US20210363199A1 - Method for soluble expression and purification of hydrophobin - Google Patents

Method for soluble expression and purification of hydrophobin Download PDF

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US20210363199A1
US20210363199A1 US16/944,078 US202016944078A US2021363199A1 US 20210363199 A1 US20210363199 A1 US 20210363199A1 US 202016944078 A US202016944078 A US 202016944078A US 2021363199 A1 US2021363199 A1 US 2021363199A1
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recombinant fusion
fusion protein
target protein
hydrophobin
protein
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Geun Joong Kim
Dae Eun Cheong
Ho-Dong LIM
Sang-Oh AHN
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Industry Foundation of Chonnam National University
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Industry Foundation of Chonnam National University
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Assigned to INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY reassignment INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LIM, HO-DONG, AHN, SANG-OH, CHEONG, DAE EUN, KIM, GEUN JOONG
Publication of US20210363199A1 publication Critical patent/US20210363199A1/en
Assigned to INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY reassignment INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: YOU, SUNG HWAN
Priority to US18/056,708 priority Critical patent/US20230119241A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand

Definitions

  • sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named Sequence_Listing_PLS20312.txt created on Jul. 30, 2020, and having a size of 6423 bytes and is filed concurrently with the specification.
  • sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
  • the following disclosure relates to a method of expressing hydrophobin in a soluble form and a method of purifying hydrophobin, and more particularly, to a method of expressing a recombinant fusion protein including hydrophobin in a soluble form in a host cell and then purifying the expressed recombinant fusion protein.
  • Hydrophobin is a small protein initially isolated from Shizophyllum commune .
  • the hydrophobin is composed of 100 to 150 amino acids, and is mainly expressed in a mycelia fungus.
  • the hydrophobin which is amphipathic forms a hydrophobic surface layer through self-assembly when a monomer thereof is secreted to the outside.
  • the hydrophobin acts to coat a surface of the hypha through an amphiphilic polymer composition.
  • the hydrophobin not only enables the hypha to effectively respond to changes in the surrounding environment but also functions as a shield between a cell wall and an air layer or at an interface between a cell wall and a solid surface during sporulation, fruiting body development, and host invasion through infection structure formation (Bayry et al., 2012).
  • the hydrophobin forms four disulfide bonds in a molecule from eight cysteine residues included in a protein molecule. These disulfide bonds play an important role in stabilization of an amphipathic three-dimensional structure that imparts activity similar to a surfactant in the hydrophobin, which enables the hydrophobin to be self-assembled into an amphipathic monolayer at a hydrophilic-hydrophobic interface. Hydrophobin is classified into Class I and Class II depending on a hydropathy plot, solubility, and a structure formed during self-assembly.
  • Class I and Class II hydrophobins form an amphipathic monolayer at a hydrophilic-hydrophobic interface; however, Class I hydrophobin forms amyloid-like rodlets which are insoluble at a hydrophilic-hydrophobic interface, whereas, Class II hydrophobin forms a monolayer having a high solubility at a hydrophilic-hydrophobic interface.
  • hydrophobin Due to the above features, hydrophobin has been spotlighted in the biomaterial industry. Accordingly, the use of hydrophobin in cosmetics or food and beverage requiring stable and uniform foam has been actively studied. In addition, hydrophobin has received increasing attention as a next-generation innovative material such as a medical coating agent or a functional particle for a nano-structure applied to a living body in various industries.
  • An embodiment of the present invention is directed to providing a recombinant fusion protein for soluble expression of hydrophobin in a heterologous host, a method of producing the same, and a method of purifying soluble hydrophobin through the production method.
  • a recombinant fusion protein expressed in a soluble form includes: a target protein; and a ramp tag for controlling a translation speed, fused at an N-terminal of the target protein, wherein a signal sequence of the N-terminal of the target protein is subjected to mutation including the deletion.
  • the target protein may be hydrophobin.
  • the target protein may be Class I hydrophobin.
  • the target protein may be DewA.
  • a wild type target protein may consist of an amino acid sequence of SEQ ID NO: 2.
  • the target protein may consist of an amino acid sequence of SEQ ID NO: 8 or 10.
