U.S. patent application number 16/944078 was filed with the patent office on 2021-11-25 for method for soluble expression and purification of hydrophobin.
The applicant listed for this patent is INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY. Invention is credited to Sang-Oh AHN, Dae Eun CHEONG, Geun Joong KIM, Ho-Dong LIM.
Application Number | 20210363199 16/944078 |
Document ID | / |
Family ID | 1000005022437 |
Filed Date | 2021-11-25 |
United States Patent
Application |
20210363199 |
Kind Code |
A1 |
KIM; Geun Joong ; et
al. |
November 25, 2021 |
METHOD FOR SOLUBLE EXPRESSION AND PURIFICATION OF HYDROPHOBIN
Abstract
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 are provided.
Inventors: |
KIM; Geun Joong; (Gwangju,
KR) ; CHEONG; Dae Eun; (Gwangju, KR) ; LIM;
Ho-Dong; (Jeongeup-si, KR) ; AHN; Sang-Oh;
(Gwangju, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY |
Gwangju |
|
KR |
|
|
Family ID: |
1000005022437 |
Appl. No.: |
16/944078 |
Filed: |
July 30, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 7/06 20130101; C07K
2319/00 20130101; C07K 14/37 20130101 |
International
Class: |
C07K 14/37 20060101
C07K014/37; C07K 7/06 20060101 C07K007/06 |
Foreign Application Data
Date |
Code |
Application Number |
May 20, 2020 |
KR |
10-2020-0060264 |
Claims
1. A recombinant fusion protein expressed in a soluble form,
comprising: a target protein; and a ramp tag for controlling a
translation rate, 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.
2. The recombinant fusion protein of claim 1, wherein the target
protein is hydrophobin.
3. The recombinant fusion protein of claim 2, wherein the target
protein is a Class I hydrophobin.
4. The recombinant fusion protein of claim 3, wherein the target
protein is DewA.
5. The recombinant fusion protein of claim 1, wherein a wild type
target protein consists of an amino acid sequence of SEQ ID NO:
2.
6. The recombinant fusion protein of claim 1, wherein the target
protein consists of an amino acid sequence of SEQ ID NO: 8 or
10.
7. The recombinant fusion protein of claim 1, wherein the ramp tag
consists of an amino acid sequence of SEQ ID NO: 5.
8. The recombinant fusion protein of claim 1, wherein the signal
sequence consists of a base sequence of SEQ ID NO: 3.
9. The recombinant fusion protein of claim 1, wherein the mutation
is 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.
10. A base sequence encoding the recombinant fusion protein of
claim 1.
11. A vector for soluble expression of the recombinant fusion
protein, the base sequence of claim 10 being introduced into the
vector.
12. A transformant for soluble expression of the recombinant fusion
protein, the transformant being transformed with the vector of
claim 11.
13. A method of purifying a recombinant fusion protein expressed in
a soluble form, the method comprising: 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 speed, fused at an N-terminal of the target protein;
and b) purifying the expressed recombinant fusion protein.
14. The method of claim 13, wherein b) includes: 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.
15. The method of claim 13, wherein the target protein is
hydrophobin.
16. The method of claim 15, wherein the target protein is Class I
hydrophobin.
17. The method of claim 16, wherein the target protein is DewA.
18. The method of claim 13, wherein a wild type target protein
consists of an amino acid sequence of SEQ ID NO: 2.
19. The method of claim 13, wherein the target protein consists of
an amino acid sequence of SEQ ID NO: 8 or 10.
20. The method of claim 13, wherein the ramp tag is obtained by
collecting a rare codon of the host cell.
21. The method of claim 13, wherein the host cell is a bacterium
belonging to Escherichia sp., Salmonellae sp., Yersinia sp.,
Shigella sp., Enterobacter sp., Pseudomonas sp., Proteus sp., or
Klebsiella sp.
22. The method of claim 14, wherein each of the first solvent and
the second solvent is isopropyl alcohol.
23. The method of claim 22, wherein a volume ratio of the isopropyl
alcohol to the first supernatant is 1:2 or 1:3.
24. The method of claim 20, wherein the ramp tag consists of an
amino acid sequence of SEQ ID NO: 5.
25. The method of claim 20, wherein the rare codon is collected by
analyzing a frequency of the codon and a number of isoacceptor tRNA
genes.
26. The method of claim 25, wherein the frequency of the codon is
0.1 to 1%.
27. The method of claim 24, wherein the number of isoacceptor tRNA
genes is 0 to 2.
28. The method of claim 13, wherein the target protein is a
physiologically active protein including hormones and receptors
thereof, biological response modifiers and receptors thereof,
cytokines and receptors thereof, enzymes, antibodies, and antibody
fragments.
29. A recombinant fusion protein expressed in a soluble form, the
recombinant fusion protein being purified by the method of claim
13.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn. 119
to Korean Patent Application No. 10-2020-0060264, filed on May 20,
2020, in the Korean Intellectual Property Office, the disclosure of
which is incorporated herein by reference in its entirety.
