Production of recombinant collagenase active in plants derived from grimontia hollisae

Abstract

The present invention relates to a technology for stably mass-producing a recombinant collagenase derived from Grimontia hollisae by utilizing a plant expression system. A collagenase variant of the present invention overcomes the limitations associated with decreases in activity and purity due to autolysis and maintains excellent collagen-degrading activity, and thus may be provided as an economical and stable supply source in various industrial fields such as medicine and tissue engineering in the future.

Description

Production of active recombinant collagenase derived from GRIMONTIA HOLLISAE in plants

[0001] The present invention relates to a technology for producing recombinant collagenase using a plant expression system, and more specifically, to a Grimontia hollisae-derived recombinant collagenase variant that inhibits autolysis and maintains high activity, and a method for producing the same using a plant expression system.

[0002] Recently, there has been a continuously increasing interest in non-invasive treatment methods in the fields of medicine and biotechnology that can minimize the physical burden on patients. As part of these treatment strategies, enzyme-based therapeutic approaches are attracting attention as an important technological tool in the treatment of various diseases and tissue regeneration, based on their high selectivity and biocompatibility. Generally, enzymes possess the advantage of exhibiting high catalytic activity even at very low concentrations, enabling them to efficiently induce biological reactions.

[0003] Among these enzymes, collagenase is known to play a role in promoting tissue regeneration and wound healing by degrading collagen through the selective cleavage of its peptide bonds within damaged tissues. Collagenase has been reported to possess therapeutic efficacy not only for wound healing but also for various conditions such as burns, triceps, herniated discs, keloids, cellulite, and lipomas; accordingly, its potential applications in the medical and pharmaceutical fields are continuously being studied. Furthermore, collagenase is widely used in tissue engineering and cell biology research as an essential enzyme for isolating primary cells from tissues.

[0004] To date, collagenase is generally produced primarily using fish-derived tissues (SM Daboor et al., 2010) or bacterial expression systems (Yaqing Zhu et al., 2011). However, these existing production methods entail various technical limitations. First, in the case of naturally derived collagenase, the isolation and purification processes are complex, yields are low, and the reproducibility of activity is poor depending on process conditions. Particularly when using bacterial expression systems, it has been reported that collagenase tends to be easily degraded by autolysis or host proteases, leading to a significant decrease in protein stability during the production process (Gree et al. (2017), Tanaka et al. (2020)).

[0005] Furthermore, since collagenase is essentially a proteolytic enzyme, it frequently causes structural damage to active proteins or partial degradation of expressed proteins into fragmented forms by non-specifically cleaving itself or host proteins during the production process. This degradation phenomenon leads to problems such as reduced total collagenase activity, increased activity variability between batches, and decreased purification efficiency, making it difficult to stably secure high-purity, high-activity collagenase suitable for therapeutic or research use.

[0006] Some prior art proposes methods such as optimizing expression conditions, using protease inhibitors, and low-temperature expression to mitigate these problems; however, these approaches have limitations as they increase process complexity or raise production costs. Furthermore, the problem remains that these approaches alone cannot fundamentally resolve the structural instability and degradation susceptibility of collagenase itself.

[0007] Meanwhile, the production of recombinant proteins using plant expression systems has the advantages of mass production potential, reduced risk of contamination by pathogenic microorganisms, and a relatively simple purification process utilizing affinity tags.

[0008] Therefore, there is a continuous demand for technology that provides a structurally stabilized form of protein that allows collagenase to maintain its original physiological activity without being degraded during the production process.

[0009] Daboor, S. M., et al. (2012). Isolation and activation of collagenase from fish processing waste. Advances in Bioscience and Biotechnology, 3, 191-203.

[0010] Zhu, Y., et al. (2022). Optimized recombinant expression and characterization of collagenase in Bacillus subtilis WB600. Fermentation, 8(9), 449.

[0011] Gree, R., et al. (2017). Effectiveness of different molecular forms of Clostridium histolyticum class I collagenase to recover islets. Islands, 9, 177-181.

[0012] Tanaka, H., et al. (2020). Recombinant collagenase from Grimontia hollisae as a tissue dissociation enzyme for isolating primary cells. Scientific Reports, 10, 3927.

[0013] The present invention aims to provide a collagenase variant that can maintain its original physiological activity without self-degradation during the production process, a nucleotide sequence encoding the same, a recombinant expression vector into which the nucleotide sequence is introduced, a transgenic plant transformed with the recombinant expression vector, and a method for mass-producing the collagenase variant using the transgenic plant.

[0014] To achieve the above objective, the present invention provides a collagenase variant in which one or more of the amino acid residues at the 622nd, 625th, 635th, 638th, 640th, 643rd, 644th, and 647th positions of the amino acid sequence of SEQ ID NO. 1 are substituted with alanine (Ala), serine (Ser), or threonine (Thr).

[0015] In the present invention, the collagenase variant may be characterized by having an N-terminal leader sequence and a prodomain deleted.

[0016] In the present invention, the collagenase variant may be characterized by having 1 to 87 amino acids deleted based on the amino acid sequence of SEQ ID NO. 1.

[0017] In the present invention, the collagenase variant may be characterized by being represented by the amino acid sequence of SEQ ID NO. 2.

[0018] In the present invention, the collagenase variant may be characterized by having an autodegradation rate improved by 50% or more compared to recombinant wild-type collagenase.

[0019] The present invention also provides a base sequence encoding the collagenase variant.

[0020] The present invention also provides a recombinant expression vector into which the above-mentioned nucleotide sequence is introduced.

[0021] The above recombinant expression vector may be characterized by including a collagenase variant expression cassette, a CRT expression cassette, and a p38 expression cassette.

[0022] In the present invention, the collagenase variant expression cassette may be characterized by having a promoter represented by the nucleotide sequence of SEQ ID NO. 3, a 5' UTR represented by the nucleotide sequence of SEQ ID NO. 4, a sequence encoding a collagenase variant represented by the amino acid sequence of SEQ ID NO. 8, and a terminator represented by the nucleotide sequence of SEQ ID NO. 12 operably connected.