  • the ramp tag may consist of an amino acid sequence of SEQ ID NO: 5.
  • the signal sequence may consist of a base sequence of SEQ ID NO: 3.
  • the mutation may be one or two or more selected from the group consisting of a substitution and a deletion of a part or all of amino acids of the signal sequence and an addition of a new amino acid.
  • a vector for soluble expression of the recombinant fusion protein the base sequence being introduced into the vector.
  • a transformant for soluble expression of the recombinant fusion protein the transformant being transformed with the vector.
  • a method of purifying a recombinant fusion protein expressed in a soluble form including: a) expressing a recombinant fusion protein in a soluble form by transforming a non-human host cell with a vector into which a base sequence encoding a recombinant fusion protein is introduced, the recombinant fusion protein including: a target protein; and a ramp tag for controlling a translation rate, fused at an N-terminal of the target protein; and b) purifying the expressed recombinant fusion protein.
  • Step b) above may include: b1) suspending the transformed cell and lysing and centrifuging the suspended cell to obtain a first supernatant; b2) shaking and then centrifuging a first mixture obtained by adding a first solvent to the first supernatant to obtain a second supernatant; and b3) shaking and then centrifuging a second mixture obtained by adding a second solvent to the second supernatant to recover the target protein.
  • the target protein may be hydrophobin.
  • the target protein may be Class I hydrophobin.
  • the target protein may be DewA.
  • a wild type target protein may consist of an amino acid sequence of SEQ ID NO: 2.
  • the target protein may consist of an amino acid sequence of SEQ ID NO: 8 or 10.
  • the ramp tag may be obtained by collecting a rare codon of the host cell.
  • the host cell may be a bacterium belonging to Escherichia sp., Salmonellae sp., Yersinia sp., Shigella sp., Enterobacter sp., Pseudomonas sp., Proteus sp., or Klebsiella sp.
  • Each of the first solvent and the second solvent may be selected from the group consisting of C3 organic solvents.
  • a volume ratio of the organic solvent to the first supernatant may be 1:2 or 1:3.
  • the ramp tag may consist of an amino acid sequence of SEQ ID NO: 5.
  • the rare codon may be collected by analyzing a frequency of the codon and the number of isoacceptor tRNA genes.
  • the frequency of the codon may be 0.1 to 1%.
  • the number of isoacceptor tRNA genes may be 0 to 2.
  • the target protein may be a physiologically active protein including hormones and receptors thereof, biological response modifiers and receptors thereof, cytokines and receptors thereof, enzymes, antibodies, and antibody fragments.
  • a recombinant fusion protein expressed in a soluble form the recombinant fusion protein being purified by the method.
  • FIG. 1 is a schematic view of a structure of a recombinant fusion protein for soluble expression of hydrophobin.
  • FIGS. 2A and 2B show SDS-PAGE analysis results after culturing at (a) 37° C. and (b) 18° C. to confirm soluble expression of hydrophobin.
  • FIG. 3 illustrates an example of a flowchart of a process of purifying the soluble hydrophobin expressed according to the present invention.
  • FIG. 4 shows an SDS-PAGE analysis result for confirming purification efficiency of the soluble hydrophobin purified according to the present invention.
  • FIG. 5 illustrates a purification procedure of hydrophobin by a two phase separation method using Triton X-114 known in the related art.
  • FIGS. 6 A and B shows SDS-PAGE analysis results for confirming purification efficiency of the hydrophobin purified by the two phase separation method using the Triton X-114.
  • target protein in the present invention is a protein intended to be produced in large quantities by those skilled in the art, and refers to any protein that can be expressed in a transformant by inserting polynucleotide encoding the protein to a recombinant expression vector.
  • recombinant protein or “fusion protein” refers to a protein to which another protein is connected to an N-terminal or a C-terminal of an initial target protein sequence, or refers to a protein to which another amino acid sequence is added.
  • the above term refers to a recombinant fusion protein of the present invention obtained by linking a fusion partner to a target protein, or refers to a recombinant protein of a target protein from which a fusion partner is removed by a protein cleaving enzyme.