SEQUENCE LISTING
[0002] The official copy of the 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. The sequence listing contained in this
ASCII formatted document is part of the specification and is herein
incorporated by reference in its entirety.
TECHNICAL FIELD
[0003] 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.
BACKGROUND
[0004] 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. In hyphae
growth of fungi, the hydrophobin which is amphipathic forms a
hydrophobic surface layer through self-assembly when a monomer
thereof is secreted to the outside. As a result, the hydrophobin
acts to coat a surface of the hypha through an amphiphilic polymer
composition. Through the above action, 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).
[0005] 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. Both 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.
[0006] 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.
[0007] Until now, methods of producing hydrophobin using a
bacterium, a plant cell, yeast, or the like have been attempted, in
addition to a method using mold which is the original strain as it
is. In particular, a protein to be produced in a bacterium in an
inclusion body form has been purified for use. However, current
methods require a denaturation/refolding process to obtain a final
hydrophobin protein, which means a significant increase in costs in
the mass production of hydrophobin.
[0008] For example, it has been reported by BASF SE in 2009 that
two recombinant Class I hydrophobins such as H*Protein A (yaaD--A.
nidulans DewA-His.sub.6) and H*Protein B (cleaved yaaD--A. nidulans
DewA-His6) are able to be produced to the extent of industrial
applicability using a bacterium as a host; however, in this case,
hydrophobin is also produced in an inclusion body form. Until now,
there have been no reports of a case in which a recombinant
hydrophobin is successfully expressed in a soluble form (Wendel et
al. 2010).
[0009] In addition to the problem above, HPLC, two phase separation
method (ATPS; aqueous two-phase systems) using a surfactant have
been mainly used for purifying hydrophobin in the related art, but
it takes a lot of time and cost during the purification due to a
complex procedure and use of chemical materials that are not easy
to handle. Therefore, these problems are required to be urgently
solved.
RELATED ART DOCUMENT
Patent Document
[0010] (Patent Document 1) Korean Patent Publication No. 1446054
(2014 Sep. 24) [0011] (Patent Document 2) Korean Patent Laid-Open
Publication No. 2014-0022839
Non-Patent Document
[0011] [0012] (Non-Patent Document 1) Bayry J. et al. PLOS Pathog
8(5):e1002700 (2012) [0013] (Non-Patent Document 2) Linder M. B. et
al. FEMS Microbiol Rev 29(5):877-896 (2005) [0014] (Non-Patent
Document 3) Kwan A H Y et al. PNAS USA 2006; 103:3621 LP 3626
(2006) [0015] (Non-Patent Document 4) Wendel W. et al. Eur.
Biophys. J. 39:457-468 (2010)
SUMMARY
[0016] 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.
[0017] In one general aspect, 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.
[0018] The target protein may be hydrophobin.
[0019] The target protein may be Class I hydrophobin.
[0020] The target protein may be DewA.
[0021] A wild type target protein may consist of an amino acid
sequence of SEQ ID NO: 2.
[0022] The target protein may consist of an amino acid sequence of
SEQ ID NO: 8 or 10.
[0023] The ramp tag may consist of an amino acid sequence of SEQ ID
NO: 5.
[0024] The signal sequence may consist of a base sequence of SEQ ID
NO: 3.
[0025] 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.
[0026] In another general aspect, there is provided a base sequence
encoding the recombinant fusion protein.
[0027] In still another general aspect, there is provided a vector
for soluble expression of the recombinant fusion protein, the base
sequence being introduced into the vector.
[0028] In still another general aspect, there is provided a
transformant for soluble expression of the recombinant fusion
protein, the transformant being transformed with the vector.
[0029] In still another general aspect, there is provided a method
of purifying a recombinant fusion protein expressed in a soluble
form, the method 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.
[0030] 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.
[0031] The target protein may be hydrophobin.
[0032] The target protein may be Class I hydrophobin.
[0033] The target protein may be DewA.
[0034] A wild type target protein may consist of an amino acid
sequence of SEQ ID NO: 2.
[0035] The target protein may consist of an amino acid sequence of
SEQ ID NO: 8 or 10.
[0036] The ramp tag may be obtained by collecting a rare codon of
the host cell.
[0037] 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.
[0038] Each of the first solvent and the second solvent may be
selected from the group consisting of C3 organic solvents.
[0039] A volume ratio of the organic solvent to the first
supernatant may be 1:2 or 1:3.
[0040] The ramp tag may consist of an amino acid sequence of SEQ ID
NO: 5.
[0041] The rare codon may be collected by analyzing a frequency of
the codon and the number of isoacceptor tRNA genes.
[0042] The frequency of the codon may be 0.1 to 1%. The number of
isoacceptor tRNA genes may be 0 to 2.
[0043] 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.
[0044] In still another general aspect, there is provided a
recombinant fusion protein expressed in a soluble form, the
recombinant fusion protein being purified by the method.
[0045] Other features and aspects will be apparent from the
following detailed description, the drawings, and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1 is a schematic view of a structure of a recombinant
fusion protein for soluble expression of hydrophobin.