[0023] In the present invention, the collagenase expression cassette may be characterized by additionally operably linked a base sequence encoding an endoplasmic reticulum targeting sequence, a base sequence encoding a purification tag, and / or a base sequence encoding an endoplasmic reticulum retention sequence.

[0024] In the present invention, the CRT expression cassette may be characterized by having a promoter represented by the nucleotide sequence of SEQ ID NO. 13, a 5' UTR represented by the nucleotide sequence of SEQ ID NO. 5, a CRT represented by the nucleotide sequence of SEQ ID NO. 14, and a terminator represented by the nucleotide sequence of SEQ ID NO. 12 operably connected.

[0025] In the present invention, the CRT expression cassette may be characterized by additionally operably connecting a base sequence encoding an endoplasmic reticulum targeting sequence, a base sequence encoding a purification tag, and / or a base sequence encoding an endoplasmic reticulum retention sequence.

[0026] In the present invention, the p38 expression cassette comprises a promoter represented by the nucleotide sequence of SEQ ID NO. 18,

[0027] It may be characterized by an L-UTR represented by the nucleotide sequence of SEQ ID NO. 19, a p38 represented by the nucleotide sequence of SEQ ID NO. 20, and a terminator represented by the nucleotide sequence of SEQ ID NO. 22 being operably connected.

[0028] The present invention also provides a transgenic plant transformed with the recombinant expression vector.

[0029] The present invention also provides a method for producing a collagenase variant, comprising the following steps:

[0030] (a) a step of culturing the above-mentioned transgenic plant; and

[0031] (b) A step of isolating and purifying collagenase variants from the above-mentioned transgenic plants.

[0032] In the present invention, the transgenic plant may be characterized by being selected from food crops including rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats, and sorghum; vegetable crops including Arabidopsis thaliana, Chinese cabbage, radish, chili pepper, strawberry, tomato, watermelon, cucumber, cabbage, Korean melon, pumpkin, green onion, onion, and carrot; specialty crops including ginseng, tobacco, cotton, sesame, sugarcane, sugar beet, perilla, peanuts, and rapeseed; fruit trees including apple trees, pear trees, jujube trees, peaches, grapes, citrus fruits, persimmons, plums, apricots, lemons, and bananas; and flowers including roses, carnations, chrysanthemums, lilies, sunflowers, cosmos, and tulips.

[0033] In the present invention, the collagenase variant produced by the above method may be characterized by having collagenase activity.

[0034] The recombinant collagenase variant according to the present invention can stably maintain the structural integrity of the protein throughout the production and purification processes by effectively suppressing autolysis and non-specific cleavage, which are inherent problems of proteolytic enzymes, through the substitution of amino acid residues at specific positions and the deletion of the prodomain. In addition, by utilizing an optimized expression system including a plant cell endoplasmic reticulum (ER) targeting and accumulation system and a gene silencing inhibitor (p38), the limitations of existing bacterial systems can be overcome to mass-produce high-purity and high-activity collagenase in high yield. Furthermore, the purified enzyme retains its excellent collagen-degrading activity, which has an outstanding effect of enabling the economical and stable supply of therapeutic proteins that can be utilized in various industrial fields such as medical, pharmaceutical, and tissue engineering.

[0035] Figure 1 shows a schematic diagram (A) of a recombinant expression vector for expressing a collagenase variant in a plant and the results (B) of analyzing whether the collagenase variant expressed in a plant using the recombinant expression vector was expressed by Western blot and Coomassie staining.

[0036] Figure 2 shows the results of analyzing the protein distribution at each purification step using SDS-PAGE after purifying the recombinant collagenase protein expressed in plants.

[0037] Figure 3 shows the results of evaluating collagen degradation activity using purified collagenase, including a reaction composition table (A) for the collagen degradation reaction and the results of analyzing the protein degradation pattern generated after the reaction using SDS-PAGE (B).

[0038] The present invention will be described in more detail below.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0040] In the numerical ranges described in this specification, “to” is used to mean including both threshold ranges (greater than or equal to and less than), and when not including both threshold ranges, the numerical range is described as “greater than” and “less than.” In this specification, the term “about” used for numerical values ​​is used to mean a range that is expected to produce an effect substantially equivalent to the stated numerical value by a person skilled in the art; for example, it may be ±20%, ±10%, ±5%, etc. of the stated numerical value, but is not limited thereto.

[0041] In the present invention, a collagenase variant was developed using a plant system that can maintain its original physiological activity without self-degradation. Specifically, it was confirmed that when a specific amino acid is substituted in wild-type collagenase derived from Grimontia hollisae, the self-degradation rate of collagenase is significantly improved, and a structurally sound, highly active enzyme can be mass-produced in a plant system.

[0042] Accordingly, in one aspect, the present invention relates to a collagenase variant that maintains physiological activity without self-degradation.

[0043] In the present invention, the collagenase relates to a collagenase variant in which one or more of the amino acid residues at the 622nd, 625th, 635th, 638th, 640th, 643rd, 644th, and 647th positions based on the amino acid sequence of SEQ ID NO. 1 are substituted with alanine (Ala), serine (Ser), or threonine (Thr).

[0044]

[0045]

[0046]

[0047] In the present invention, the collagenase variant may be characterized by having an N-terminal leader sequence and a prodomain deleted, and in one embodiment, 1 to 87 amino acids may be deleted (ΔN87) based on the amino acid sequence of SEQ ID NO. 1.

[0048] In the present invention, the collagenase variant may be represented by SEQ ID NO. 2, and the eight amino acid residues represented by X may each independently be any one of glycine, alanine, serine, or threonine. However, at least one of the eight amino acid residues may be an amino acid residue other than glycine.

