Protein soluble expression using peptide tags

文档序号：788882 发布日期：2021-04-09 浏览：28次中文

阅读说明：本技术 利用肽标签的蛋白质可溶性表达 (Protein soluble expression using peptide tags ) 是由小池和好泷田英司松井健史于 2019-08-28 设计创作，主要内容包括：通过将目的蛋白与具有下述氨基酸序列的肽标签连接并表达,将目的蛋白高效蓄积在可溶性级分中：X(PY)-qPZ,P表示脯氨酸,X表示由0～5个氨基酸组成的氨基酸序列,所述氨基酸独立选自由精氨酸(R),甘氨酸(G),丝氨酸(S),赖氨酸(K),苏氨酸(T),亮氨酸(L),天冬酰胺(N),谷氨酰胺(Q)及甲硫氨酸(M)组成的组,Y表示由1～4个氨基酸组成的氨基酸序列,所述氨基酸独立选自由R,G,K,T,L,N,Q及M组成的组,q表示1～10的整数,Z表示由0～10个氨基酸组成的氨基酸序列,所述氨基酸独立选自由R,G,S,K,T,L,N,Q及M组成的组,在所述肽标签的氨基酸序列中,Q,M,L,N及T合计包含3个以上。(The target protein is efficiently accumulated in the soluble fraction by linking and expressing the target protein with a peptide tag having the following amino acid sequence: x (PY) q PZ, P represents proline, X represents an amino acid sequence consisting of 0-5 amino acidsWherein the amino acids are independently selected from the group consisting of arginine (R), glycine (G), serine (S), lysine (K), threonine (T), leucine (L), asparagine (N), glutamine (Q) and methionine (M), Y represents an amino acid sequence consisting of 1 to 4 amino acids, the amino acids are independently selected from the group consisting of R, G, K, T, L, N, Q and M, Q represents an integer of 1 to 10, Z represents an amino acid sequence consisting of 0 to 10 amino acids, the amino acids are independently selected from the group consisting of R, G, S, K, T, L, N, Q and M, and in the amino acid sequence of the peptide tag, Q, M, L, N and T together comprise 3 or more.)

1. A soluble fraction obtained by introducing a polynucleotide encoding a fusion protein comprising a target protein and a peptide tag linked to the target protein and having the following amino acid sequence, into an expression system containing the polynucleotide, producing the fusion protein derived from the polynucleotide, and accumulating the fusion protein,

X(PY)_qPZ

p represents a proline, and P represents a proline,

x represents an amino acid sequence consisting of 0 to 5 amino acids independently selected from the group consisting of arginine (R), glycine (G), serine (S), lysine (K), threonine (T), leucine (L), asparagine (N), glutamine (Q) and methionine (M),

y represents an amino acid sequence consisting of 1 to 4 amino acids independently selected from the group consisting of R, G, K, T, L, N, Q and M,

q represents an integer of 1 to 10,

z represents an amino acid sequence consisting of 0 to 10 amino acids independently selected from the group consisting of R, G, S, K, T, L, N, Q and M,

in the amino acid sequence of the peptide tag, Q, M, L, N and T are contained in total in 3 or more.

2. The soluble fraction of claim 1, wherein PY is 1 or more selected from PGQ, PGM, PGT, PGL, PQQ, PGN, PGQG, PGMG, PGTG, PGLG, PGNG, PQQQ.

3. The soluble fraction of claim 1 or 2, wherein the peptide tag is 10-30 amino acids in length.

4. The soluble fraction of any one of claims 1 to 3, wherein the peptide tag has the amino acid sequence of SEQ ID NO: 7, 10, 12, 15, 17, 19, 21 or 23.

5. The soluble fraction of any one of claims 1 to 4, wherein the protein of interest is an enzyme.

6. The soluble fraction of any one of claims 1 to 5, wherein the fusion protein comprises a secretion signal.

7. The soluble fraction of any one of claims 1 to 6, wherein the peptide tag is attached to the C-terminal side of the protein of interest.

8. A method for producing a fusion protein, wherein the soluble fraction of any one of claims 1 to 7 is recovered and the fusion protein is extracted.

9. An expression system for introducing a polynucleotide encoding a fusion protein comprising a target protein and a peptide tag linked to the target protein and having the following amino acid sequence, and producing the fusion protein undenatured,

X(PY)_qPZ

p represents a proline, and P represents a proline,

y represents an amino acid sequence consisting of 1 to 4 amino acids independently selected from the group consisting of R, G, K, T, L, N, Q and M,

q represents an integer of 1 to 10,

z represents an amino acid sequence consisting of 0 to 10 amino acids independently selected from the group consisting of R, G, S, K, T, L, N, Q and M,

in the amino acid sequence of the peptide tag, Q, M, L, N and T are contained in total in 3 or more.

10. A solution produced from the expression system of claim 9 and comprising the fusion protein undenatured.

11. A method for improving the efficiency of substance production by utilizing a metabolic system involving an enzyme, wherein the fusion protein is produced in the form of an undenatured protein whose target protein is an enzyme using the expression system according to claim 9, and the substrate conversion reaction is carried out using the undenatured enzyme fusion protein obtained.

12. A fusion protein comprising a target protein and a peptide tag comprising the following amino acid sequence linked to the target protein,

X(PY)_qPZ

p represents a proline, and P represents a proline,

y represents an amino acid sequence consisting of 1 to 4 amino acids independently selected from the group consisting of R, G, K, T, L, N, Q and M,

q represents an integer of 1 to 10,

z represents an amino acid sequence consisting of 0 to 10 amino acids independently selected from the group consisting of R, G, S, K, T, L, N, Q and M,

in the amino acid sequence of the peptide tag, Q, M, L, N and T are 3 or more in total, and at least 1M is included therein.

13. The fusion protein of claim 12, wherein PY is selected from 1 or more of PGM and PGMG.

14. The fusion protein of claim 12 or 13, wherein the peptide tag is 10-30 amino acids in length.

15. The fusion protein of any one of claims 12-14, wherein the peptide tag has the amino acid sequence of SEQ ID NO: 7, 10, 12, 15, 17, 19, 21 or 23.