[0047] FIGS. 2A and 2B show SDS-PAGE analysis results after
culturing at (a) 37.degree. C. and (b) 18.degree. C. to confirm
soluble expression of hydrophobin.
[0048] FIG. 3 illustrates an example of a flowchart of a process of
purifying the soluble hydrophobin expressed according to the
present invention.
[0049] FIG. 4 shows an SDS-PAGE analysis result for confirming
purification efficiency of the soluble hydrophobin purified
according to the present invention.
[0050] FIG. 5 illustrates a purification procedure of hydrophobin
by a two phase separation method using Triton X-114 known in the
related art.
[0051] 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.
DETAILED DESCRIPTION
[0052] Hereinafter, a method of expressing hydrophobin in a soluble
form and a method of purifying hydrophobin according to the present
invention will be described in detail with reference to the
accompanying drawings.
[0053] The drawings to be described below are provided by way of
example so that the spirit of the present invention can be
sufficiently transferred to those skilled in the art. Therefore,
the present invention is not limited to the drawings suggested
below but may be modified in many different forms. In addition, the
drawings suggested below will be exaggerated in order to clarify
the spirit of the present invention.
[0054] Technical terms and scientific terms used in the
specification of the present invention have the general meanings
understood by those skilled in the art to which the present
invention pertains unless otherwise defined, and a description for
the known function and configuration obscuring the present
invention will be omitted in the following description and the
accompanying drawings. In addition, singular forms used in the
specification of the present invention may be intended to include
plural forms unless otherwise specified. In addition, a unit used
in the specification of the present invention unless otherwise
specified is based on a weight. As an example, % or a unit of a
ratio refers to wt % or a weight ratio.
[0055] The term "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.
[0056] In addition, the term "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. In addition, 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.
[0057] The term "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.
[0058] 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. In addition, 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. In addition, the expression
"consist(s) of" means the presence of only the defined element,
material, and step.
[0059] The terms "sample" and "specimen" in the present invention
refer to subjects for analysis and are interchangeably used
throughout the specification.
[0060] Hereinafter, a recombinant fusion protein expressed in a
soluble form according to the present invention will be described
in detail.
[0061] 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.
[0062] In the recombinant fusion protein expressed in a soluble
form according to an exemplary embodiment of the present invention,
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. According to an exemplary embodiment of the present
invention, 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.
[0063] In the recombinant fusion protein expressed in a soluble
form according to an exemplary embodiment of the present invention,
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. In the recombinant fusion protein according to
an exemplary embodiment of the present invention, the ramp tag may
consist of an amino acid sequence of SEQ ID NO: 5.
[0064] In the present invention, 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. In a case where the contents
corresponding to the detailed description of the present invention
are not particularly described, 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.
[0065] As a specific example related to the content disclosed in
Korean Patent Publication No. 1446054, 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. Then, a protein that has a high added
value but is difficult to be expressed, for example, esterase,
.beta.-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.
[0066] In the present invention, 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. By doing so, the ramp tag may be included in the
recombinant fusion protein.
[0067] In the recombinant fusion protein expressed in a soluble
form according to an exemplary embodiment of the present invention,
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. Jose Juan Almagro Armenteros, Konstantinos D.
Tsirigos, Casper Kaae Sonderby, Thomas Nordahl Petersen, Ole
Winther, Soren Brunak, Gunnar von Heijne and Henrik Nielsen. Nature
Biotechnology, 37, 420-423 (2019)) which is an exocrine signal
sequence prediction program, but the present invention is not
limited thereto. According to an exemplary embodiment of the
present invention, the signal sequence may consist of a base
sequence of SEQ ID NO: 3.
[0068] In the recombinant fusion protein expressed in a soluble
form according to an exemplary embodiment of the present invention,
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.
[0069] According to an exemplary embodiment of the present
invention, 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.sup.th amino acid of a sequence may be deleted. According
to an exemplary embodiment of the present invention, as a specific
example, 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.
[0070] According to an exemplary embodiment of the present
invention, 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. Preferably, codons
with respect to tags coded with six histidines (6.times.His tag)
may be connected, but the present invention is not limited
thereto.
[0071] From examples to be described below, it was newly confirmed
that, in the case of the recombinant fusion protein expressed in a
soluble form according to the present invention, 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.
[0072] The present invention provides a base sequence encoding the
recombinant fusion protein. According to an exemplary embodiment of
the present invention, for example, the base sequence may have a
base sequence of SEQ ID NO: 1.
[0073] In addition, the present invention provides a vector for
soluble expression of the protein, the base sequence being
introduced into the vector.
[0074] In addition, the present invention provides a transformant
for soluble expression of the recombinant fusion protein, the
transformant being transformed with the vector. According to an
exemplary embodiment of the present invention, the type of the
transformant is not particularly limited as long as it may achieve
the object of the present invention. However, an individual
facilitated for a gene expression, for example, Escherichia coli or
the like may be used.
