer Inverted Repeat Triplication + Linker 2 (expressed in E. coli)):

[0258] ATGGGTGAACGTGGTCCTCCTGGTCCTGCTGGTGAACGTGGTCTGCCGGGTGTTGCTGGTCGTCCTGGTGCTCAGGGTCCAGAAGGTCCTCCTGGTCCTACTGGTCGTCAGGGTGAACGTGGTGAACCGGGTCCACGTGGTGATCCAGGTGTTCCAGGTGGTCCTGATGGTCGTCCAGGTCCTGAAGGTCGTGAAGGTCAGCGTGGTACTCCAGGTCCTCCGGGTGAACCGGGTCAAGCAGGTCCACGTGGTGCGGTAGGTCCTCTGGGTCGTGAAGGCGCTCCTGGTCCACCAGGTCGTGAAGGTGTTCCAGGTGGTGGTGAACGTGGTCCACCAGGTCCGGCTGGTGAACGTGGTCTGCCAGGTGTGGCTGGTCGTCCAGGTGCTCAAGGTCCAGAAGGTCCACCTGGTCCGACTGGTCGTCAAGGCGAACGTGGCGAACCAGGTCCACGTGGTGATCCGTAA

[0259] Seq 123 (3 - fold direct repeat of Seq 17 (expressed in E. coli)):

[0260] ATGGGTCCGCAGGGTGAAGTAGGCGATCCAGGTGCTGAGGGTCCGATGGGTCAGCGTGGTCGTGAAGGTCCACGTGGTGAAGCAGGCCCTCCAGGTGACCGTGGTGAACGTGGTCCTCAGGGCGAAGTAGGTGATCCGGGTGCTGAAGGTCCGATGGGTCAACGTGGTCGTGAAGGTCCGCGTGGTGAAGCAGGTCCTCCTGGTGATCGTGGTGAACGTGGCCCGCAAGGCGAAGTTGGTGATCCGGGTGCTGAAGGCCCAATGGGTCAGCGTGGTCGTGAAGGTCCGCGTGGTGAAGCAGGTCCACCTGGTGATCGTGGTGAACGCTAA

[0261] Seq 124 (3 - fold spaced inverted repeat of Seq 17 (expressed in E. coli)):

[0262] ATGGGTCCGCAAGGTGAGGTTGGTGATCCGGGCGCTGAGGGTCCAATGGGTCAACGTGGTCGTGAAGGTCCTCGTGGTGAAGCAGGTCCACCAGGTGACCGTGGTGAACGTGGCCGTGAAGGTCGTGATGGTCCGCCAGGTGCTGAAGGTCGTCCTGGTGAACGTGGTCGTCAGGGTATGCCGGGTGAAGCAGGTCCGGATGGTGTTGAAGGTCAGCCAGGCGGCCCGCAAGGTGAAGTAGGTGATCCAGGTGCTGAGGGTCCGATGGGTCAGCGTGGTCGTGAGGGTCCTCGTGGTGAAGCGGGTCCTCCTGGTGATCGTGGTGAACGTTAA

[0263] Seq 125 (Seq 17 spacer inverted repeat 3x + linker 2 (expressed in E. coli)):

[0264] ATGGGTCCACAAGGTGAAGTTGGTGATCCTGGCGCTGAAGGTCCTATGGGTCAACGTGGTCGTGAAGGTCCACGTGGTGAAGCTGGTCCGCCAGGTGACCGTGGTGAACGTGGTGTACCGGGTGGTCGTGAAGGTCGTGATGGTCCTCCAGGCGCTGAAGGTCGTCCAGGTGAACGTGGTCGTCAGGGTATGCCTGGTGAAGCTGGTCCGGACGGTGTTGAAGGTCAACCGGGTGTTCCAGGTGGTGGTCCGCAAGGTGAAGTAGGCGATCCGGGTGCGGAAGGTCCAATGGGTCAGCGTGGTCGTGAAGGTCCTCGTGGTGAAGCTGGTCCTCCTGGTGATCGTGGTGAACGTTAA

[0265] Fermentation of recombinant collagen expression strain and treatment of fermentation broth

[0266] The recombinant collagen expression strains were inoculated into appropriate seed culture medium, grown to the logarithmic growth phase, and then inoculated into a shake flask containing a suitable fermentation medium. After a certain period of fermentation, an inducer was added to induce expression, and the fermentation was stopped when the bacterial biomass or expression product no longer increased. The fermentation broth was centrifuged at high speed to collect the supernatant and bacterial cells. The supernatant was directly used for subsequent purification. The bacterial cells were resuspended in phosphate buffer, then crushed with a high-pressure homogenizer, and then centrifuged at high speed to collect the supernatant for subsequent purification.