16. The fusion protein according to any one of claims 12 to 15, wherein the protein of interest is an enzyme.

17. The fusion protein of any one of claims 12-15, wherein the fusion protein comprises a secretion signal.

18. The fusion protein of any one of claims 12-17, wherein the peptide tag is attached to the C-terminal side of the protein of interest.

19. A polynucleotide encoding the fusion protein of any one of claims 12 to 18.

20. A recombinant vector comprising the polynucleotide of claim 19.

Technical Field

The present invention relates to a technique for producing a recombinant protein, and more particularly, to a technique for expressing a recombinant protein in a soluble fraction with high efficiency.

Background

With the development of gene recombination technology, the possibility of heterologous expression of useful proteins derived from higher organisms using Escherichia coli, yeast, or the like has been realized. The production of useful proteins expressed heterologously has now reached a position of more widespread technology. As measures for improving the expression and accumulation of useful proteins, the selection of promoters and terminators, the use of translation enhancers, the codon change of introduced genes, the intracellular transport and localization of proteins, and the like have been studied. In addition to this, a number of techniques have been developed to increase the expression of useful proteins by attaching peptide tags thereto. For example, patent document 1 discloses that expression of a useful protein in a soluble fraction is enhanced by linking and expressing an Endo Britle DOMAIN (EBD) peptide to the useful protein. However, since serine is contained between proline and proline in the EBD peptide, the total yield of the produced protein may be low.

Documents of the prior art

Patent document

Patent document 1: U.S. patent publication No. 20090137004

Disclosure of Invention

Problems to be solved by the invention

The present invention addresses the problem of efficiently producing a target protein in a soluble fraction in a gene recombinant protein production technique.

Means for solving the problems

The present inventors have conducted intensive studies to solve the above problems. As a result, it was found that the expression level of the target protein in the soluble fraction was significantly increased by using a peptide tag having the following amino acid sequence. That is, the amino acid composition and sequence of the peptide tag to be ligated are drastically changed to dramatically increase the expression level in the soluble fraction while maintaining the expression level of the target protein, and thus the success has been achieved. The present invention has been completed based on such findings.

The gist of the present invention is as follows.

[1] A soluble fraction obtained by introducing a polynucleotide encoding a fusion protein comprising a target protein and a peptide tag linked to the target protein and having the following amino acid sequence, into an expression system containing the polynucleotide, producing the fusion protein derived from the polynucleotide, and accumulating the fusion protein,

X(PY)_qPZ

p represents a proline, and P represents a proline,

y represents an amino acid sequence consisting of 1 to 4 amino acids independently selected from the group consisting of R, G, K, T, L, N, Q and M,

q represents an integer of 1 to 10, Z represents an amino acid sequence consisting of 0 to 10 amino acids independently selected from the group consisting of R, G, S, K, T, L, N, Q and M,

in the amino acid sequence of the peptide tag, Q, M, L, N and T are contained in total in 3 or more.

[2] The soluble fraction according to [1], wherein PY is 1 or more selected from PGQ, PGM, PGT, PGL, PQQ, PGN, PGQG, PGMG, PGTG, PGLG, PGNG, PQQQ.

[3] The soluble fraction according to [1] or [2], wherein the peptide tag has a length of 10 to 30 amino acids.

[4] The soluble fraction according to any one of [1] to [3], wherein the peptide tag has an amino acid sequence of SEQ ID NO: 7, 10, 12, 15, 17, 19, 21 or 23.

[5] The soluble fraction according to any one of [1] to [4], wherein the target protein is an enzyme.

[6] The soluble fraction according to any one of [1] to [5], wherein the fusion protein comprises a secretion signal.

[7] The soluble fraction according to any one of [1] to [6], wherein the peptide tag is linked to the C-terminal side of the target protein.

[8] A process for producing a fusion protein, which comprises recovering the soluble fraction according to any one of [1] to [7] and extracting the fusion protein.

[9] An expression system for introducing a polynucleotide encoding a fusion protein comprising a target protein and a peptide tag linked to the target protein and having the following amino acid sequence, and producing the fusion protein undenatured,

X(PY)_qPZ

p represents a proline, and P represents a proline,

y represents an amino acid sequence consisting of 1 to 4 amino acids independently selected from the group consisting of R, G, K, T, L, N, Q and M,

q represents an integer of 1 to 10,

z represents an amino acid sequence consisting of 0 to 10 amino acids independently selected from the group consisting of R, G, S, K, T, L, N, Q and M,

in the amino acid sequence of the peptide tag, Q, M, L, N and T are contained in total in 3 or more.

[10] A solution produced from the expression system of [9] and comprising the fusion protein undenatured.

[11] A method for improving the efficiency of substance production by utilizing a metabolic system involving an enzyme, wherein the fusion protein is produced as an undenatured protein using the expression system described in [9], the target protein of the fusion protein is an enzyme, and a substrate conversion reaction is carried out using the obtained undenatured enzyme fusion protein.

[12] A fusion protein comprising a target protein and a peptide tag comprising the following amino acid sequence linked to the target protein,

X(PY)_qPZ

p represents a proline, and P represents a proline,

y represents an amino acid sequence consisting of 1 to 4 amino acids independently selected from the group consisting of R, G, K, T, L, N, Q and M,

q represents an integer of 1 to 10,

z represents an amino acid sequence consisting of 0 to 10 amino acids independently selected from the group consisting of R, G, S, K, T, L, N, Q and M, and Q, M, L, N and T in the amino acid sequence of the peptide tag are 3 or more in total, including at least 1M.

[13] The fusion protein according to [12], wherein PY is at least 1 selected from PGM and PGMG.

[14] The fusion protein according to [12] or [13], wherein the peptide tag is 10 to 30 amino acids in length.

[15] The fusion protein according to any one of [12] to [14], wherein the peptide tag has an amino acid sequence of SEQ ID NO: 7, 10, 12, 15, 17, 19, 21 or 23.

[16] The fusion protein according to any one of [12] to [15], wherein the target protein is an enzyme.