[0075] Hereinafter, a method of purifying a recombinant fusion
protein expressed in a soluble form according to the present
invention will be described in detail.
[0076] The method of purifying a recombinant fusion protein
expressed in a soluble form according to the present invention
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.
[0077] In the method of purifying a recombinant fusion protein
expressed in a soluble form according to the present invention,
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.
[0078] In the method of purifying a recombinant fusion protein
expressed in a soluble form according to an exemplary embodiment of
the present invention, the target protein may consist of an amino
acid sequence of SEQ ID NO: 2. According to an exemplary embodiment
of the present invention, 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.
[0079] In the method of purifying a recombinant fusion protein
expressed in a soluble form according to an exemplary embodiment of
the present invention, the ramp tag may be obtained by collecting a
rare codon of the host cell, but is not limited thereto.
[0080] In the method of purifying a recombinant fusion protein
expressed in a soluble form according to an exemplary embodiment of
the present invention, 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. According to an exemplary embodiment of
the present invention, as a specific example, Escherichia sp. may
be used, and more specifically, E. coli BL21 or the like may be
used.
[0081] In the method of purifying a recombinant fusion protein
expressed in a soluble form according to an exemplary embodiment of
the present invention, 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.
[0082] In a case where the organic solvent is used in the
purification, 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. In addition, according to an exemplary
embodiment of the present invention, as a specific example, in a
case where isopropyl alcohol is used as each of the first solvent
and the second solvent and a two phase separation method is
applied, a target protein expressed in a soluble form may be
separated with high efficiency, which is preferable. However, the
scope of the present invention is not limited thereto.
[0083] The purification of the recombinant fusion protein expressed
in a soluble form by the two phase separation method according to
the present invention is a new attempt that has not been applied in
the related art (refer to Korean Patent Laid-Open Publication No.
2014-0022839, there have been attempts to purify hydrophobin using
C.sub.1-C.sub.3 alcohol, but it was not a purification method for
soluble expression). Thus, it was confirmed that a protein
expressed in a soluble form may be separated and purified with high
efficiency from specific examples to be described below. 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.
[0084] In the method of purifying a recombinant fusion protein
expressed in a soluble form according to an exemplary embodiment of
the present invention, 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.
[0085] In the method of purifying a recombinant fusion protein
expressed in a soluble form according to an exemplary embodiment of
the present invention, the centrifugation in the sub-step b1) may
be performed in a temperature range of 1 to 10.degree. C.,
preferably 2 to 8.degree. C., and more preferably 3 to 6.degree. 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.
[0086] In addition, the centrifugation in each of the sub-step b2)
and b3) may be performed in a temperature range of 15 to 28.degree.
C., preferably 17 to 25.degree. C., and more preferably 19 to
23.degree. 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.
[0087] In the method of purifying a recombinant fusion protein
expressed in a soluble form according to an exemplary embodiment of
the present invention, the centrifugation may be performed at 3,000
to 20,000 rpm for 1 to 90 minutes. According to an exemplary
embodiment of the present invention, 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.
[0088] In addition, 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.
[0089] In addition, 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.
[0090] In the method of purifying a recombinant fusion protein
expressed in a soluble form according to an exemplary embodiment of
the present invention, 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.
[0091] In addition, in the method of purifying a recombinant fusion
protein expressed in a soluble form according to an exemplary
embodiment of the present invention, 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.
[0092] In the method of purifying a recombinant fusion protein
expressed in a soluble form according to an exemplary embodiment of
the present invention, 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. According to an exemplary embodiment of
the present invention, 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.
[0093] In the method of purifying a recombinant fusion protein
expressed in a soluble form according to an exemplary embodiment of
the present invention, 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. In the recombinant fusion
protein according to an exemplary embodiment of the present
invention, the ramp tag may consist of an amino acid sequence of
SEQ ID NO: 5.
[0094] In the method of purifying a recombinant fusion protein
expressed in a soluble form according to an exemplary embodiment of
the present invention, the rare codon may be collected by analyzing
a frequency of the codon and the number of isoacceptor tRNA
genes.
[0095] In the method of purifying a recombinant fusion protein
expressed in a soluble form according to an exemplary embodiment of
the present invention, the frequency of the codon may be 0.05 to
3%, and preferably 0.1 to 1%, but is not limited thereto.
[0096] In the method of purifying a recombinant fusion protein
expressed in a soluble form according to an exemplary embodiment of
the present invention, the number of isoacceptor tRNA genes may be
0 to 2.
[0097] In the method of purifying a recombinant fusion protein
expressed in a soluble form according to an exemplary embodiment of
the present invention, 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.
[0098] In the method of purifying a recombinant fusion protein
expressed in a soluble form according to an exemplary embodiment of
the present invention, as a specific example, 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.
[0099] Hereinafter, the present invention will be described in
detail with reference to examples. The examples are provided only
to explain the present invention in more detail, but the scope of
the present invention is not limited by these examples.
Reagent, Material, and Protocol
[0100] 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.