[0267] The seed culture medium and fermentation culture medium of the recombinant collagen protein expression strain based on Escherichia coli of the present invention are LB culture medium (yeast powder 5.0g / l, peptone 10.0g / l, NaCl 10.0g / l), the growth temperature is 37°C, and the shaking speed is 150-300rpm. The inducer is 0.02mM-1mM IPTG, and the fermentation temperature after adding the inducer is 10°C, or 16°C, or 20°C, or 25°C, or 30°C, or 37°C. The fermentation liquid is centrifuged to collect the bacterial cells, and then the bacterial cells are treated according to the above-described crushing method, and then the supernatant and the precipitate are collected respectively for purification.

[0268] The seed culture medium of the recombinant collagen protein expression strain based on Pichia pastoris of the present invention is YPD culture medium (yeast powder 10 g / l, peptone 20 g / l, glucose 20 g / l), and the fermentation culture medium before induction is BMG culture medium (yeast powder 10 g / l, peptone 20 g / l, dipotassium hydrogen phosphate trihydrate 3 g / l, potassium dihydrogen phosphate 11.8 g / l, 4×10 -5 % Biotin, 1% glycerol), the fermentation medium after induction was BMM medium (yeast powder 10 g / l, peptone 20 g / l, potassium dihydrogen phosphate trihydrate 3 g / l, potassium dihydrogen phosphate 11.8 g / l, 4×10 -5 % Biotin, 0.5% methanol), the growth and fermentation temperature is 25-32°C, the shaking speed is 150-300 rpm, and the inducer is methanol with a final concentration of 0.2%-1% added every 24 hours. Only the supernatant of the fermentation broth is collected for subsequent purification.

[0269] Purification of recombinant collagen

[0270] The liquid containing the target protein treated above is precipitated and salted out with 0% to 30% ammonium sulfate, and then purified by Ni column affinity chromatography under 20 to 40mM imidazole conditions, washed with impurities under 150 to 200mM imidazole conditions, and eluted under 300 to 400mM imidazole conditions, and the sample purity can reach more than 70%. In order to further improve the sample purity and remove impurities such as pigments, ion exchange chromatography is used for purification, and the equilibrium conditions are 50 to 100mM NaCl, pH 5.0 to 6.5, and the elution conditions are 200 to 300mM NaCl, pH5.0 to 6.5.

[0271] Example 4: Determination of molecular weight and protein content of recombinant collagen

[0272] The apparent molecular weight of the recombinant collagen was determined by SDS-PAGE. The theoretical molecular weight of the recombinant collagen was determined by HPLC analysis under the following conditions: SEC 120 column, 0.15 M phosphate buffer as the mobile phase, flow rate of 1 ml / min, and column temperature of 30°C.