[17] The fusion protein according to any one of [12] to [15], wherein the fusion protein comprises a secretion signal.

[18] The fusion protein according to any one of [12] to [17], wherein the peptide tag is linked to the C-terminal side of the target protein.

[19] A polynucleotide encoding the fusion protein according to any one of [12] to [18 ].

[20] A recombinant vector comprising the polynucleotide of [19 ].

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, the target protein can be expressed efficiently by linking to a peptide tag having a specific sequence, and the amount of the target protein accumulated in the soluble fraction can be increased, thereby facilitating the isolation and purification of the target protein. When the target protein contains a secretion signal sequence, the efficiency of secretory production of the target protein into a medium can be improved.

In addition, when the target protein is an enzyme, a solution containing an undenatured enzyme fusion protein can be easily prepared from a culture medium, a cell disruption solution, or the like, and can be applied to an enzymatic reaction based on a metabolic system in which an enzyme participates, and thus can contribute to substance production by an efficient conversion reaction using a substrate.

Further, since the introduced enzyme is less aggregated and insoluble in the cell, the enzyme reaction in the cell is efficiently promoted.

Unlike the peptide tag described in patent document 1, the peptide tag used in the present invention does not contain serine between proline, and therefore, the yield of protein in the soluble fraction can be expected to be improved. In addition, even in the case of being linked to the C-terminal side of the target protein, the expression of the fusion protein in the soluble fraction can be improved.

Drawings

FIG. 1 shows the construction sequence of a plasmid for expressing a fusion protein in Bacillus pumilus (Brevibacillus) to which various peptide tags have been ligated at the N-terminus of esterase.

FIG. 2 is a diagram showing the procedure for constructing a plasmid for expressing a fusion protein in Bacillus pumilus, the fusion protein being obtained by ligating esterase to the C-terminus of various peptide tags.

FIG. 3 is a graph showing a comparison of the expression levels of esterases in the soluble fraction, each of which peptide tags were ligated to the N-terminal side.

FIG. 4 is a graph showing a comparison of the expression levels of esterases in the soluble fraction, each of which peptide tags were ligated to the C-terminal side.

FIG. 5 shows the results of comparison of the activity of an esterase labeled with a label (A: PX12-32, B: PX12-34) with that of an esterase labeled with no label. S represents a fraction secreted into the medium, and P represents a soluble fraction in the cell.

FIG. 6 is a diagram showing comparison of the expression levels of human growth hormone in each fraction in which each peptide tag is ligated to the N-terminal side (yeast).

Detailed Description

The fusion protein may be extracted by producing the fusion protein derived from the polynucleotide in an expression system into which a polynucleotide encoding the fusion protein comprising a target protein and a peptide tag linked to the target protein and having the following amino acid sequence is introduced, accumulating the fusion protein to form a soluble fraction, and recovering the soluble fraction.

The peptide tag used in the present invention has the following sequence.

X(PY)_qPZ

X represents an amino acid sequence consisting of 0 to 5 amino acids independently selected from the group consisting of arginine (R), glycine (G), serine (S), lysine (K), threonine (T), leucine (L), asparagine (N), glutamine (Q) and methionine (M).

When X is 0 amino acids, the N-terminal amino acid of the peptide tag is P.

In case X is 1 amino acid, the N-terminus of the peptide tag is an amino acid selected from R, G, S, K, T, L, N, Q and M.

In the case where X is 1 amino acid, Q, L, N, M or T is preferable, and Q, M or T is more preferable.

When X is2 to 5 amino acids, 2 to 5X may be the same amino acid residue selected from R, G, S, K, T, L, N, Q, M, or may be different amino acid residues.

The number of X is preferably 1 to 5, more preferably 2 to 5, further preferably 2 to 3, and particularly preferably 2.

When X is2 to 5 amino acids, 2 amino acids on the C-terminal side, i.e., (PY)_qPreferably, the immediately preceding 2 amino acids of (a) are RQ, RL, RN, RM or RT, more preferably RQ, RM or RT.

(PY)_qWherein Y represents an amino acid sequence comprising 1 to 4 amino acids independently selected from R, G, K, T, L, N, Q and M, and Q represents an integer of 1 to 10.

That is, (PY)_qRefers to the introduction of PY, i.e., Y is 1 amino acid (PY)_a)2 amino acids (PY)_aY_b) 3 amino acids (PY)_aY_bY_c) Or 4 amino acids (PY)_aY_bY_cY_d) Thus PY is added_a，PY_aY_b，PY_aY_bY_cAnd PY_aY_bY_cY_dAny one or more of them are continued for a total of q consecutive times (P represents proline). Preferably, PY is2 amino acids (PY)_aY_b) Or 3 amino acids (PY)_aY_bY_c). q is an integer of 1 to 10, preferably an integer of 2 to 10, more preferably an integer of 2 to 5, further preferably an integer of 2 to 3, and particularly preferably 2.

Each Y may be the same amino acid residue selected from R, G, K, T, L, N, Q and M, or may be different amino acid residues, however, preferably at least 1 of all Y (all Y included in the peptide tag) included in PY Q consecutive times is Q, N, L, M or T, more preferably 2 or more are Q, N, L, M or T. Preferably, at least 1 of all Y (all Y included in the peptide tag) included in PY Q consecutive times is Q, M or T is preferable, and more preferably 2 or more are Q, M or T.

In addition, for each PY (PY) of q consecutive times₁，PY₁Y₂，PY₁Y₂Y₃And PY₁Y₂Y₃Y₄) In the case of Y, it is preferable that the amino acid other than Q, N, L, M and T is G.

For example, in Y2 amino acids (PY)₁Y₂) In the case where PY is preferably PGQ, PGN, PGL, PGM or PGT, more preferably PGQ, PGM or PGT, and Y is 3 amino acids (PY)₁Y₂Y₃) In the case of (2), PY is preferably PGQG, PGNG, PGLG, PGMG or PGTG, more preferably PGQG, PGMG or PGTG, and a sequence obtained by arbitrarily combining these and continuing q times is a preferred embodiment.