[0049]

[0050]

[0051]

[0052] In the present invention, the collagenase variant may have a significantly improved autolysis rate compared to recombinant wild-type collagenase, for example, an improvement of about 10% or more. In some embodiments, the degree of improvement in the autolysis rate may be any numerical value or any numerical range within the range of about 50% to about 1000%. For example, the degree of improvement in the autolysis rate may be in the range of about 50% to about 100%, about 80% to about 200%, about 100% to about 300%, about 150% to about 500%, or about 200% to about 800%, and exemplarily, may be improved by about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, about 150% or more, about 200% or more, about 250% or more, or about 300% or more.

[0053] In the present invention, the autolysis rate may be evaluated based on the change in total protein amount during the process of expressing and producing the collagenase variant or recombinant wild-type collagenase in a plant cell or plant body. In this case, “the autolysis rate is improved compared to recombinant wild-type collagenase” may mean a state in which, under the same expression conditions, the collagenase variant maintains a higher amount of protein quantified at the time of recovery after expression compared to the corresponding recombinant wild-type collagenase. Here, the amount of protein may be measured by Western blot, ELISA, total protein quantification analysis, or other known methods equivalent thereto.

[0054] In the present invention, the collagenase variant may have improved enzyme activity compared to recombinant wild-type collagenase. In this specification, “improved enzyme activity” means a state in which, under the same conditions, the initial enzyme activity or residual enzyme activity after a certain period of time is relatively increased when the collagenase variant is expressed in a plant (optional, purified after expression) compared to the corresponding recombinant wild-type collagenase.

[0055] The degree of improvement in enzyme activity may be expressed as the ratio of increase in enzyme activity or the ratio of increase in residual enzyme activity relative to recombinant wild-type collagenase, and this degree of improvement may be converted into a percentage (%) for comparison. For example, enzyme activity may be increased by about 10% or more, about 20% or more, about 30% or more, about 50% or more, or about 100% or more, but is not limited thereto.

[0056] The above enzyme activity can be measured through a collagen degradation reaction, substrate conversion rate, amount of product formed, or a known method for evaluating enzyme activity equivalent thereto, and such evaluation can be performed independently or together with the improvement of the above autolysis rate.

[0057] In the present invention, the improvement of enzyme activity may include cases where the enzyme structure is stably maintained by inhibiting the autolysis of the collagenase variant during the enzyme reaction process, and as a result, the decrease in enzyme activity is prevented or the active state is maintained for a long time. That is, as the autolysis of the collagenase variant is reduced compared to the recombinant wild-type collagenase, the proportion of effective active enzyme increases, and consequently, the measured enzyme activity may appear relatively high.

[0058] In another aspect, the present invention relates to a base sequence encoding the collagenase variant.

[0059] In another aspect, the present invention relates to a recombinant expression vector into which the above-mentioned nucleotide sequence is introduced.

[0060] As a specific embodiment, the recombinant expression vector may include a collagenase variant expression cassette, a CRT expression cassette, and a p38 expression cassette.

[0061] In the present invention, the collagenase variant expression cassette may be one in which a sequence encoding a collagenase variant is operably linked to a promoter, an expression control sequence, and a terminator.

[0062] In one embodiment, the collagenase variant expression cassette may be operably connected to a promoter represented by the nucleotide sequence of SEQ ID NO. 3, a 5' UTR represented by the nucleotide sequence of SEQ ID NO. 4, a sequence encoding a collagenase variant represented by the amino acid sequence of SEQ ID NO. 8, and a terminator represented by the nucleotide sequence of SEQ ID NO. 12.

[0063] In the present invention, the CRT expression cassette may have a sequence encoding a CRT operably connected to a promoter, an expression control sequence, and a terminator.

[0064] In the present invention, calreticulin (CRT) is a chaperone protein present in the lumen of the endoplasmic reticulum of plant cells, and is involved in the folding and stability of recombinant proteins that are targeted and expressed in the endoplasmic reticulum. When recombinant proteins are expressed in the endoplasmic reticulum in plants, CRT can contribute to improving the accumulation or production yield of proteins by inhibiting protein misfolding and degradation.

[0065] In one embodiment, the CRT expression cassette may be operably connected to a promoter represented by the nucleotide sequence of SEQ ID NO. 13, a 5' UTR represented by the nucleotide sequence of SEQ ID NO. 5, a CRT represented by the nucleotide sequence of SEQ ID NO. 14, and a terminator represented by the nucleotide sequence of SEQ ID NO. 12.

[0066] In another embodiment, the collagenase expression cassette and / or CRT expression cassette may additionally operably link a base sequence encoding an endoplasmic reticulum targeting sequence, a base sequence encoding a purification tag, and / or a base sequence encoding an endoplasmic reticulum retention sequence.

[0067] In the present invention, the endoplasmic reticulum targeting sequence refers to an amino acid sequence used for the purpose of transporting the collagenase variant into the endoplasmic reticulum, and any endoplasmic reticulum targeting signal sequence known in the art may be used without limitation. The endoplasmic reticulum targeting sequence may generally be a signal peptide located at the N-terminus of a protein, and may include, for example, a binding immunoglobulin protein (BiP) signal sequence, a calreticulin signal sequence, a calnexin signal sequence, or a modified sequence functionally equivalent thereto, but is not limited thereto.

[0068] In the present invention, the purification tag may use any tag peptide known in the art for the purpose of isolating and purifying collagenase variants without limitation. Specifically, the protein tag may be one or more selected from the group consisting of Avi tag, Calmodulin tag, polyglutamate tag, E tag, FLAG tag, HA tag, His tag, Myc tag, S tag, SBP tag, IgG-Fc tag, CTB tag, Softag 1 tag, Softag 3 tag, Strep tag, TC tag, V5 tag, VSV tag, and Xpress tag. In one embodiment, the purification tag may be a sequence encoding the His tag represented by SEQ ID NO. 10 and may be represented by the nucleotide sequence of SEQ ID NO. 9. In another embodiment, the purification tag may be a sequence encoding the HA tag represented by SEQ ID NO. 17 and may be represented by the nucleotide sequence of SEQ ID NO. 16.