[0273] The apparent molecular weight of the recombinant collagen obtained based on the 17 GXY core unit sequences mentioned above is higher than the theoretical molecular weight. The reason may be related to the hydrophilicity of the collagen core sequence, which has been confirmed in the SDS-PAGE diagram of the recombinant collagen. Based on this, it can be judged that the target protein is correctly expressed. Figure 3 When Seq 1 shown in (a) was expressed in E. coli in duplicate for 3 times, the theoretical molecular weight was 11 kD, and the apparent molecular weight obtained by SDS-PAGE was about 16 kD (see Figure 3 (a) indicated by the arrow). Figure 3 Lane 1 of (a): supernatant sample of bacterial cell fragmentation expressed by Seq 1 forward repeat 3 times (Seq 18); Lane 2: supernatant sample of bacterial cell fragmentation expressed by Seq 1 reverse repeat 3 times (Seq 19); Lane 3: supernatant sample of bacterial cell fragmentation expressed by Seq 1 reverse repeat 3 times with interval plus adapter 1 (Seq 20); Lane 4: supernatant sample of bacterial cell fragmentation expressed by Seq 1 reverse repeat 3 times with interval plus adapter 2 (Seq 21); Lane 5: supernatant sample of bacterial cell fragmentation expressed by Seq 1 reverse repeat 3 times with interval plus adapter 3 (Seq 22); Lane 6: supernatant sample of bacterial cell fragmentation expressed by Seq 1 reverse repeat 3 times with interval plus adapter 4 (Seq 23); Lanes 1' to 6': bacterial cell precipitate samples of corresponding expression; Lane M: protein marker; the sample loading volume is 5 μl (sample before purification). For another example Figure 3 (b) shows the SDS-PAGE electrophoresis of the purified samples of Seq 1 to Seq 17, which were expressed by three times of reverse repetition and adapter 2. Lanes 1 to 17 correspond to samples of Seq 1 to Seq 17 (seq21, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71), and the loading volume is 5 to 10 μl. Except for Seq 9 (Seq 47) which was expressed in Pichia pastoris, the rest were expressed in Escherichia coli. Figure 3 From the electrophoresis diagram in (b), some degradation occurred in some samples. It is speculated that this is mainly related to the core unit sequence itself and its expression host. It may also be related to the instability of some samples themselves or inappropriate conditions during the storage process.

[0274] Combined with the SDS-PAGE electrophoresis diagram described above, Quantity One software was used to scan and analyze the proportion of the target protein, and then the Bradford method was used to determine the total protein content of the bacteria, so as to calculate the content of the recombinant collagen based on the proportion of the target protein and the total protein content.

[0275] Taking Seq 1 as an example, the target protein is basically not expressed directly without repeating the sequence. According to the general method, the target protein can be produced by tandem expression in Escherichia coli after forward repeating 3 times (see electrophoresis diagram). Figure 3 (a)), after the sequence interval was reversely repeated 3 times according to the method provided by the present invention, the expression level of the target protein shown by SDS-PAGE electrophoresis scanning increased by 2.2 times (see Table 2), which indicates that the method provided by the present invention obtains a higher expression level when the core unit sequence is repeated for the same multiples.

[0276] The carboxyl end of the Seq 1 core unit sequence contains a GPNG motif, which has the potential to form a β-fold or a turn, and helps to guide the core unit sequence of the intervening reverse repeat to form a folded structure. The folded protein relies on the GXY unit itself to easily form a triple helix structure through hydrogen bonds, covalent or non-covalent cross-linking, so it is possible to form a triple helix or triple helix-like folded structure. The stability of the protein with a folded structure is higher than that of a long single-chain protein, so the ability to resist environmental degradation is stronger, and the expression level is higher under the same conditions. Further, based on the design concept of the present invention, a linker 1, a linker 2, a linker 3 or a linker 4 that helps to form a β-fold or a turn is added between two adjacent Seq 1 core unit sequences of the intervening reverse repeat, and the expression level is increased by 2.2 to 2.8 times compared with the expression method of the forward repeat.

[0277] Table 2 Comparison of expression levels of core unit sequence Seq 1 in different expression strategies in Escherichia coli

[0278] Seq1 expression strategy Expression level increased by fold Repeat 3 times in the forward direction 1.0 Repeat 3 times with interval reverse 2.2 Interval reverse repeat 3 times + connector 1 2.6 Interval reverse repeat 3 times + connector 2 2.8 Interval reverse repeat 3 times + connector 3 2.2 Interval reverse repeat 3 times + connector 4 2.6

[0279] Example 5: Cell proliferation activity assay of purified recombinant collagen

[0280] Use recombinant collagen to coat cell culture plates or other validated methods, use appropriate cells that may be encountered in the expected clinical application, such as skin fibroblasts, with a seeding density of about 50%, detect cell proliferation after 1 to 3 days of routine culture, and examine the dose-effect relationship of the gradient concentration of recombinant collagen coating.

[0281] The present invention adopts NIH / 3T3 cells or HSF cells with a seeding density of about 50%, inoculated in a 96-well plate, and 100μl of cell suspension is added to each well, and cultured overnight. On the second day, 500μg / ml, 100μg / ml, 50μg / ml, 10μg / ml, 5μg / ml, 1μg / ml, 0.1μg / ml, and 0.01μg / ml are added according to the gradient dilution. After 24h, 48h or 96h of culture, CCK8 is used to detect cell proliferation, 10μl per well, and its light absorption value is measured at a wavelength of 450nm using an enzyme-linked immunosorbent assay, which can indirectly reflect the number of living cells. Cell viability is defined as the cell proliferation survival rate compared with the control without drug addition, and EC50 refers to the drug addition concentration required when the cell proliferation reaches half of the highest platform value.