Z represents an amino acid sequence consisting of 0-10 amino acids independently selected from the group consisting of R, G, S, K, T, L, N, Q and M.

When Z is 0 amino acids, the amino acid at the C-terminus of the peptide tag is P.

In case Z is 1 amino acid, an amino acid selected from R, G, S, K, T, L, N, Q and M forms the C-terminus of the peptide tag.

When Z is2 to 10 amino acids, 2 to 10 of Z may be the same amino acid residue selected from R, G, S, K, T, L, N, Q and M, or may be different amino acid residues.

The number of Z is preferably 1 to 10, more preferably 1 to 5, even more preferably 2 to 5, further preferably 2 to 3, and particularly preferably 2.

When Z is 1 amino acid, it is preferably R or S, and more preferably R.

When Z is2 to 10 amino acids, RS is preferably the 2 amino acids on the C-terminal side, that is, the last 2 amino acids of the peptide tag.

Among the amino acids contained in the peptide tag, that is, the amino acid contained in X, the amino acid contained in Y, and the amino acid contained in Z, Q, N, L, M, and T preferably contain 3 or more in total, and more preferably Q, M, or T contains 3 or more in total. The total proportion of Q, N, L, M and T among the amino acids contained in the peptide tag is preferably 20 to 50%, more preferably 20 to 30%.

In addition, among the amino acids contained in the peptide tag, i.e., the amino acid contained in X, the amino acid contained in Y, and the amino acid contained in Z, Q, N, L, M, and T are contained in total in 3 or more, and the above-mentioned peptide tag in which M contains at least 1 is a novel peptide tag, a fusion protein containing the novel peptide tag, a polynucleotide encoding the fusion protein, and a recombinant vector containing the polynucleotide are included in the scope of the present invention as they are.

The length of the peptide tag used in the present invention is preferably 6 to 50 amino acids, more preferably 6 to 40 amino acids, still more preferably 8 to 40 amino acids, yet more preferably 10 to 30 amino acids, yet more preferably 12 to 25 amino acids, and particularly preferably 12 to 20 amino acids.

Preferred examples of the peptide tag used in the present invention include peptide tags having the following sequences. [ tables 1-1]

RXPY₁Y₂Y₃PY₄Y₅PRS

No.

Y₁

Y₂

Y₃

Y₄

Y₅

1-1 (SEQ ID NO. 33)

1-2 (SEQ ID NO: 34)

1-3 (SEQ ID NO: 35)

1-4 (SEQ ID NO: 36)

1-5 (SEQ ID NO: 37)

1-6 (SEQ ID NO: 38)

1-7 (SEQ ID NO: 39)

1-8 (SEQ ID NO: 40)

1-9 (SEQ ID NO: 41)

1-10 (SEQ ID NO: 42)

1-11 (SEQ ID NO: 43)

1-12 (SEQ ID NO: 44)

1-13 (SEQ ID NO: 45)

1-14 (SEQ ID NO: 46)

1-15 (SEQ ID NO: 47)

1-16 (SEQ ID NO: 48)

1-17 (SEQ ID NO: 49)

1-18 (sequence)Number 50)

t-19 (SEQ ID NO: 51)

1-20 (SEQ ID NO: 52)

1-21 (SEQ ID NO: 53)

1-22 (SEQ ID NO: 54)

1-23 (SEQ ID NO: 55)

1-24 (SEQ ID NO: 56)

1-25 (SEQ ID NO: 57)

1-26 (SEQ ID NO: 58)

1-27 (SEQ ID NO: 59)

1-28 (SEQ ID NO: 60)

1-29 (SEQ ID NO: 61)

1-30 (SEQ ID NO: 62)

1-31 (SEQ ID NO: 63)

1-32 (SEQ ID NO: 64)

1-33 (SEQ ID NO. 65)

1-34 (SEQ ID NO: 66)

1-35 (SEQ ID NO: 67)

1-36 (SEQ ID NO: 68)

1-37 (SEQ ID NO: 69)

1-38 (SEQ ID NO: 70)

1-39 (SEQ ID NO: 71)

1-40 (SEQ ID NO: 72)

[ tables 1-2]

RXPY₁Y₂Y₃PY₄Y₅PRS

No.

Y₁

Y₂

Y₃

Y₄

Y₅

1-41 (SEQ ID NO: 73)

1-42 (SEQ ID NO: 74)

1-43 (SEQ ID NO: 75)

1-44 (SEQ ID NO: 76)

1-45 (SEQ ID NO: 77)

1-46 (SEQ ID NO: 78)

1-47 (SEQ ID NO. 79)

1-48 (SEQ ID NO: 80)

1-49 (SEQ ID NO: 81)

1-50 (SEQ ID NO: 82)

1-51 (SEQ ID NO: 83)

1-52 (SEQ ID NO: 84)

1-53 (SEQ ID NO: 85)

1-54 (SEQ ID NO: 86)

1-55 (SEQ ID NO: 87)

1-56 (SEQ ID NO: 88)

1-57 (SEQ ID NO. 89)

1-58 (SEQ ID NO: 90)

1-59 (SEQ ID NO: 91)

1-60 (SEQ ID NO: 92)

1-61 (SEQ ID NO: 93)

1-62 (SEQ ID NO: 94)

1-63 (SEQ ID NO: 95)

1-64 (SEQ ID NO: 96)

1-65 (SEQ ID NO: 97)

1-66 (SEQ ID NO: 98)

1-67 (SEQ ID NO. 99)

1-68 (Serial number 100)

1-69 (SEQ ID NO: 101)

1-70 (SEQ ID NO: 102)

[ tables 1 to 3]

RXPY₁Y₂Y₃PY₄Y₅GRS

No.