[0101] The ramp tag was prepared by the method of analyzing codon
usage of E. coli described in Korean Patent Publication No.
1446054.
[0102] The pET24a plasmid vector was obtained from New England Labs
(UK).
[0103] Other reagents were obtained from Sigma Aldrich (US).
Experimental Example 1
Gene Manipulation for Recombinant Fusion Protein Expression
[0104] In order to confirm soluble expression of a target protein
expressed in an insoluble inclusion body form in the related art,
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.
TABLE-US-00001 TABLE 1 Base Sequence and Amino Acid Sequence of
Class I Hydrophobin DewA SEQ ID NO: Classification Sequence (5'
.fwdarw. 3') 1 Base sequence of
atgcgcttcatcgtctctctcctcgccttcactgccgcggccaccgcaa DewA
ccgccctcccggcctctgccgcaaagaacgcgaagctggccacctc
ggcggccttcgccaagcaggctgaaggcaccacctgcaatgtcggc
tcgatcgcttgctgcaactcccccgctgagaccaacaacgacagtctg
ttgagcggtctgctcggtgctggccttctcaacgggctctcgggcaac
actggcagcgcctgcgccaaggcgagcttgattgaccagctgggtct
gctcgctctcgtcgaccacactgaggaaggccccgtctgcaagaaca
tcgtcgcttgctgccctgagggaaccaccaactgtgttgccgtcgaca
acgctggcgccggtaccaaggctgagtaa 2 Amino acid
MRFIVSLLAFTAAATATALPASAAKNAKLATS sequence of DewA
AAFAKQAEGTTCNVGSIACCNSPAETNNDSL LSGLLGAGLLNGLSGNTGSACAKASLIDQLG
LLALVDHTEEGPVCKNIVACCPEGTTNCVAV DNAGAGTKAE 3 Signal sequence of
atgcgcttcatcgtctctctcctcgccttcactgccgcggccaccgcaa DewA (entire base
ccgccctcccggcctctgccgca sequence).sup.a .sup.aThe signal sequence
is a sequence predicted by using SignalP (Ver 4.0) which is an
exocrine signal sequence prediction program.
A part or all of the signal sequence was deleted by using a general
polymerase chain reaction (PCR) method well known to those skilled
in the art.
[0105] In addition, 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.
TABLE-US-00002 TABLE 2 Base Sequence and Amino Acid Sequence of
Ramp Tag, and Base Sequence of His-Tag SEQ ID Sequence NO:
Classification (5' .fwdarw. 3') 4 Base sequence of ramp tag
cttcacagtcctaatccc 5 Amino acid sequence of LPASAA ramp tag 6 Base
sequence of 6 .times. His Tag caccaccaccaccaccac
[0106] An experimental group in which a part or all of the signal
sequence in the base sequence of the Class I hydrophobin DewA of
Table 1 was deleted was prepared, and then sequences of hydrophobin
DewA of which a signal sequence was mutated were given from SEQ ID
NO 7 as shown in Table 3.
TABLE-US-00003 TABLE 3 Mutated Hydrophobin DewA according to Signal
Sequence Mutation Experimental Base sequence and amino acid
sequence of mutated SEQ ID NO: group hydrophobin (5' .fwdarw. 3') 7
Example 1 aagaacgcgaagctggccacctcggcggccttcgccaagcaggctgaa
ggcaccacctgcaatgtcggctcgatcgcttgctgcaactcccccgctga
gaccaacaacgacagtctgttgagcggtctgctcggtgctggccttctcaa
cgggctctcgggcaacactggcagcgcctgcgccaaggcgagcttgatt
gaccagctgggtctgctcgctctcgtcgaccacactgaggaaggccccgt
ctgcaagaacatcgtcgcttgctgccctgagggaaccaccaactgtgttgc
cgtcgacaacgctggcgccggtaccaaggctgagtaa 8
KNAKLATSAAFAKQAEGTTCNVGSIACCNSPAE TNNDSLLSGLLGAGLLNGLSGNTGSACAKASLI
DQLGLLALVDHTEEGPVCKNIVACCPEGTTNCV AVDNAGAGTKAE 9 Example 2
ctcccggcctctgccgcaaagaacgcgaagctggccacctcggcggcct
tcgccaagcaggctgaaggcaccacctgcaatgtcggctcgatcgcttgc
tgcaactcccccgctgagaccaacaacgacagtctgttgagcggtctgct
cggtgctggccttctcaacgggctctcgggcaacactggcagcgcctgc
gccaaggcgagcttgattgaccagctgggtctgctcgctctcgtcgacca
cactgaggaaggccccgtctgcaagaacatcgtcgcttgctgccctgagg
gaaccaccaactgtgttgccgtcgacaacgctggcgccggtaccaaggc tgagtaa 10
LPASAAKNAKLATSAAFAKQAEGTTCNVGSIAC CNSPAETNNDSLLSGLLGAGLLNGLSGNTGSAC
AKASLIDQLGLLALVDHTEEGPVCKNIVACCPE GTTNCVAVDNAGAGTKAE 1 Comparative
Atgcgcttcatcgtctctctcctcgccttcactgccgcggccaccgcaacc Example 1
gccctcccggcctctgccgcaaagaacgcgaagctggccacctcggcg
gccttcgccaagcaggctgaaggcaccacctgcaatgtcggctcgatcg
cttgctgcaactcccccgctgagaccaacaacgacagtctgttgagcggt
ctgctcggtgctggccttctcaacgggctctcgggcaacactggcagcgc
ctgcgccaaggcgagcttgattgaccagctgggtctgctcgctctcgtcga
ccacactgaggaaggccccgtctgcaagaacatcgtcgcttgctgccctg
agggaaccaccaactgtgttgccgtcgacaacgctggcgccggtaccaa ggctgagtaa
Example 1
[0107] 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.times.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.times.His).