  • vector or “expression vector” is a linear or circular DNA molecule consisting of fragments encoding polypeptide, the polypeptide consisting of elements and additional fragments provided for gene transcription and translation and being linked to the DNA molecule to be operable.
  • the additional fragment includes a promoter and a transcription termination signal sequence.
  • the vector or the expression vector includes one or more replication origins, one or more selection markers, and the like. In general, the vector or the expression vector is derived from plasmid or virus DNA or from both of them.
  • the expression “comprise(s)” is intended to be open-ended transitional phrase having an equivalent meaning to an expression such as “include(s)”, “contain(s)”, “have(has)”, or “is (are) characterized”, and does not exclude elements, materials, or steps, which are not further recited.
  • the expression “substantially consist(s) of” means that one specific element, material, and step, which are not recited with a specific element, material, and step, may be present at an amount having no unacceptably significant influence on at least one basic and novel technical idea of the invention.
  • the expression “consist(s) of” means the presence of only the defined element, material, and step.
  • sample and “specimen” in the present invention refer to subjects for analysis and are interchangeably used throughout the specification.
  • the recombinant fusion protein expressed in a soluble form is a recombinant fusion protein including a target protein and a ramp tag for controlling a translation rate, fused at an N-terminal of the target protein.
  • a signal sequence of the N-terminal of the target protein is subjected to mutation.
  • the target protein may be hydrophobin, more preferably a Class I hydrophobin, and still more preferably a Class I DewA, and a wild type target protein may consist of an amino acid sequence of SEQ ID NO: 2.
  • the target protein of which the N-terminal has the mutated signal sequence may have an amino acid sequence of SEQ ID NO: 8 or 10, but the scope of the present invention is not limited thereto.
  • the ramp tag may be composed of 1 to 20 amino acids, preferably 6 to 12 amino acids, and more preferably 6 to 8 amino acids, but is not limited thereto.
  • the ramp tag may consist of an amino acid sequence of SEQ ID NO: 5.
  • a ramp tag for controlling a translation rate for expressing a recombinant fusion protein, a method used for an application thereof, and the like is disclosed in Korean Patent Publication No. 1446054 registered by the present applicant, the contents of which are incorporated herein by reference in their entirety.
  • the method used in the above patent publication or a method appropriately modified to meet the object of the present invention, and the like may be applied.
  • a ramp tag for controlling a translation rate is prepared by using a method including: making a rare codon table according to a host cell; converting a DNA sequence of a target gene into codons; analyzing a frequency and position at which rare codons in the rare codon table appear in an open reading frame (ORF) of the target gene; and collecting and arranging the rare codons.
  • a protein that has a high added value but is difficult to be expressed for example, esterase, ⁇ -glucosidase, cytolysin A, single chain Fv (scFv), asparaginase B, tetra-cell adhesion molecule (T-CAM), or B3(Fv)PE38, is effectively expressed by using the ramp tag.
  • the ramp tag may be prepared in a form in which the ramp tag for controlling a translation rate of the above patent publication is fused at the N-terminal of the target protein.
  • the ramp tag may be included in the recombinant fusion protein.
  • the signal sequence may be confirmed by a general method such as SignalP (see SignalP 5.0 improves signal peptide predictions using deep neural networks.
  • SignalP see SignalP 5.0 improves signal peptide predictions using deep neural networks.
  • the signal sequence may consist of a base sequence of SEQ ID NO: 3.
  • the mutation may be one or two or more selected from the group consisting of a substitution and a deletion of a part or all of amino acids of the signal sequence and an addition of a new amino acid.
  • the mutation may be a deletion of a part or all of amino acids of the signal sequence, and for example, the 1st amino acid to the 18 th amino acid of a sequence may be deleted.
  • the entire signal sequence corresponding to the underlined part of SEQ ID NO: 1 shown in Table 1, below, may be deleted, but the present invention is not limited thereto.
  • FIG. 1 illustrates an example of a structure of a recombinant fusion protein for soluble expression of a target protein.
  • the ramp tag is fused at an N-terminal of the hydrophobin used as the target protein.
  • the entire signal sequence of the hydrophobin is deleted.