[0069] In the present invention, the endoplasmic reticulum retention sequence refers to an amino acid sequence used for the purpose of maintaining the collagenase variant within the endoplasmic reticulum, and any endoplasmic reticulum retention signal sequence known in the art may be used without limitation. The endoplasmic reticulum retention sequence may generally be located at the C-terminus of a protein and serves to inhibit the protein that has moved to the endoplasmic reticulum from moving to other cellular organelles, such as the Golgi apparatus. For example, the endoplasmic reticulum retention sequence may include KDEL, HDEL, or a functionally equivalent modified sequence, but is not limited thereto.

[0070] In the present invention, the p38 expression cassette may be one in which a sequence encoding p38 is operably linked to a promoter, an expression control sequence, and a terminator.

[0071] In the present invention, p38 is a gene silencing suppressor derived from a plant virus, which means a protein that increases the expression level of foreign proteins by inhibiting the RNA silencing pathway in plant cells.

[0072] In one embodiment, the p38 expression cassette may be operably connected to a promoter represented by the nucleotide sequence of SEQ ID NO. 18, an L-UTR represented by the nucleotide sequence of SEQ ID NO. 19, a p38 represented by the nucleotide sequence of SEQ ID NO. 20, and a terminator represented by the nucleotide sequence of SEQ ID NO. 22.

[0073] In the present invention, the term “expression construct” refers to a combination of genes designed for the expression of a specific target protein. This term may be used interchangeably with the commonly used terms “gene construct” or “expression cassette.” Such an expression construct refers to a combination of DNA sequences designed to induce efficient and stable protein expression within a specific host cell or tissue, comprising one or more regulatory sequences (e.g., promoter, 5' UTR, intron, leader sequence, 3' UTR, terminator, etc.) and a target gene.

[0074] The amino acid sequences and nucleic acid sequences described in this specification may be interpreted to extend to sequences having homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with the provided sequences.

[0075] The above "% of sequence homology" can be verified by comparing two optimally arranged sequences with a comparison region, and a portion of the nucleotide sequence in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for the optimal arrangement of the two sequences.

[0076] In the present invention, the collagenase variant may additionally have a conservative amino acid substitution introduced at an amino acid residue other than the amino acid residue to which the variant was introduced.

[0077] The term “conservative amino acid substitution” denotes any amino acid substitution for a given amino acid residue, wherein the surrogate residue is chemically very similar to that of the given residue, such that no substantial reduction in polypeptide function (e.g., enzyme activity) occurs. Conservative amino acid substitutions are commonly known in the prior art, and examples thereof are described, for example, in U.S. Patent Nos. 6,790,639, 6,774,107, 6,194,167, or 5,350,576. In a preferred embodiment, the conservative amino acid substitution will be any one occurring within one of the following six groups.

[0078] Amino acids may be grouped as follows according to their common side chain properties, except where otherwise stated in this specification:

[0079] (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

[0080] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0081] (3) Acids: Asp, Glu;

[0082] (4) Basics: His, Lys, Arg;

[0083] (5) Residues affecting chain orientation: Gly, Pro;

[0084] (6) Aromatic: Trp, Tyr, Phe.

[0085] For example, conservative substitutions may be the amino acid substitutions listed in Table 1 below.

[0086] [Table 1]

[0087]

[0088] In this specification, the term “recombinant” means a result (e.g., nucleic acid, vector) in which nucleic acid is introduced, modified, or rearranged by artificial (experimental) manipulation, unlike in its naturally occurring state, an organism configured to express the result (e.g., cell, microorganism, plant, animal, or virus), or the product thereof (e.g., protein).

[0089] For example, “recombinant cell” refers to a cell that contains heterologous nucleic acid or engineered nucleic acid (e.g., mutation, substitution, deletion, insertion, rearrangement, synthetic sequence, etc.), or is produced by transformation, transduction, or other genetic engineering methods to express heterologous polypeptide as a result thereof.

[0090] Here, foreign nucleic acids may include (i) sequences that do not exist in the natural state of the cell species, or (ii) sequences that, even if naturally present, have been artificially introduced or manipulated to regulate expression (e.g., same-species genes driven by heterologous promoter / regulatory sequences, codon optimization sequences, tag addition sequences, variant sequences, etc.).

[0091] Additionally, “recombinant protein” refers to a protein encoded by recombinant nucleic acid and expressed or manufactured in a recombinant host, which may have the same sequence as the natural protein (e.g., heterologous expression of the same sequence) or may include modifications (e.g., substitution / deletion / insertion / fusion / tag addition).

[0092] In this specification, the term “recombinant expression vector” means a vector artificially configured to contain foreign nucleic acid to enable the transcription and / or translation of said nucleic acid within a host cell. The recombinant expression vector may be, for example, a bacterial plasmid, a phage, a yeast plasmid, a plant virus, a plant expression vector, a mammalian cell virus, or other equivalent vectors, but is not limited thereto.

[0093] Generally, any plasmid or vector may be used as the recombinant expression vector of the present invention as long as it can be replicated or maintained stably within a host cell. The recombinant expression vector preferably comprises an origin of replication, a promoter that regulates transcription, a selectable marker gene, and a translation control element.

[0094] The above translation regulatory elements may include, for example, a ribosome binding site, a 5′ untranslated region (5′ UTR), a sequence around the start codon, or a combination thereof, and serve to regulate the efficiency of protein expression in a host cell. Additionally, the expression vector may additionally include a transcription termination sequence, a 3′ untranslated region (3′ UTR), a polyadenylation signal, or a combination thereof as needed.

[0095] A recombinant expression vector containing appropriate transcription and / or translation regulatory signals may be constructed by methods known to those skilled in the art, and such methods include, but are not limited to, in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombinant techniques.