Example 2
[0108] 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.
Comparative Example 1
[0109] A recombinant expression vector
(pET24a-Ramp-DewA-6.times.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.
Experimental Example 2
Confirmation of Soluble Expression of Recombinant Fusion
Protein
[0110] In order to confirm soluble expression of the recombinant
fusion protein in each experimental group of Table 3, 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.degree. C. for 12
hours.
[0111] Thereafter, in each experimental group, three single
colonies (#1, #2, and #3) were inoculated into each of 3.5 mL of a
liquid LB medium and then pre-cultured at 37.degree. C. and 220 rpm
for 5 or 6 hours.
[0112] 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.degree. C. and 220 rpm, and then, when an absorbance
(OD.sub.600) at 600 nm has reached about 0.7, 0.2 mM isopropyl
.beta.-D-thiogalactopyranoside (IPTG) was added to induce
expression of protein.
[0113] After the addition of IPTG, each experimental group was
separated into two temperature experimental groups of 37.degree. C.
and 18.degree. 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.
[0114] Thereafter, the target protein expressed by the following
method was recovered from the E. coli cultured at each
temperature.
[0115] a) 3.5 mL of each culture medium was centrifuged at
4.degree. C. and 13,200 rpm for 2 minutes to obtain a precipitate,
and the precipitate was suspended in 200 mM Tris-HCl (pH 8.0) and
then lysed by ultrasonic waves using a pulse of 2 seconds for 20
seconds.
[0116] b) The recovered cell lysate was centrifuged at 4.degree. C.
and 13,200 rpm for 20 minutes, thereby obtaining a first
supernatant from which cell debris was removed. In this case, after
the cell lysing, an analysis sample for SDS-PAGE analysis (total
fraction: T) was obtained, and after the centrifugation of the cell
lysate, an analysis sample (soluble fraction: S) was also obtained
to perform the SDS-PAGE analysis. A residual fraction after the
centrifugation indicates an insoluble fraction I, and the results
thereof are illustrated in FIGS. 2A and 2B.
[0117] It was confirmed from the results of FIGS. 2A and 2B that,
in Comparative Example 1, when the expression was induced through
IPTG and then the protein was expressed at 37.degree. C., the
expression amount (95 wt %) of the target protein DewA in the
insoluble fraction I was significantly larger than that in the
soluble fraction S (5 wt %).
[0118] On the other hand, it was confirmed that, in 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.
[0119] 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. In
addition, it was confirmed that, in particular, since high
expression of 15% or more of a total protein of the E. coli was
exhibited, when a high cell density culture was performed, 1 g/L of
a soluble protein was produced.
[0120] In addition, it was confirmed that in Comparative Example 1,
when the culture was performed at 18.degree. C., the expression
amount (to 100 wt %) of the target protein DewA in the insoluble
fraction I was mostly occupied as compared in the soluble fraction
S (hardly occupied).
[0121] On the other hand, it was confirmed that in 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.
[0122] This is similar to the culture at 37.degree. C., and it
could be confirmed that the target protein was expressed in a
soluble form by deletion of the signal sequence and fusion of the
ramp tag in the recombinant fusion protein, regardless of the
culture temperature.
[0123] Meanwhile, it was confirmed that in 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).
[0124] It should be understood that it is obvious to those skilled
in the art that 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.
Experimental Example 3
Purification of Class I Hydrophobin DewA Expressed in Soluble
Form
[0125] In each experimental group of Experimental Example 1, a
recombinant fusion protein was expressed in E. coli in the same
manner as that of Experimental Example 2, and then Class I
hydrophobin DewA used as the target protein expressed in a soluble
form was purified by the following purification process.
[0126] a) The Class I hydrophobin DewA was expressed in the same
manner as that in each experimental group of Table 3.
[0127] b) A culture medium in which DewA was expressed was
centrifuged at 4.degree. C. and 6,000 rpm to recover cells.
[0128] c) The recovered cells were re-suspended with 200 mM
Tris-HCl (pH 8.0) and were sonicated to lyse the cells. In this
case, the entire fraction sample was obtained.
[0129] d) The lysed cells were centrifuged at 4.degree. C. and
10,000 rpm for 1 hour to recover a first supernatant. In this case,
a soluble fraction sample was obtained.