Y₁

Y₂

Y₃

Y₄

Y₅

2-1 (SEQ ID NO: 103)

2-2 (Serial number 104)

2-3 (SEQ ID NO: 105)

2-4 (SEQ ID NO: 106)

2-5 (SEQ ID NO: 107)

2-6 (SEQ ID NO: 108)

2-7 (SEQ ID NO: 109)

2-8 (Serial number 110)

2-9 (SEQ ID NO: 111)

2-10 (Serial number 112)

2-11 (SEQ ID NO: 113)

2-12 (SEQ ID NO: 114)

2-13 (SEQ ID NO: 115)

2-14 (Serial number 116)

2-15 (SEQ ID NO: 117)

2-16 (SEQ ID NO: 118)

2-17 (SEQ ID NO: 119)

2-18 (SEQ ID NO: 120)

2-19 (SEQ ID NO: 121)

2-20 (SEQ ID NO: 122)

2-21 (SEQ ID NO. 123)

2-22 (SEQ ID NO: 124)

2-23 (SEQ ID NO. 125)

2-24 (SEQ ID NO: 126)

2-25 (SEQ ID NO: 127)

2-26 (SEQ ID NO: 128)

2-27 (SEQ ID NO: 129)

2-28 (Serial number 130)

2-29 (SEQ ID NO. 131)

2-30 (SEQ ID NO: 132)

2-31 (SEQ ID NO: 133)

2-32 (SEQ ID NO: 134)

2-33 (SEQ ID NO: 135)

2-34 (SEQ ID NO: 136)

2-35 (SEQ ID NO. 137)

2-36 (SEQ ID NO: 138)

2-37 (SEQ ID NO: 39)

2-38 (SEQ ID NO: 140)

2-39 (SEQ ID NO: 141)

2-40 (SEQ ID NO: 142)

[ tables 1 to 4]

RXPY₁Y₂Y₃PY₄Y₅GRS

No.

Y₁

Y₂

Y₃

Y₄

Y₅

2-41 (SEQ ID NO. 143)

2-42 (SEQ ID NO: 144)

2-43 (SEQ ID NO. 145)

2-44 (SEQ ID NO: 146)

2-45 (SEQ ID NO: 147)

2-46 (SEQ ID NO: 148)

2-47 (SEQ ID NO: 149)

2-48 (SEQ ID NO: 150)

2-49 (SEQ ID NO. 151)

2-50 (SEQ ID NO: 152)

2-51 (SEQ ID NO: 153)

2-52 (Serial number 154)

2-53 (SEQ ID NO: 155)

2-54 (Serial number 156)

2-55 (SEQ ID NO: 157)

2-56 (SEQ ID NO: 158)

2-57 (Serial number 159)

2-58 (Serial number 160)

2-59 (Serial number 161)

2-60 (SEQ ID NO: 162)

2-61 (SEQ ID NO. 163)

2-62 (SEQ ID NO: 164)

2-63 (Serial number 165)

2-64 (SEQ ID NO: 166)

2-65 (SEQ ID NO: 167)

2-66 (Serial number 168)

2-67 (SEQ ID NO. 169)

2-68 (SEQ ID NO: 170)

2-69 (SEQ ID NO. 171)

2-70 (SEQ ID NO: 172)

[ tables 1 to 5]

RXPY₁Y₂PY₃Y₄PY₅RS

No.

Y₁

Y₂

Y₃

Y₄

Y₅

3-1 (Serial number 173)

3-2 (Serial number 174)

3-3 (SEQ ID NO: 175)

3-4 (SEQ ID NO. 176)

3-5 (Serial number 177)

3-6 (SEQ ID NO. 178)

3-7 (SEQ ID NO: 170)

3-8 (SEQ ID NO. 180)

3-9 (SEQ ID NO. 181)

3-10 (Serial number 182)

3-11 (SEQ ID NO: 183)

3-12 (SEQ ID NO: 184)

3-13 (SEQ ID NO. 185)

3-14 (Serial number 186)

3-15 (SEQ ID NO. 187)

3-16 (SEQ ID NO: 188)

3-17 (SEQ ID NO. 189)

3-18 (Serial number 190)

3-19 (SEQ ID NO. 191)

3-20 (Serial number 192)

3-21 (SEQ ID NO: 193)

3-22 (SEQ ID NO. 194)

3-23 (SEQ ID NO: 195)

3-24 (SEQ ID NO: 196)

3-25 (SEQ ID NO. 197)

3-26 (Serial number 198)

3-27 (Serial number 199)

3-28 (SEQ ID NO: 200)

3-29 (SEQ ID NO: 201)

3-30 (SEQ ID NO: 202)

3-31 (SEQ ID NO. 203)

3-32 (SEQ ID NO: 204)

3-33 (SEQ ID NO: 205)

3-34 (Serial number 206)

3-35 (SEQ ID NO: 207)

3-36 (SEQ ID NO: 208)

3-37 (SEQ ID NO. 209)

3-38 (Serial number 210)

3-39 (SEQ ID NO: 211)

3-40 (Serial number 212)

[ tables 1 to 6]

RXPY₁Y₂PY₃Y₄PY₅RS

No.

Y₁

Y₂

Y₃

Y₄

Y₅

3-41 (SEQ ID NO: 213)

3-42 (SEQ ID NO: 214)

3-43 (Serial number 215)

3-44 (SEQ ID NO: 216)

3-45 (Serial number 217)

3-46 (SEQ ID NO: 218)

3-47 (SEQ ID NO: 219)

3-48 (Serial number 220)

3-49 (SEQ ID NO. 221)

3-50 (SEQ ID NO: 222)

3-51 (SEQ ID NO: 223)

3-52 (SEQ ID NO: 224)

3-53 (SEQ ID NO: 225)

3-54 (Serial number 226)

3-55 (SEQ ID NO: 227)

3-56 (SEQ ID NO: 228)

3-57 (SEQ ID NO: 229)

3-58 (Serial number 230)

3-59 (Serial number 231)

3-60 (Serial number 232)

3-61 (SEQ ID NO. 233)

3-62 (SEQ ID NO: 234)

3-63 (SEQ ID NO. 235)

3-64 (SEQ ID NO: 236)

3-65 (SEQ ID NO: 237)

3-66 (SEQ ID NO: 238)

3-67 (SEQ ID NO: 239)

3-68 (SEQ ID NO: 240)

3-69 (Serial number 241)

3-70 (SEQ ID NO: 242)

In the present invention, the fusion protein is a protein in which the above-described peptide tag is linked to a target protein. The peptide tag may be linked to the N-terminus, the C-terminus, or both the N-terminus and C-terminus of the target protein. The peptide tag may be directly linked to the N-terminus and/or C-terminus of the target protein, or may be bound to the target protein via a sequence of 1 to several amino acids (e.g., 1 to 5 amino acids). The sequence of 1 to several amino acids may be any sequence as long as it does not adversely affect the function and expression level of the target protein, but by using the protease recognition sequence, the peptide tag can be cleaved from the target protein after expression and purification. Examples of the protease recognition sequence include a factor Xa recognition sequence and the like. The fusion protein may contain other tag sequences necessary for detection, purification, and the like, such as a His tag, HN tag, and FLAG tag.