[0096] The above "operably linked" may be a gene and an expression control sequence linked in such a manner that gene expression is enabled when an appropriate molecule binds to the expression control sequence. An "expression control sequence" means a DNA sequence that regulates the expression of an operably linked sequence in a specific host cell. Such a control sequence includes a promoter for carrying out transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0097] The expression construct of the present invention can be effectively linked to a suitable promoter within an expression vector to lead mRNA synthesis. Suitable vectors for expressing the DNA fragment and the gene encoding the target protein according to the present invention in plant cells include, but are not limited to, the pCAMBIA1300 series, pRTVn, pRI 101, pGreenII series, pBI series, pBIN19, pPZP series, pEAQ series, pH7WG2, pK7WG2, Geminiviral vector, etc.

[0098] A preferred example of a recombinant expression vector of the present invention is a Ti-plasmid vector capable of transferring a portion of itself, the so-called T-region, into a plant cell when present in a suitable host, such as Agrobacterium toumafaciens and Rhizobium rhizogenes. Other types of Ti-plasmid vectors are currently used to transfer hybrid DNA sequences into plant cells or protoplasts from which new plants can be produced by appropriately inserting the hybrid DNA into the plant genome. A particularly preferred form of a Ti-plasmid vector is a so-called binary vector as claimed in EP 0120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce DNA according to the present invention into a plant host may be selected from viral vectors, such as those derived from double-stranded plant viruses (e.g., CaMV) and single-stranded viruses, Gemini viruses, etc., such as incomplete plant viral vectors. The use of such vectors can be advantageous, especially when it is difficult to properly transform plant hosts.

[0099] As a specific embodiment, the recombinant expression vector may be a recombinant expression vector in which an expression construct according to the present invention is sequentially and operably connected using a conventional vector used for protein expression as a basic framework.

[0100] In addition, the recombinant expression vector may include a ribosome binding site and a transcription terminator as a translation initiation site.

[0101] The recombinant expression vector will preferably include one or more selectable markers. The markers are nucleic acid sequences having characteristics that can typically be selected by chemical methods, and include all genes capable of distinguishing transformed cells from non-transformed cells. Examples include, but are not limited to, herbicide resistance genes such as glyphosate or phosphinothricin, antibiotic resistance genes such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol, and the aadA gene.

[0102] In the recombinant expression vector of the present invention, the promoter may be, but is not limited to, Glu13a, GluB1, Glub4, Prolamin, Fmm, FM'M-UD, CSVMV, de35s, CaMV 35S, actin, ubiquitin, pEMU, amylase, or Clp promoters. The term “promoter” refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which RNA polymerase binds to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. A “constitutive promoter” is a promoter that is active under most environmental conditions, developmental states, or cell differentiation. Since the selection of transformants can be made by various tissues at various stages, constitutive promoters may be preferred in the present invention. Therefore, constitutive promoters do not limit the selectivity.

[0103] In the present invention, the term “terminator” refers to a regulatory sequence that improves gene expression efficiency by inducing the precise termination and stabilization of transcribed mRNA. The terminator functions to prevent unnecessary transcriptional prolongation by providing a transcription termination signal, and to maintain a constant protein expression level by increasing the stability of the generated mRNA. In the recombinant expression vector of the present invention, the terminator may include, but is not limited to, a nopaline synthase (NOS) terminator, an octopine synthase (OCS) terminator, a Cauliflower mosaic virus 35S (CaMV 35S) terminator, a rice actin (Act1) terminator, an Arabidopsis thaliana heat shock protein (HSP) terminator, a rubisco small subunit (rbcS) terminator, a pea rbcS E9 terminator, an Arabidopsis ubiquitin (UBQ) terminator, a protease inhibitor, a tobacco extensin terminator, and other complex constituent terminators (e.g., terminator 9 used in the present invention).

[0104] In another aspect, the present invention relates to a transgenic plant transformed with the recombinant expression vector.

[0105] The above plants are a concept encompassing plant cells and plant bodies, and the plants may be selected from food crops including rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats, and sorghum; vegetable crops including Arabidopsis thaliana, Chinese cabbage, radish, chili peppers, strawberries, tomatoes, watermelons, cucumbers, cabbage, Korean melons, pumpkins, leeks, onions, and carrots; specialty crops including ginseng, tobacco, cotton, sesame, sugarcane, sugar beets, perilla, peanuts, and rapeseed; fruit trees including apple trees, pear trees, jujube trees, peaches, grapes, citrus fruits, persimmons, plums, apricots, lemons, and bananas; and flowers including roses, carnations, chrysanthemums, lilies, sunflowers, cosmos, and tulips, but are not limited thereto.

[0106] In another aspect, the present invention provides a method for producing a transgenic plant cell or a transgenic plant, comprising the step of introducing the recombinant expression vector into a plant cell or a plant body.

[0107] The above-mentioned recombinant expression vector may be introduced into plant cells or plants, and the introduction method may be any one of the following: Agrobacterium sp.-mediated transformation method, particle bombardment, sonication, electroporation, and PEG (polyethylene glycol)-mediated transformation method, but is not limited thereto. These introduction methods are well known in the art.

[0108] In another aspect, the present invention relates to a method for producing a collagenase variant comprising the following steps.

[0109] (a) a step of culturing the above-mentioned transgenic plant; and

[0110] (b) A step of recovering (e.g., isolating and purifying) collagenase variants from the above-mentioned transgenic plants.

[0111] The method for producing a target protein from the above-described transformed plant can be obtained by transforming plant cells with a recombinant expression vector according to the present invention, then culturing for a suitable period of time to express a collagenase variant, and then obtaining the protein from the transformed cells.

[0112] At this time, any method known in the art for expressing the collagenase variant can be used.

[0113] In the production method of the present invention, in order to introduce the recombinant expression vector into a plant, a culture of a transformant into which the recombinant expression vector has been introduced may be introduced into a plant cell or a plant body. For example, the culture of the transformant may be introduced by syringe infiltration into the leaves of the plant or by vacuum infiltration. The Agrobacterium injected in this manner receives a signal from the acetosyringone substance and delivers the expression construct of the recombinant expression vector into the plant cell.