  • Various types of amino acid sequences that may be used for detecting and purifying a protein may be fused at a C-terminal of the hydrophobin used as the target protein, and for example, one or two or more tags or proteins selected from the group consisting of His tag, T7 tag, S-tag, Flag-tag, HA-tag, V5 epitope, PelB, and Xpress epitope may be used.
  • codons with respect to tags coded with six histidines (6 ⁇ His tag) may be connected, but the present invention is not limited thereto.
  • a target protein expressed only in an insoluble inclusion body form in the related art is expressed in a soluble protein form. Accordingly, the present inventors solved the fundamental problem such as protein refolding which was the existing problem, and confirmed that a target protein having the original activity may be obtained in a high yield. Further, the present inventors have established a process capable of purifying a target protein with a significantly simplified procedure and high separation yield as compared with a process of purifying the target protein in the insoluble inclusion body form according to the related art.
  • the present invention provides a base sequence encoding the recombinant fusion protein.
  • the base sequence may have a base sequence of SEQ ID NO: 1.
  • the present invention provides a vector for soluble expression of the protein, the base sequence being introduced into the vector.
  • the present invention provides a transformant for soluble expression of the recombinant fusion protein, the transformant being transformed with the vector.
  • the type of the transformant is not particularly limited as long as it may achieve the object of the present invention.
  • an individual facilitated for a gene expression for example, Escherichia coli or the like may be used.
  • the method of purifying a recombinant fusion protein expressed in a soluble form includes: a) expressing a recombinant fusion protein in a soluble form by transforming a non-human host cell with a vector into which a base sequence encoding a recombinant fusion protein is introduced, the recombinant fusion protein including: a target protein; and a ramp tag for controlling a translation rate, fused at an N-terminal of the target protein; and b) purifying the expressed recombinant fusion protein.
  • step b) above may include: b1) suspending the transformed cell and lysing and centrifuging the suspended cell to obtain a first supernatant; b2) shaking and then centrifuging a first mixture obtained by adding a first solvent to the first supernatant to obtain a second supernatant; and b3) shaking and then centrifuging a second mixture obtained by adding a second solvent to the second supernatant to recover the target protein.
  • the target protein may consist of an amino acid sequence of SEQ ID NO: 2.
  • the target protein may be hydrophobin, more preferably a Class I hydrophobin, and still more preferably a Class I DewA, but the scope of the present invention is not limited thereto.
  • the ramp tag may be obtained by collecting a rare codon of the host cell, but is not limited thereto.
  • the host cell may be a bacterium belonging to Escherichia sp., Salmonellae sp., Yersinia sp., Shigella sp., Enterobacter sp., Pseudomonas sp., Proteus sp., or Klebsiella sp, but is not limited thereto.
  • Escherichia sp. may be used, and more specifically, E. coli BL21 or the like may be used.
  • a two phase separation method using an organic solvent may be used in the purification, but the present invention is not limited thereto. Any method may be appropriately introduced as long as it is a purification method capable of separating the soluble protein according to the present invention with high efficiency.
  • each of the first solvent and the second solvent may be selected from the group consisting of C3 organic solvents.
  • Specific examples of the first solvent and the second solvent include isopropyl alcohol.
  • a target protein expressed in a soluble form may be separated with high efficiency, which is preferable.
  • the scope of the present invention is not limited thereto.
  • FIG. 3 illustrates an example of the method of purifying a recombinant fusion protein expressed in a soluble form by the two phase separation method using the organic solvent according to the present invention. In FIG. 3 , it is confirmed that the protein expressed in a soluble form may be purified by the above method with high efficiency.
  • processes such as recovery, suspension, and lysing of cells that are performed after the soluble expression of the target protein may be performed by appropriately introducing a reagent, method, and the like known to those skilled in the art in a range in which the object of the present invention may be achieved.
  • the centrifugation in the sub-step b1) may be performed in a temperature range of 1 to 10° C., preferably 2 to 8° C., and more preferably 3 to 6° C. Since the first supernatant may be effectively separated from the lysed cell in the above range, the centrifugation is preferably performed in the above range.