[0282] The cell proliferation activity of the recombinant collagen protein related to the Seq 1 core unit sequence expressed by the present invention is shown in Table 3 and Attached Figure 4 (Results after 48 hours of drug addition and culture), the cells used were NIH / 3T3 cells. Compared with the forward repeat expression, the cell proliferation activity of the recombinant collagen obtained by the interspersed reverse repeat expression was significantly improved, indicating that the improvement of cell proliferation activity was mainly caused by the interspersed reverse repeat expression method, which is related to the fact that the recombinant collagen expressed by this method can form a possible folding structure. The effect of adding a linker on cell activity is relatively small because Seq 1 itself contains a motif that is easy to form folds or turns, and the linker only plays an auxiliary role in the formation of the secondary structure of the recombinant collagen. However, the linker sequence is derived from fibrous proteins such as natural human collagen or natural human elastin, which itself has certain cell functions, thereby causing a smaller difference in cell activity of the recombinant collagen. Figure 4 Expression strategies in, A: forward repeat 3 times (seq 18); B: spaced inverted repeat 3 times (seq 19); C: spaced inverted repeat 3 times + adapter 1 (seq 20); D: spaced inverted repeat 3 times + adapter 2 (seq 21); E: spaced inverted repeat 3 times + adapter 3 (seq 22); F: spaced inverted repeat 3 times + adapter 4 (seq 23).

[0283] Table 3 Cell proliferation activity of recombinant collagen proteins related to the core unit sequence of Seq 1

[0284]

[0285] Other core unit sequences expressed by the present invention also have similar cell activity results as mentioned above, see Table 4 (gradient dilution and drug addition 500μg / ml, 100μg / ml, 50μg / ml, 10μg / ml, 5μg / ml, 1μg / ml, 0.1μg / ml, 0.01μg / ml cultured for 48h) for the Cell viability platform value (i.e., Cell viability when reaching the proliferation platform phase) and EC50 value. In general, the cell activity of most recombinant collagen proteins obtained by the intermittent reverse repeat expression provided by the present invention is higher than that of the recombinant collagen proteins obtained by the general forward repeat expression, which is reflected in a higher platform value, or a smaller EC50 value, or both.

[0286] Table 4 Comparison of cell proliferation activity of recombinant collagens expressed in two strategies for core unit sequences Seq 2 to Seq 17

[0287]

[0288] Example 6: Size Exclusion Chromatography Analysis of Recombinant Collagen

[0289] Size Exclusion Chromatography (SEC), also known as Molecular Sieve HPLC, is often used to analyze protein purity and determine the molecular weight of proteins. Proteins with small molecular weights will enter the molecular sieve gel pores more, with longer retention times and later peak times, while proteins with large molecular weights will have earlier peak times.

[0290] The mobile phase for SEC detection of recombinant collagen of the present invention is 150mM phosphate buffer, the sample loading is not less than 10μg, and the peak is detected at a wavelength of 215nm. The present invention first determines the peak time of a series of standard proteins of known molecular weight, obtains a standard curve, and then calculates the relative molecular weight by the peak time of the recombinant collagen to be tested under the same conditions. The peak times of two 44kD and 13.7kD standard proteins used in the present invention are 15.314min and 17.599min respectively.