[0114] Collagenase variants can be recovered through various separation and purification methods known in the art. Typically, to remove cell debris, the cell lysate may be centrifuged, followed by precipitation, e.g., salting out (precipitation of ammonium sulfate and sodium phosphate), solvent precipitation (precipitation of protein fractions using acetone, ethanol, etc.), and dialysis, electrophoresis, and various column chromatographs may be performed. Purification may be achieved by applying techniques such as ion exchange chromatography, gel-permeation chromatography, HPLC, reverse-phase HPLC, affinity column chromatography, or ultrafiltration, either individually or in combination.

[0115] Collagenase variants produced by the above method have their autodegradation inhibited, so collagenase activity can be effectively maintained.

[0116]

[0117] The present invention will be described in more detail below through examples. However, as the present invention is susceptible to various modifications and may take various forms, the specific examples and descriptions provided below are intended only to aid in understanding the invention and are not intended to limit the invention to the specific disclosed forms. The scope of the present invention should be understood to include all modifications, equivalents, and substitutions that fall within the spirit and technical scope of the invention.

[0118]

[0119] Example 1. Preparation of expression cassettes and expression analysis for high expression of Grimontia hollisae-derived collagenase in Nicotiana benthamiana plants

[0120]

[0121] 1-1. Collagenase Recombinant Gene Design

[0122] For plant expression, a variant Collagenase-8G / A was first designed by replacing eight glycine residues (amino acid positions 622, 625, 635, 638, 640, 643, 644, and 647) in the collagenase sequence derived from Grimontia hollisae with alanine residues.

[0123] In addition, to ensure stable protein expression, a variant of collagenase ΔN87[8G / A] with the N-terminal leader sequence and prodomain removed was designed.

[0124] A BamHI restriction enzyme recognition sequence was introduced to the N-terminus of the above collagenase ΔN87[8G / A] gene sequence, and a His-tag consisting of six histidine residues was introduced to the C-terminus. In addition, to ensure that collagenase expressed in plant cells accumulates in the endoplasmic reticulum, an ER retention signal (HDEL) was added to the C-terminus of the His-tag, and a recombinant gene was designed to include a stop codon and an XhoI restriction enzyme recognition sequence. The designed recombinant gene was chemically synthesized (Genuniversal, USA).

[0125] 1-2. Construction of Expression Vector

[0126] The synthesized collagenase ΔN87[8G / A] DNA fragment was cleaved with BamHI and XhoI restriction enzymes, and then ligated into the expression vector p38-CRT-99 TEX1L, which was cleaved with the same restriction enzymes, to construct the recombinant expression vector pTEX1L::GHΔN87[8G / A].

[0127] The above expression vector p38-CRT-99 TEX1L was completed by cutting the chemically synthesized XhoI-MluI-CsVMV promoter-ApaI fragment with XhoI and ApaI, cutting the chemically synthesized ApaI-5'-UTR-BiP-CRT-HA-HDEL-StuI-terminator #9-EcoRI fragment with ApaI and EcoRI, then triple ligating it into the pTEX1L vector (method for mass production of target protein in plants; KR20210117808A) cut with XhoI and EcoRI, cutting it into XhoI and MluI, and then ligating it into the chemically synthesized XhoI-terminator 9-MluI fragment cut with XhoI and MluI.

[0128] The above recombinant expression vector is designed to include p38, a gene silencing suppressor, and CRT, an endoplasmic reticulum chaperone protein, within a single vector, with the purpose of enhancing the expression level of foreign proteins in plant cells. In addition, an Arabidopsis thaliana BiP-derived ER targeting signal sequence derived from pTEX1L (KR20210117808A) is located at the N-terminus of the expression vector, so that the expressed collagenase is efficiently targeted and accumulated in the endoplasmic reticulum.

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[0151] 1-3. Agrobacterium-mediated plant infiltration and expression

[0152] The constructed expression vector was introduced into Agrobacterium tumefaciensEHA105 (Japan Tobacco Inc.). The transformed Agrobacterium was cultured overnight and then resuspended in a suspension containing 10 mM MgCl2, 10 mM MES (pH 5.6), and acetosyringone. The concentration of the Agrobacterium suspension was OD 600 The value was adjusted to 0.8.

[0153] The modified Agrobacterium suspension was applied to Nicotiana benthamiana leaves by an infiltration method using a syringe, so that a collagenase expression cassette was delivered into the plant leaf cells in the form of T-DNA.

[0154] 1-4. Protein Extraction and Expression Analysis

[0155] Nicotiana benthamiana (Lehle Seeds, USA) leaves were harvested on days 3 and 5 after infiltration treatment, respectively, ground under liquid nitrogen, and total protein extracts were prepared. Proteins were extracted using an extraction buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and a protease inhibitor cocktail (Sigma-Aldrich, USA).

[0156] 6× sample buffer (500 mM Tris-HCl [pH 6.8], 10% SDS, 0.5% bromophenol blue, 30% (v / v) glycerol, 100 mM DTT) was added to the extracted protein supernatant to adjust the concentration to a final 1× level, and the mixture was heated for 10 minutes to prepare a protein sample for electrophoresis. The sample was separated via 10% SDS-PAGE and then analyzed using western blot analysis with an anti-His antibody (Sigma-Aldrich, USA) and a secondary antibody conjugated with the corresponding HRP (goat anti-mouse IgG-HRP, Sigma-Aldrich, USA).

[0157] As a result, a protein band corresponding to collagenase was identified at approximately 75 kDa, and a relatively higher expression level was observed in the sample on day 3 compared to the sample on day 5.

[0158]

[0159] Example 2. Isolation and purification of Grimontia hollisae-derived collagenase expressed in Nicotiana benthamiana

[0160]

[0161] 2-1. Sample Preparation and Protein Extraction

[0162] 20 g of leaf tissue from Nicotiana benthamiana plants expressing Grimontia hollisae-derived collagenase was collected and powdered under liquid nitrogen. The powdered tissue was suspended in protein extraction buffer (100 mM NaCl, 25 mM Tris-HCl, pH 7.5, 0.2% Tween-20) at a ratio of 1 g : 10 mL and adjusted to a final volume of 200 mL.