[0130] e) Isopropyl alcohol whose volume is greater by 2.5 times
than that of the first supernatant was slowly added to the first
supernatant to obtain a first mixture.
[0131] f) The first mixture was shaken at 30.degree. C. and 200 to
250 rpm for 15 minutes.
[0132] g) After the shaking, the first mixture was centrifuged at
20.degree. C. (room temperature) and 10,000 rpm for 10 minutes to
recover a second supernatant.
[0133] h) Isopropyl alcohol whose volume is the same as that of the
second supernatant was slowly added to the second supernatant to
obtain a second mixture.
[0134] i) The second mixture was shaken at 30.degree. C. and 200 to
250 rpm for 15 minutes.
[0135] j) Thereafter, after the shaking, the second mixture was
centrifuged at 20.degree. C. (room temperature) and 10,000 rpm for
10 minutes to separate Class I hydrophobin DewA protein used as the
target protein purified in a white precipitated form.
[0136] Each of the total fraction sample and the soluble fraction
samples obtained in c) and d) and the target protein DewA in the
white precipitated form obtained in j) was dissolved in 1 mL of 200
mM Tris-HCl (pH 8.0) used as a re-suspension solvent, 1 mL of
Tris-HCl was continuously and sequentially added, and then SDS-PAGE
analysis of the diluted sample was performed. The results thereof
are illustrated in FIG. 4.
[0137] In FIG. 4, 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, and
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.
[0138] As can be seen from the above results, it is shown that 80%
or more of the total fraction sample of lane 1 is also observed in
the soluble fraction sample of lane 2, and, in each of lanes 3 to
7, the target protein expressed in a soluble form may be
efficiently purified by the two phase separation method using the
organic solvent of the present invention.
[0139] It was confirmed from the results that the hydrophobin
expressed in a soluble form was efficiently purified by a simple
purification method without a complex physical and chemical
purification process.
[0140] In addition, it should be understood that it is obvious to
those skilled in the art that, similarly to Experimental Example 2,
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.
Comparative Example 2
[0141] 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.
Comparative Example 3
[0142] 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.
Comparative Example 4
[0143] 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.
[0144] As a result, it was confirmed that, when the purification
was performed by a method outside the scope of the present
invention in each of Comparative Examples 2 to 4, the Class I
hydrophobin DewA expressed in a soluble form was not efficiently
purified (data not shown).
[0145] Therefore, it should be understood that it is obvious to
those skilled in the art that the 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.
Comparative Example 5
Purification of Hydrophobin According to Related Art (Triton X-114
Used as Nonionic Surfactant)
[0146] 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.
[0147] The recombinant expression vector
(pET24a-Ramp-DewA(-ss)-6.times.His) of Example 1 was cultured at
37.degree. 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.
[0148] 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.
[0149] The total fraction sample was centrifuged at 4.degree. C.
and 10,000 rpm for 30 minutes to obtain a soluble fraction sample
(supernatant).
[0150] Then, 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.
[0151] Then, only a lower part containing the hydrophobin was left
by removing an upper part, isobutyl alcohol whose volume is the
same as that of the lower part was added to the lower part,
vortexing was performed for 1 minute to mix them, and the mixture
was centrifuged at 4.degree. C. and 5,000 rpm while confirming
phase separation.
[0152] The upper part removed in the previous step was also
extracted by using isobutyl alcohol in the same manner as described
above.
[0153] The above procedure and results are illustrated in FIG.
5.
[0154] In FIG. 5, 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.
[0155] A tube 2 shows a state after a phase is separated into an
upper part 3 and a lower part 4.
[0156] 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.
[0157] 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.
[0158] 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, and lane 6
represents a sample of the lower part in the tube 4.
[0159] As described above, 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. However, it was
confirmed that 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.
[0160] As set forth above, 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.