  • each centrifugation in each of the sub-step b2) and b3) may be performed in a temperature range of 15 to 28° C., preferably 17 to 25° C., and more preferably 19 to 23° C. Since the second supernatant and the target protein may be effectively separated from the first mixture and the second mixture, respectively, in the above range, each centrifugation is preferably performed in the above range.
  • the centrifugation may be performed at 3,000 to 20,000 rpm for 1 to 90 minutes.
  • the centrifugation in the sub-step b1) may be performed at 3,000 to 10,000 rpm, preferably 4,000 to 8,000 rpm, and more preferably 5,000 to 7,000 rpm, for 40 to 80 minutes, preferably 45 to 75 minutes, and more preferably 50 to 70 minutes, but is not limited thereto. Since the first supernatant may be effectively separated from the lysed cell in the above range, the centrifugation is preferably performed in the above range.
  • the centrifugation in the sub-step b2) may be performed at 6,000 to 15,000 rpm, preferably 7,500 to 13,500 rpm, and more preferably 8,500 to 11,500 rpm, for 40 to 80 minutes, preferably 45 to 75 minutes, and more preferably 50 to 70 minutes, but is not limited thereto. Since the second supernatant may be effectively separated from the first mixture in the above range, the centrifugation is preferably performed in the above range.
  • the centrifugation in the sub-step b3) may be performed at 6,000 to 15,000 rpm, preferably 7,500 to 13,500 rpm, and more preferably 8,500 to 11,500 rpm, for 1 to 20 minutes, preferably 4 to 16 minutes, and more preferably 7 to 13 minutes, but is not limited thereto. Since the target protein may be effectively separated from the second supernatant in the above range, the centrifugation is preferably performed in the above range.
  • a volume ratio of the organic solvent to the first supernatant may be 1:1.5 or 1:4, preferably 1:2 or 1:3.5, and more preferably 1:2 or 1:3, but is not limited thereto. According to an exemplary embodiment of the present invention, as a specific example, the volume ratio thereof may be 1:2.5.
  • a volume ratio of the organic solvent to the second supernatant may be 1:0.5 or 1:1.5, preferably 1:0.6 or 1:1.4, and more preferably 1:0.8 or 1:1.2, but is not limited thereto. According to an exemplary embodiment of the present invention, as a specific example, the volume ratio thereof may be 1:1.
  • the target protein may be hydrophobin, more preferably Class I hydrophobin, and still more preferably Class I DewA, and a wild type target protein may consist of an amino acid sequence of SEQ ID NO: 2.
  • the target protein of which the N-terminal has the mutated signal sequence may have an amino acid sequence of SEQ ID NO: 8 or 10, but the scope of the present invention is not limited thereto.
  • the ramp tag may consist of 1 to 20 amino acids, preferably 6 to 12 amino acids, and more preferably 6 to 8 amino acids, but is not limited thereto.
  • the ramp tag may consist of an amino acid sequence of SEQ ID NO: 5.
  • the rare codon may be collected by analyzing a frequency of the codon and the number of isoacceptor tRNA genes.
  • the frequency of the codon may be 0.05 to 3%, and preferably 0.1 to 1%, but is not limited thereto.
  • the number of isoacceptor tRNA genes may be 0 to 2.
  • the target protein may be a physiologically active protein including hormones and receptors thereof, biological response modifiers and receptors thereof, cytokines and receptors thereof, enzymes, antibodies, and antibody fragments.
  • the target protein may be selected from the group consisting of hydrophobin, GST, MBP, NusA, CBP, GFP, Thioredoxin, Mistic, Sumo, and DSB, and more specifically, the target protein may be hydrophobin, but is not limited thereto.
  • Target protein GenBank ID: gi67902037, Class I hydrophobin DewA (BASF, 2009) derived from Aspergillus nidulans , the target protein was synthesized by Bioneer Inc. (Daejeon, Korea) based on the genetic information provided by GenBank.
  • the ramp tag was prepared by the method of analyzing codon usage of E. coli described in Korean Patent Publication No. 1446054.
  • the pET24a plasmid vector was obtained from New England Labs (UK).
  • Class I hydrophobin DewA derived from A. nidulans was used as a target protein.
  • a base sequence and an amino acid sequence thereof are shown in Table 1.