[0163] To prevent oxidation and protein degradation of the sample during the extraction process, 0.3% (w / v) of PVPP (Sigma-Aldrich, USA), 0.3% (w / v) of activated charcoal powder (Sigma-Aldrich, USA), and 2 mM of PMSF (Sigma-Aldrich, USA) were added immediately before extraction. The mixture was incubated at 4°C for 15 minutes and mixed uniformly to ensure sufficient protein extraction.

[0164] 2-2. Removal of Solids and Clarification

[0165] The extract was centrifuged in stages to remove plant tissue residues and insoluble impurities. After each centrifugation stage, the supernatant was filtered using a gauze cloth before proceeding to the next step.

[0166] 1st-2nd centrifugation: 12,000 RPM, 15 min, 4℃ (250 mL bottle)

[0167] Tertiary centrifugation: 14,000 RPM, 30 min, 4℃ (50 mL bottle)

[0168] 4th centrifugation: 14,000 RPM, 10 min, 4℃ (2 mL tube)

[0169] 2-3. Affinity Purification

[0170] Ni in the purified supernatant 2+ 1 mL of NTA agarose beads (Qiagen, Germany) was added, and the final concentration was adjusted to 10 mM using imidazole (Sigma-Aldrich, USA) to minimize non-specific binding. The supernatant and beads were mixed at a ratio of approximately 100:1 (v / v), and then stirred at 4–9°C for 90 minutes to bind His-tag attached collagenase to the beads.

[0171] After the binding reaction, the beads were recovered and washed twice with 100 mL of washing buffer (25 mM Tris-HCl, pH 7.5, 20 mM imidazole) to remove unbound proteins and non-specific binding proteins.

[0172] 2-4. Elution and Concentration

[0173] Ni 2+ Collagenase bound to -NTA agarose beads was eluted using elution buffer (100 mM NaCl, 25 mM Tris-HCl, pH 7.5, 400 mM imidazole). To increase protein recovery, elution was performed twice with 1 mL aliquots as one set, and this was repeated a total of two times to obtain the eluted fraction.

[0174] The recovered eluent was concentrated at 14,000 rpm using a centrifugal filter unit (10 kDa MWCO, Amicon Ultra or Centricon, Merck Millipore, USA).

[0175] 2-5. Buffer Replacement and Protein Recovery

[0176] To remove imidazole during the concentration process, the buffer was changed twice using 400 μL of washing buffer (25 mM Tris-HCl, pH 7.5). Afterward, 1 mL of PBS buffer was added to adjust the final buffer conditions, and the protein solution buffer was completely replaced with PBS by centrifuging twice at 14,000 rpm for 20 minutes each time.

[0177] The finally purified collagenase was recovered by concentrating it in 1 mL of PBS buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4) and subsequently used for activity analysis and additional experiments.

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[0180] Example 3. Identification and quantification of isolated collagenase

[0181] The collagenase recombinant protein isolated, purified, and concentrated in Example 2 was identified, and its concentration was quantified.

[0182] To this end, a protein sample for electrophoresis was prepared by adding 6× sample buffer (500 mM Tris-HCl [pH 6.8], 10% SDS, 0.5% bromophenol blue, 30% (v / v) glycerol, 100 mM DTT) to the protein solution obtained in Example 2 and mixing to a final concentration of 1×. The prepared protein sample was boiled for 10 minutes to denature the protein, then separated using 10% SDS-PAGE, and the protein bands were confirmed by Coomassie Brilliant Blue staining.

[0183] In the analysis, each sample was defined as follows.

[0184] FT (flow-through): Ni 2+ Total protein that leaked out without binding to NTA beads

[0185] W (wash): Protein recovered during the washing process

[0186] B (bound): Ni even after leaching 2+ - Protein remaining on NTA beads

[0187] E (elution): Using imidazole Ni 2+ Protein eluted from NTA resin

[0188] C (concentrated): The final protein obtained through the concentration process after elution.

[0189] BSA (bovine serum albumin): Control group for protein quantification

[0190] SDS-PAGE analysis revealed a main band of collagenase protein corresponding to the expected molecular weight in the elution fraction (E) and the concentrated final protein (C). Protein quantification was calculated by comparing the loading amount of the concentrated protein (C) fraction on SDS-PAGE with BSA standards (0.1, 0.25, and 0.5 μg). As a result, it was confirmed that a total of 100 μg of recombinant collagenase protein was present in 1 mL of PBS buffer, which means that 100 ng of collagenase protein is contained per 1 μL.

[0191] In addition, no significant non-specific cleavage or degradation products were observed in the collagenase protein throughout the separation, purification, and concentration processes, and SDS-PAGE analysis confirmed that most of the protein existed in a form maintaining its expected molecular weight.

[0192] Therefore, through this example, it was confirmed that a total of 100 μg of Grimontia hollisae-derived collagenase recombinant protein can be stably obtained from 20 g of Nicotiana benthamiana plant leaf tissue without degradation, and this result demonstrates that the protein cleavage problem, which has been repeatedly pointed out as a problem in the existing collagenase production process, can be substantially improved.

[0193]

[0194] Example 4. Confirmation of Collagenase Activity Derived from Grimontia hollisae through Confirmation of Collagen Degradation

[0195] The collagen degradation activity of the collagenase recombinant protein isolated, purified, and concentrated in Example 2 was confirmed.

[0196] For the activity analysis, control and experimental groups were formed under the following conditions. Control group B used 2 μg of collagenase isolated and purified in Example 2 alone, and control group A used 4.4 μg of collagen Type (Sigma-Aldrich, USA) alone. Experimental groups 1 through 4 were prepared by mixing 100 ng, 200 ng, 400 ng, and 800 ng of collagenase isolated and purified in Example 2, respectively, with 4.4 μg (1 μL) of collagen and reacting at 37°C for 50 minutes. Each reaction was performed using TNC buffer to ensure a total reaction volume of 20 μL, and all samples were processed under identical reaction conditions.