Sequence CWU 1
1
101408DNAArtificial SequenceDewA nucleotide 1atgcgcttca tcgtctctct
cctcgccttc actgccgcgg ccaccgcaac cgccctcccg 60gcctctgccg caaagaacgc
gaagctggcc acctcggcgg ccttcgccaa gcaggctgaa 120ggcaccacct
gcaatgtcgg ctcgatcgct tgctgcaact cccccgctga gaccaacaac
180gacagtctgt tgagcggtct gctcggtgct ggccttctca acgggctctc
gggcaacact 240ggcagcgcct gcgccaaggc gagcttgatt gaccagctgg
gtctgctcgc tctcgtcgac 300cacactgagg aaggccccgt ctgcaagaac
atcgtcgctt gctgccctga gggaaccacc 360aactgtgttg ccgtcgacaa
cgctggcgcc ggtaccaagg ctgagtaa 4082135PRTArtificial SequenceDewA
amino acid 2Met Arg Phe Ile Val Ser Leu Leu Ala Phe Thr Ala Ala Ala
Thr Ala1 5 10 15Thr Ala Leu Pro Ala Ser Ala Ala Lys Asn Ala Lys Leu
Ala Thr Ser 20 25 30Ala Ala Phe Ala Lys Gln Ala Glu Gly Thr Thr Cys
Asn Val Gly Ser 35 40 45Ile Ala Cys Cys Asn Ser Pro Ala Glu Thr Asn
Asn Asp Ser Leu Leu 50 55 60Ser Gly Leu Leu Gly Ala Gly Leu Leu Asn
Gly Leu Ser Gly Asn Thr65 70 75 80Gly Ser Ala Cys Ala Lys Ala Ser
Leu Ile Asp Gln Leu Gly Leu Leu 85 90 95Ala Leu Val Asp His Thr Glu
Glu Gly Pro Val Cys Lys Asn Ile Val 100 105 110Ala Cys Cys Pro Glu
Gly Thr Thr Asn Cys Val Ala Val Asp Asn Ala 115 120 125Gly Ala Gly
Thr Lys Ala Glu 130 135372DNAArtificial SequenceDewA signal
sequence 3atgcgcttca tcgtctctct cctcgccttc actgccgcgg ccaccgcaac
cgccctcccg 60gcctctgccg ca 72418DNAArtificial SequenceRamp Tag
nucleotide 4cttcacagtc ctaatccc 1856PRTArtificial SequenceRamp Tag
amino acid 5Leu Pro Ala Ser Ala Ala1 5618DNAArtificial Sequence6 x
His Tag nucleotide 6caccaccacc accaccac 187336DNAArtificial
SequenceDewA nucleotide - Example 1 7aagaacgcga agctggccac
ctcggcggcc ttcgccaagc aggctgaagg caccacctgc 60aatgtcggct cgatcgcttg
ctgcaactcc cccgctgaga ccaacaacga cagtctgttg 120agcggtctgc
tcggtgctgg ccttctcaac gggctctcgg gcaacactgg cagcgcctgc
180gccaaggcga gcttgattga ccagctgggt ctgctcgctc tcgtcgacca
cactgaggaa 240ggccccgtct gcaagaacat cgtcgcttgc tgccctgagg
gaaccaccaa ctgtgttgcc 300gtcgacaacg ctggcgccgg taccaaggct gagtaa
3368111PRTArtificial SequenceDewA amino acid - Example 1 8Lys Asn
Ala Lys Leu Ala Thr Ser Ala Ala Phe Ala Lys Gln Ala Glu1 5 10 15Gly
Thr Thr Cys Asn Val Gly Ser Ile Ala Cys Cys Asn Ser Pro Ala 20 25
30Glu Thr Asn Asn Asp Ser Leu Leu Ser Gly Leu Leu Gly Ala Gly Leu
35 40 45Leu Asn Gly Leu Ser Gly Asn Thr Gly Ser Ala Cys Ala Lys Ala
Ser 50 55 60Leu Ile Asp Gln Leu Gly Leu Leu Ala Leu Val Asp His Thr
Glu Glu65 70 75 80Gly Pro Val Cys Lys Asn Ile Val Ala Cys Cys Pro
Glu Gly Thr Thr 85 90 95Asn Cys Val Ala Val Asp Asn Ala Gly Ala Gly
Thr Lys Ala Glu 100 105 1109354DNAArtificial SequenceDewA
nucleotide - Example 2 9ctcccggcct ctgccgcaaa gaacgcgaag ctggccacct
cggcggcctt cgccaagcag 60gctgaaggca ccacctgcaa tgtcggctcg atcgcttgct
gcaactcccc cgctgagacc 120aacaacgaca gtctgttgag cggtctgctc
ggtgctggcc ttctcaacgg gctctcgggc 180aacactggca gcgcctgcgc
caaggcgagc ttgattgacc agctgggtct gctcgctctc 240gtcgaccaca
ctgaggaagg ccccgtctgc aagaacatcg tcgcttgctg ccctgaggga
300accaccaact gtgttgccgt cgacaacgct ggcgccggta ccaaggctga gtaa
35410117PRTArtificial SequenceDewA amino acid - Example 2 10Leu Pro
Ala Ser Ala Ala Lys Asn Ala Lys Leu Ala Thr Ser Ala Ala1 5 10 15Phe
Ala Lys Gln Ala Glu Gly Thr Thr Cys Asn Val Gly Ser Ile Ala 20 25
30Cys Cys Asn Ser Pro Ala Glu Thr Asn Asn Asp Ser Leu Leu Ser Gly
35 40 45Leu Leu Gly Ala Gly Leu Leu Asn Gly Leu Ser Gly Asn Thr Gly
Ser 50 55 60Ala Cys Ala Lys Ala Ser Leu Ile Asp Gln Leu Gly Leu Leu
Ala Leu65 70 75 80Val Asp His Thr Glu Glu Gly Pro Val Cys Lys Asn
Ile Val Ala Cys 85 90 95Cys Pro Glu Gly Thr Thr Asn Cys Val Ala Val
Asp Asn Ala Gly Ala 100 105 110Gly Thr Lys Ala Glu 115
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