  • a sequence of a ramp tag linked to an N-terminal of the Class I hydrophobin DewA used as the target protein was produced by applying the method of analyzing a rare codon disclosed in Korean Patent Publication No. 1446054.
  • a base sequence and an amino acid sequence thereof are shown in Table 2.
  • the base sequence of SEQ ID NO: 7 obtained by deleting the entire signal sequence in the base sequence of SEQ ID NO: 1, the base sequence of SEQ ID: 4 of the ramp tag, and the sequence of SEQ ID NO: 6 of 6 ⁇ His Tag were synthesized as in the order illustrated in the schematic view of FIG. 1 , and then the pET24a plasmid vector was used by a general cloning method, thereby preparing a recombinant expression vector (pET24a-Ramp-DewA(-ss)-6 ⁇ His).
  • a recombinant expression vector was prepared in the same procedure and condition as those of Example 1, except that the base sequence of the mutated hydrophobin was changed to a base sequence of SEQ ID NO: 8.
  • a recombinant expression vector (pET24a-Ramp-DewA-6 ⁇ His) was prepared in the same procedure and condition as those of Example 1, except that the signal sequence (SEQ ID NO: 1) was not mutated.
  • E. coli BL21(DE3) was transformed with each recombinant expression vector, and the transformed E. coli BL21(DE3) was inoculated into a solid LB medium (10 g/L of NaCl, 10 g/L of tryptone, 5 g/L of yeast extract) containing agar to which 100 ug/mL of kanamycin was added and then cultured in a solid state at 37° C. for 12 hours.
  • a solid LB medium (10 g/L of NaCl, 10 g/L of tryptone, 5 g/L of yeast extract) containing agar to which 100 ug/mL of kanamycin was added
  • 100 uL of the pre-cultured medium was sub-cultured in 3.5 mL of a new LB medium in which 100 ug/mL of kanamycin was added and cultured at 37° C. and 220 rpm, and then, when an absorbance (OD 600 ) at 600 nm has reached about 0.7, 0.2 mM isopropyl ⁇ -D-thiogalactopyranoside (IPTG) was added to induce expression of protein.
  • OD 600 absorbance at 600 nm has reached about 0.7
  • IPTG isopropyl ⁇ -D-thiogalactopyranoside
  • each experimental group was separated into two temperature experimental groups of 37° C. and 18° C., and the expression of the Class I hydrophobin DewA used as the target protein was induced at each temperature and 250 rpm for 3 hours.
  • the target protein expressed by the following method was recovered from the E. coli cultured at each temperature.
  • Example 1 the expression amount (7 wt %) of the target protein DewA in the insoluble fraction I was significantly reduced as compared in the soluble fraction S (93 wt %), and thus, most of the DewA protein was expressed in a soluble form.
  • the above results mean that the Class I hydrophobin DewA used as the target protein of the present invention was expressed in a soluble form with high efficiency by the preparation of the recombinant fusion protein according to the present invention.
  • Example 1 the expression amount (5 wt %) of the target protein DewA in the insoluble fraction I was significantly reduced as compared in the soluble fraction S (95 wt %), and thus, most of the DewA protein was expressed in a soluble form.
  • Example 2 the soluble expression of the target protein DewA was somewhat improved as compared in Comparative Example 1. However, it was evaluated that it was not applicable to the purification method of the present invention premised on the soluble expression of the target protein (data not shown).
  • the first mixture was centrifuged at 20° C. (room temperature) and 10,000 rpm for 10 minutes to recover a second supernatant.
  • lane 1 represents an total fraction sample T
  • lane 2 represents a soluble fraction sample S
  • lane 3 represents a sample obtained by dissolving Class I hydrophobin DewA used as a target protein purified by the purification method in 1 mL of 200 mM Tris-HCl (pH 8.0)
  • lane 4 represents a sample obtained by additionally adding 1 mL of Tris-HCl to the sample of lane 3
  • each of lanes 5 to 7 represents a diluted sample obtained by adding 1 mL of 200 mM Tris-HCl to the previous sample in the same manner as described above.