The type of the target protein contained in the fusion protein is not particularly limited, but is preferably a protein used for medical use, diagnostic use, or substance production, and examples thereof include: growth factors, hormones, cytokines, blood proteins, enzymes, antigens, antibodies, transcription factors, receptors, fluorescent proteins or their partial peptides, etc.

As the enzyme, there may be mentioned, for example, lipase, protease, steroid synthase, kinase, phosphatase, xylanase, esterase, methylase, demethylase, oxidase, reductase, cellulase, aromatase, collagenase, transglutaminase, glycosidase and chitinase.

Examples of the growth factor include Epidermal Growth Factor (EGF), insulin-like growth factor (IGF), Transforming Growth Factor (TGF), Nerve Growth Factor (NGF), brain-derived neurotrophic factor (BDNF), Vascular Endothelial Growth Factor (VEGF), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), platelet-derived growth factor (PDGF), Erythropoietin (EPO), Thrombopoietin (TPO), Fibroblast Growth Factor (FGF), and Hepatocyte Growth Factor (HGF).

Examples of hormones include insulin, glucagon, somatostatin, growth hormone, parathyroid hormone, prolactin, leptin and calcitonin.

Examples of the cytokine include interleukins, interferons (IFN α, IFN β, IFN γ), and Tumor Necrosis Factors (TNF).

Examples of the blood protein include thrombin, serum albumin, factor VII, factor VIII, factor IX, factor X, and tissue plasminogen activator.

Examples of the antibody include complete antibody, Fab, F (ab')₂Fc, Fc fusion protein, heavy chain (H chain),light chain (L chain), Single chain fv (scFv), sc (fv)₂Disulfide bond binding fv (sdfv), Diabody (Diabody).

The antigen protein used as a vaccine is not particularly limited as long as it is a protein capable of inducing an immune response, and may be appropriately selected according to the object of the immune response to be assumed, and examples thereof include proteins derived from pathogenic bacteria and proteins derived from pathogenic viruses.

The fusion protein may be supplemented with a secretory signal peptide that functions in the host cell for secretory production use. The secretion signal peptide includes a invertase secretion signal, a P3 secretion signal, an alpha factor secretion signal and the like in the case of yeast as a host, a PelB secretion signal in the case of Escherichia coli as a host, a P2 secretion signal, a P22 secretion signal and the like in the case of Bacillus pumilus as a host. In addition, when a plant is used as a host, the plant is derived from a plant belonging to the genus Nicotiana (A)Nicotiana tabacum) Arabidopsis genus (A), (B)Arabidopsis thaliana) Dutch strawberry genus (A)Fragaria×ananassa) Lactuca genus (lettuce)Lactuca sativa) And the like.

Furthermore, a transport signal peptide such as an endoplasmic reticulum retention signal peptide or a vacuolar transport signal peptide may be added to express the fusion protein in a specific cell compartment.

The fusion protein can be produced by genetic engineering by introducing a polynucleotide encoding the fusion protein into an expression system.

In the present invention, a polynucleotide refers to a substance having a nucleotide sequence that carries genetic information encoding a fusion protein, and includes DNA, RNA, and the like. That is, the polynucleotide encoding the fusion protein includes a polynucleotide encoding the protein of interest, and a polynucleotide encoding the peptide tag. The polynucleotide encoding the protein of interest, and the polynucleotide encoding the peptide tag are linked in a manner that combines the reading frames.

The polynucleotide encoding the target protein can be obtained, for example, by a general genetic engineering technique based on a known nucleotide sequence.

In addition, it is also preferable that the codon corresponding to the amino acid constituting the fusion protein is appropriately changed depending on the host cell producing the fusion protein so as to increase the amount of translation of the fusion protein. As a method of altering codons, for example, the method of Kang et al (Protein Expr Purif.2004 Nov; 38 (1): 129-35) is referred to. Further, there may be mentioned a method of selecting a codon which is frequently used in a host cell, a method of selecting a codon having a high GC content, or a method of selecting a codon which is frequently used in a housekeeping gene of a host cell.

The polynucleotide encoding the fusion protein may also include an enhancer sequence or the like which functions in the host cell in order to enhance expression in the host cell.

The polynucleotide encoding the fusion protein can be prepared by a general genetic engineering technique, and can be constructed by ligating a DNA encoding the above-mentioned peptide tag and a DNA encoding the target protein with PCR, DNA ligase or the like, for example.

The polynucleotide encoding the fusion protein may be introduced directly into the expression system, preferably in the form of a recombinant vector comprising the polynucleotide.

The recombinant vector may be any vector in which a polynucleotide encoding the above-described fusion protein is inserted into a vector so as to be expressed in a host cell into which the vector is introduced. The vector is not particularly limited as long as it can replicate in a host cell, and examples thereof include plasmid DNA and viral DNA. The vector preferably contains a selection marker such as a drug resistance gene. Specific examples of the plasmid vector include pTrcHis2 vector, pUC119, pBR322, pBluescript II KS +, pYES2, pAUR123, pQE-Tri, pET, pGEM-3Z, pGEX, pMAL, pRI909, pRI910, pBI221, pBI121, pNCMO2, pBI101, pIG121Hm, pTrc99A, pKK223, pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo, p3 XFLAG-CMV-14, pCAT3, pcDNA3.1, pCMV, and the like.