[0197] After the reaction was completed, 6× sample buffer (500 mM Tris-HCl [pH 6.8], 10% SDS, 0.5% bromophenol blue, 30% (v / v) glycerol, 100 mM DTT) was added to each sample to adjust the concentration to a final 1× level, and then boiled for 10 minutes to prepare protein samples for electrophoresis. The prepared samples were separated by SDS-PAGE using a 7.5% acrylamide gel, and protein bands were identified by Coomassie Brilliant Blue staining.

[0198] Collagen consists of α1 chain, α2 chain, and β chain, and these bands were clearly observed in control group B (Fig. 3B). In contrast, in experimental groups 1-4 treated with collagenase, the collagen bands were observed to decrease or disappear in stages as the treatment concentration increased. This indicates that the collagenase isolated and purified according to the present invention has enzymatic activity that effectively degrades collagen.

[0199] Therefore, through this example, it was confirmed that the recombinant collagenase protein derived from Grimontia hollisae, produced and isolated from plants, stably expresses collagen degradation activity while maintaining structural integrity.

[0200]

[0201] Although the present invention has been described above with specific details such as specific components, limited embodiments, and drawings, this is provided merely to aid in a more comprehensive understanding of the invention and the invention is not limited to the above embodiments. A person skilled in the art to which the invention pertains can make various modifications and variations from this description. Accordingly, the scope of the present invention should not be limited to the embodiments described above, and all things equivalent to or equivalently modified from the claims set forth below, as well as the claims themselves, shall be considered to fall within the scope of the concept of the present invention.

Claims

1. A collagenase variant in which one or more of the amino acid residues at the 622nd, 625th, 635th, 638th, 640th, 643rd, 644th, and 647th positions of the amino acid sequence of SEQ ID NO. 1 are substituted with alanine (Ala), serine (Ser), or threonine (Thr).

2. In claim 1, the collagenase variant is a collagenase variant in which the N-terminal leader sequence and the prodomain are deleted.

3. In paragraph 2, the collagenase variant is a collagenase variant in which 1 to 87 amino acids are deleted based on the amino acid sequence of SEQ ID NO.

1.

4. In Paragraph 3, The above collagenase variant is a collagenase variant represented by the amino acid sequence of SEQ ID NO.

2.

5. In claim 1, the collagenase variant is a collagenase variant in which the autolysis rate is improved by 50% or more compared to recombinant wild-type collagenase.

6. A nucleotide sequence encoding a collagenase variant of any one of paragraphs 1 to 5.

7. A recombinant expression vector into which the nucleotide sequence of claim 6 has been introduced.

8. The recombinant expression vector of Paragraph 7 is A recombinant expression vector comprising a collagenase variant expression cassette, a CRT expression cassette, and a p38 expression cassette.

9. In claim 8, the collagenase variant expression cassette is A promoter represented by the nucleotide sequence of SEQ ID NO. 3, 5' UTR represented by the nucleotide sequence of SEQ ID NO. 4, A base sequence encoding a collagenase variant represented by the amino acid sequence of SEQ ID NO. 8 and A recombinant expression vector having a terminator operably linked thereto, represented by the nucleotide sequence of SEQ ID NO.

12.

10. In claim 9, the collagenase expression cassette is A recombinant expression vector having a base sequence encoding an endoplasmic reticulum targeting sequence, a base sequence encoding a purification tag, and / or a base sequence encoding an endoplasmic reticulum retention sequence additionally operably linked.

11. In paragraph 8, the CRT expression cassette is A promoter represented by the nucleotide sequence of SEQ ID NO. 13, 5' UTR represented by the nucleotide sequence of SEQ ID NO. 5, CRT represented by the nucleotide sequence of SEQ ID NO. 14, A recombinant expression vector having a terminator operably linked thereto, represented by the nucleotide sequence of SEQ ID NO.

12.

12. In Clause 11, the CRT expression cassette is A recombinant expression vector having a base sequence encoding an endoplasmic reticulum targeting sequence, a base sequence encoding a purification tag, and / or a base sequence encoding an endoplasmic reticulum retention sequence additionally operably linked.

13. In paragraph 8, the p38 expression cassette is A promoter represented by the nucleotide sequence of SEQ ID NO. 18, L-UTR represented by the nucleotide sequence of SEQ ID NO. 19, p38, represented by the nucleotide sequence of SEQ ID NO. 20, A recombinant expression vector having a terminator operably linked thereto, represented by the nucleotide sequence of SEQ ID NO.

22.

14. Transgenic plants transformed with the recombinant expression vector of paragraph 7.

15. A method for producing a collagenase variant comprising the following steps: (a) a step of culturing the transgenic plant of claim 7; and (b) A step of isolating and purifying collagenase variants from the above-mentioned transgenic plants.

16. In paragraph 15, the above-mentioned transgenic plant is Food crops including rice, wheat, barley, corn, soybeans, potatoes, red beans, oats, and sorghum; vegetable crops including Arabidopsis thaliana, Chinese cabbage, radish, chili pepper, strawberry, tomato, watermelon, cucumber, cabbage, Korean melon, pumpkin, green onion, onion, and carrot; Specialty crops including ginseng, tobacco, cotton, sesame, sugarcane, sugar beet, perilla, peanuts, and rapeseed; Fruit trees including apple trees, pear trees, jujube trees, peaches, grapes, citrus fruits, persimmons, plums, apricots, lemons, and bananas; and A method for producing a collagenase variant selected from floricultural plants including roses, carnations, chrysanthemums, lilies, sunflowers, cosmos, and tulips.

17. A method for producing a collagenase variant according to claim 15, wherein the collagenase variant produced by the above method has collagenase activity.

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