  • the recombinant fusion protein expressed through Example 1 was purified in the same procedure and condition as those of Experimental Example 3, except that each of methanol, ethanol, and isobutyl alcohol was used as the organic solvent instead of the isopropyl alcohol.
  • the recombinant fusion protein expressed through Example 1 was purified in the same procedure and condition as those of Experimental Example 3, except that the amount of isopropyl alcohol to be mixed with the second supernatant was set to be the same as the volume of the first supernatant.
  • the recombinant fusion protein expressed through Example 1 was purified in the same procedure and condition as those of Experimental Example 3, except that a concentration of the Tris-HCl (pH 8.0) used as a re-suspension solvent was set to be 50 mM and 400 mM.
  • hydrophobin expressed in a soluble form may be efficiently purified only by performing the purification under the purification process of the present invention and the corresponding condition, and as described in Experimental Examples 2 and 3, the above results were shown by way of example only for the Class I hydrophobin DewA; however, the above results can be reasonably generalized by being extended to at least Class I hydrophobin and broadly to the whole hydrophobin, because hydrophobin has a common feature to form amphipathic structure with four disulfide bonds (S—S) in a molecule due to eight cysteines included in the molecule.
  • S—S disulfide bonds
  • a fusion protein was purified using Triton X-114 used as a nonionic surfactant by a method described in the document (Clifton N. J. et al. Article in Methods in molecular biology, 2012) including the corresponding contents.
  • the recombinant expression vector (pET24a-Ramp-DewA(-ss)-6 ⁇ His) of Example 1 was cultured at 37° C. and 200 rpm and 0.2 mM IPTG was added at the time at which an absorbance at 600 nm has reached about 0.5 to induce expression of the recombinant fusion protein.
  • the expressed cells were centrifuged at 10,000 rpm to separate the medium and the cells from each other, and the recovered cells were sonicated, thereby obtaining a total fraction sample.
  • the total fraction sample was centrifuged at 4° C. and 10,000 rpm for 30 minutes to obtain a soluble fraction sample (supernatant).
  • Triton X-114 used as a nonionic surfactant was added thereto in an amount of 5% of a total volume and mixed with the supernatant for 1 minute, and then the mixture was allowed to stand at room temperature to induce phase separation.
  • the upper part removed in the previous step was also extracted by using isobutyl alcohol in the same manner as described above.
  • a tube 1 shows a state immediately after the cells are lysed, a total fraction sample 1 and a soluble fraction sample 2 after centrifugation of the total fraction sample 1 are obtained, a soluble fraction sample (supernatant) is mixed with Triton X-114, and then the mixture is subjected to vortexing.
  • a tube 2 shows a state after a phase is separated into an upper part 3 and a lower part 4.
  • a tube 3 shows a state after the upper part 3 is mixed with isobutyl alcohol and a phase of the mixture is separated into an upper part and a lower part 5.
  • a tube 4 shows a state after the lower part 4 is mixed with isobutyl alcohol and a phase of the mixture is separated into an upper part and a lower part 6.
  • FIGS. 6 A and B Results of performing two times of SDS-PAGE analysis on a total of six samples obtained in each step are illustrated in FIGS. 6 A and B.
  • lane 1 represents a total fraction sample
  • lane 2 represents a soluble fraction sample
  • lane 3 represents a sample of the upper part in the tube 2
  • lane 4 represents a sample of the lower part in the tube 2
  • lane 5 represents a sample of the lower part in the tube 3
  • lane 6 represents a sample of the lower part in the tube 4.
  • hydrophobin expressed in a soluble form to be expected in lane 6 was not observed, and the reason was that expression and purification of soluble hydrophobin were not performed by the purification method using Triton X-114 used as a nonionic surfactant according to the related art.
  • hydrophobin protein expressed in an insoluble inclusion body form in the related art was expressed in a soluble form, and then the hydrophobin protein was purified with high efficiency through a simple process by the configuration of the recombinant fusion protein and the purification method according to the present invention.
  • soluble expression and its simple purification of hydrophobin expressed in an inclusion body form in the related art is achieved by the soluble expression and the purification method according to the present invention, such that protein refolding in the related art is not required, thereby easily purifying and obtaining hydrophobin expressed in a soluble form with high efficiency.

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