The promoter used in the vector may be appropriately selected depending on the host cell into which the vector is to be introduced. For example, in the case of expression in yeast, GAL1 promoter, PGK1 promoter, TEF1 promoter may be usedThe promoter includes ADH1 promoter, TPI1 promoter, PYK1 promoter, etc. In the case of expression in mammalian cells, a CMV promoter, SV40 promoter, EF 1. alpha. promoter or the like can be used. In the case of expression in plants, the cauliflower virus 35S promoter, the rice actin promoter, the maize ubiquitin promoter, the lettuce ubiquitin promoter, and the like can be used. In the case of expression in Escherichia coli, the T7 promoter may be used, and in the case of expression in Bacillus pumilus, the P2 promoter, the P22 promoter, and the like may be used. Inducible promoters may be used, for example, lac, tac, trc as promoters inducible by IPTG, trp inducible by IAA, ara inducible by L-arabinose, Pzt-1 inducible by tetracycline, P inducible at high temperature (42 ℃ C.)_LA promoter of cspA gene which is a kind of cold shock gene, and the like.

Further, if necessary, a terminator sequence may be included in the host cell.

The recombinant vector can be produced by, for example, inserting the DNA encoding the fusion protein into a restriction enzyme site or a multiple cloning site of the vector by cleaving it with an appropriate restriction enzyme or adding a restriction enzyme site by PCR.

In the present invention, the polynucleotide encoding the fusion protein or the recombinant vector containing the polynucleotide is introduced into an expression system, and the fusion protein is expressed in the expression system, whereby the fusion protein can be efficiently accumulated in the soluble fraction.

The expression system herein refers to a system which has translation factors necessary for expression of proteins such as ribosomes, tRNAs, and amino acids, and can produce a fusion protein derived from a polynucleotide encoding the fusion protein or a recombinant vector containing the same, and examples thereof include prokaryotic cells of bacterial cells such as Escherichia coli, Bacillus subtilis (Bacillus bacteria), Bacillus pumilus bacteria, actinomycetes, coryneform bacteria, and cyanobacteria, yeast cells such as baker's yeast, Pichia pastoris, and Schizosaccharomyces cerevisiae, and eukaryotic cells such as Aspergillus, insect cells, mammalian cells, and plant cells. Further, cell-free protein expression systems derived from prokaryotic cells such as Escherichia coli, and cell-free protein expression systems derived from eukaryotic cells such as reticulocytes, wheat germ, and insect cell extract can also be exemplified.

The expression system is preferably a transformed cell transformed with a polynucleotide encoding the fusion protein or a recombinant vector containing the same using the expression system. The transformed cell can be prepared by introducing a polynucleotide or a recombinant vector encoding the fusion protein into a host cell by a general genetic engineering technique. For example, electroporation (Tada, et al, 1990, Theor. appl. Genet, 80: 475), protoplast method (Gene, 39, 281-286(1985)), polyethylene glycol method (Lazzeri, et al, 1991, Theor. appl. Genet.81: 437), methods using Agrobacterium introduction (Hood, et al, 1993, Transgenic, Res.2: 218, Hiei, et al, 1994Plant J.6: 271), particle gun method (Sanford, et al, 1987, J.Part. Sci.tech.5: 27), polycation method (Ohtsuki, et al, FEBS Lett.1998May 29; 428 (3): 235-40), and the like can be used. The gene expression may be transient expression or stable expression incorporated into a chromosome. In selecting transformants, a selection marker such as a drug resistance gene may be used.

In the expression systems such as the transformed cell and the cell-free protein expression system, the fusion protein is accumulated in the soluble fraction. The conditions of the medium, temperature, time and the like for protein expression may be appropriately selected depending on the kind of the expression system.

The soluble fraction refers to a fraction that can recover the fusion protein in the form of a solution, including extracellular medium, intracellular liquid fractions other than nuclei and organelles, and the like.

The fusion protein is preferably produced in the form of an undenatured protein. Here, the undenatured protein refers to a protein that retains the higher structure of the protein and does not undergo a change such as loss of activity or insolubilization, or a protein that undergoes such a small change.

By recovering the soluble fraction, a solution containing undenatured protein can be obtained.

Here, the solution containing undenatured protein means that the protein in an undenatured state is dissolved in water inside and outside the cell. The expressed protein is in a state in which it is not aggregated and is located in a suitable position in the cytosol or the cell, and the original function and activity are easily exhibited, and the target protein is in a state in which it is dissolved in a solvent mainly composed of water without aggregation outside the cell, and the dissolved protein is in a state in which it has the original function and activity.

The fusion protein accumulated in the soluble fraction may be used as it is for an enzymatic reaction or the like, or may be isolated and purified by a method known to those skilled in the art. For example, separation and purification can be carried out by using a known suitable method such as salting out, ethanol precipitation, ultrafiltration, gel filtration chromatography, ion exchange column chromatography, affinity chromatography, medium-high pressure liquid chromatography, reverse phase chromatography, hydrophobic chromatography, or the like, or by combining these methods.

Examples

Hereinafter, examples of the present invention will be described, but the present invention is not limited to these examples.

(1) Construction and transformation of plasmid for gene expression encoding esterase with peptide tag for Bacillus pumilus

Encoding the polypeptide from Bacillus subtilis(Bacillus subtilis)The plasmid 1 was obtained by inserting pUC19 into the EcoRV recognition site of the modified plasmid pUCFA (FASMAC) of the artificially synthesized DNA (SEQ ID NO: 26) of esterase (EstA, SEQ ID NO: 25).

pNCMO2(TAKARABio) (plasmid 2) was used as a plasmid for expression of Bacillus pumilus.

Plasmids expressing fusion proteins in B.pumilus, each of which had a peptide tag added to the N-terminus of esterase and a 6XHis tag for detection and purification added to the C-terminus, or fusion proteins having a peptide tag added to the N-terminus of esterase and a 6XHis tag for detection and purification added to the C-terminus, were constructed according to the following procedures (FIGS. 1 and 2).