[0291] The SEC spectra of the three expression strategies of the core unit sequence Seq 1 of the present invention are shown in Figure 5 . Figure 5(a): forward repeat 3 times (seq 18); (b): intermittent reverse repeat 3 times (seq 19); (c): intermittent reverse repeat 3 times + linker 2 (seq 21). Although these recombinant proteins show similar sizes and single bands on SDS-PAGE electrophoresis, the SEC spectra show large differences. The recombinant collagen protein obtained by forward repeat expression has a main peak of 15.456min and a relative molecular weight of about 40kD. It is speculated that this peak is the state of the recombinant protein forming a trimer. The protein expressed by intermittent reverse repeat has three main peaks at 15.341min, 16.542min, and 17.582min, respectively. The first two main peaks should be the recombinant protein forming a trimer and dimer, respectively. The relative molecular weight of the latter peak is about 14kD, which is comparable to the monomer of the recombinant protein. Collagen has a tendency to spontaneously form aggregates under high purity and high concentration conditions, otherwise it is very unstable as a long-chain monomer. After the expression of the interspaced inverted repeat, an obvious peak of monomer molecular weight appeared, which is consistent with the concept of the present invention, that is, a stable folding structure inside the monomer is formed. Further, the recombinant collagen obtained by expressing the interspaced inverted repeat plus linker 2 showed two main peaks at 16.141min and 17.327min, respectively. It is also speculated that the former is a dimer and the latter is a monomer forming an internal folding structure. There is no peak of trimer, indicating that the recombinant protein of the interspaced inverted repeat plus linker 2 has a tendency to form a folding structure inside the monomer.

[0292] Example 7: Secondary structure of recombinant collagen

[0293] The circular dichroism spectra of protein secondary structures such as α helix, β fold, random coil, polyproline II helix, etc. have their own typical characteristic absorption peaks. Polyproline II helix is ​​a triple helix structure, which mainly exists in natural collagen. The circular dichroism characteristics of proteins with triple helix structure generally have a strong negative absorption peak at 190-200nm and a weak positive absorption peak at 215-225nm. The position of the absorption peak sometimes shifts with the length of the protein molecule and its amino acid sequence. According to experience, compared with the characteristic absorption peak of the triple helix structure, if the positive absorption peak is not obvious or shows a weak negative value, the protein structure is more likely to be a random coil. If the positions of the positive absorption peak and the negative absorption peak are approximately reversed, the protein structure is more likely to be a β fold.

[0294] The circular dichroism detection conditions of the recombinant collagen of the present invention are as follows: scanning wavelength 260-190nm, scanning speed 100nm / min, bandwidth 1nm, response value 1s, buffer 0.05M acetic acid, sample concentration not less than 0.1mg / ml.

[0295] The circular dichroism spectra of the three expression strategies of the core unit sequence Seq 1 of the present invention are shown in Figure 6 . Figure 6 (a): forward repeat 3 times (seq 18); (b): interspaced reverse repeat 3 times (seq 19); (c): interspaced reverse repeat 3 times + linker 2 (seq21). The recombinant collagen obtained by forward repeat expression has basically no observable characteristic secondary structure. The recombinant collagen obtained by interspaced reverse repeat expression has a significant negative absorption peak at 200nm, forming a triple helix-like structure. Compared with the characteristic peak of the typical triple helix structure, there is no positive absorption peak at around 220nm. The recombinant collagen obtained by expressing interspaced reverse repeat plus linker 2 is even closer to the triple helix structure, and the absorption peak after exceeding 220nm is close to 0. Most of the other core unit sequences expressed in the present invention also showed a similar formation of this folded secondary structure, which is consistent with the aforementioned results of high expression level, high cell activity and molecular weight.

Claims

1. A recombinant collagen, comprising at least three peptide segments in its structure, wherein the peptide segments are peptide segment A and reverse peptide segment A, and the peptide segment A and the reverse peptide segment A are arranged alternately, and the peptide segment A comprises the following structure: (GXY) n , the reverse A peptide segment contains the following structure: (YXG) n , wherein G is glycine, X and Y are any natural or non-natural imino group or amino acid other than cysteine ​​independently in each repeating unit, n is selected from an integer greater than or equal to 3, and its sequence is selected from SEQ ID NO: 19-23, 25, 26, 28, 29, 31, 32, 34, 35, 37, 38, 40, 41, 43, 44, 46, 47, 49, 50, 52, 53, 55, 56, 58, 59, 61, 62, 64, 65, 67, 68, 70, 71.

2. A polynucleotide encoding the recombinant collagen according to claim 1.

3. An expression vector incorporating the polynucleotide according to claim 2. A host cell transformed with the polynucleotide according to claim 2 and expressing the polynucleotide.

5. A method for producing recombinant collagen in a host cell, the method comprising culturing the host cell of claim 4 under culture conditions such that the recombinant collagen is produced. A composition comprising the recombinant collagen according to claim 1 .

7. Use of the recombinant collagen according to claim 1 in the preparation of biomedical materials.

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