TABLE 2 amino acid sequence of each peptide tag

	No.	Label (R)	Attachment location	Amino acid sequence
					Comparative example 1	-	Label (-) -N	-	-
Comparative example 2	-	PG12-N	N-terminal	RSPGSGPGSPRS (Serial number 1)
					Comparative example 3	-	PX12-20-N	N-terminal	RKPGKGPGKPRS (Serial number 4)
Example 1	1-1	PX12-32-N	N-terminal	RQPGQGPGQPRS (Serial number 7)
					Example 2	1-2	PX12-33-N	N-terminal	RNPGNGPGNPRS (Serial number 10)
Example 3	1-3	PX12-34-N	N-terminal	RMPGMGPGMPRS (Serial number 12)
					Example 4	1-4	PX12-35-N	N-terminal	RTPGTGPGTPRS (Serial number 15)
Example 5	1-5	PX12-36-N	N-terminal	RLPGLGPGLPRS (Serial number 17)
					Example 6 of a praecox	1-66	PX1290-N	N-terminal	RQPQQQPQQPRS (sequence number 23)
Example 7	2-1	PX12-89-N	N-terminal	RQPGQGPGQGRS (Serial number 21)
					Example 8	3-3	PX12-83-N	N-terminal	RMPGMPGMPGRS (Serial number 19)
Comparative example 4	-	Label (-) -C	-	-
					Comparative example 5	-	PG12-C	C terminal	RSPGSGPGSPRS (Serial number 1)
Comparative example 6	-	PX12-20-C	C terminal	RKPGKGPGKPRS (Serial number 4)
					Example 9	1-1	PX12-32-C	C terminal	RQPGQGPGQPRS (Serial number 7)
Example 10	1-3	PX12-34-C	C terminal	RMPGMGPGMPRS (Serial number 10)

First, PCR was performed using a combination of the template plasmid shown in Table 3, the forward primer and the reverse primer in order to attach various peptide tags to the N-or C-terminus of esterase.

The 5' -end of each primer was added with the same sequence as that of plasmid 2. In addition, the forward primer was designed in such a manner that 2 amino acid residues AD were added after the signal peptide. KOD-PLUS-Ver.2 (Toyobo Co.) was used for PCR to achieve 2 pg/. mu.l template plasmid, 0.3. mu.M forward primer, 0.3. mu.M reverse primer, 0.2mM dNTPs, 1 Xbuffer for KOD-PLUS-Ver.2, 1.5mM MgSO₄50. mu.l of the reaction solution was prepared in the manner of 0.02U/. mu.l KOD-PLUS-Ver.2, and after heating at 94 ℃ for 5 minutes, 30 cycles of heating treatment at 98 ℃ for 10 seconds, at 60 ℃ for 30 seconds, at 68 ℃ for 40 seconds, and finally at 68 ℃ for 5 minutes were carried out.

The resulting amplified fragment was purified using QIAquick PCR purification kit (Qiagen).

Plasmid 2 was digested with NcoI and HindIII, separated by electrophoresis using 0.8% SeaKem GTG agarose, and extracted from the gel using a QIAquick gel recovery kit (Qiagen).

About 50ng of the amount of extracted plasmid 2 was mixed with 2. mu.l of the purified PCR product, and after adjusting the liquid amount to 5. mu.l, it was mixed with 5. mu.l of a 2 Xenzyme mixture (Enzyme Mix) attached to a Gene Art Seamless PLUS Cloning and Assembly Kit (Gene Art sample PLUS Cloning and Assembly Kit, Thermo Fisher Scientific), allowed to stand at room temperature for 30 minutes, and then allowed to stand on ice for 5 minutes.

Mu.l of the reaction mixture was mixed with competent cell DH 5-alpha, and after 30 minutes of standing on ice, heated at 42 ℃ for 45 seconds and allowed to stand on ice for 2 minutes, 250. mu.l of SOC was added and shaken at 37 ℃ and 200rpm for 1 hour. Thereafter, 50. mu.l of the shake was spread on 2 XYT agar medium containing 100mg/l ampicillin, and then cultured at 37 ℃ overnight to obtain a transformed colony. The colonies were transplanted into 2 XYT liquid medium containing 100mg/l ampicillin, cultured overnight at 37 ℃ with shaking at 200rpm, and then plasmids for gene expression were extracted and the nucleotide sequence was confirmed, and used for transformation of Bacillus pumilus.

Bacillus pumilus (Brevibacillus choshinensis) (II)Brevibacillus choshinensis) Competent cells of SP3 strain were rapidly thawed by allowing them to stand at 37 ℃ for 30 seconds in a heat block, centrifuged (12,000rpm, room temperature, 1 minute), and then the supernatant was removed, and then 1. mu.l of the plasmid solution for gene expression and 50. mu.l of solution A were mixed to give a total solution, and the particles of the competent cells were suspended completely by vortexing, and allowed to stand for 5 minutes. Mu.l of solution B was added, mixed by vortexing for 10 seconds, centrifuged (5,000rpm, room temperature, 5 minutes) and the supernatant removed, and centrifuged again (5,000rpm, room temperature, 30 seconds) to completely remove the supernatant. To this was added 1ml of MT medium, and after complete suspension using a micropipette, the mixture was cultured at 37 ℃ for 1 hour with shaking at 200 rpm. The culture solution was plated on MTNm plates and cultured at 37 ℃ for 1 night to obtain transformed Bacillus pumilus.

(2) Protein expression culture of Bacillus pumilus

The single colony transformed with Bacillus pumilus was spread on MTNm plates and incubated at 30 ℃ overnight. The cells were scraped from the plate medium after the culture using 1. mu.l of a sterile disposable loop, and the scraped cells were implanted into 5ml of TMNm medium previously transferred to a 50ml tube made of sterile polystyrene, and cultured at 30 ℃ for 48 hours with shaking at 120 rpm. After the completion of the culture, a culture solution containing the cells was sampled.

Each 100. mu.l of a culture solution containing the cells was dispensed into a 1.5ml Eppendorf tube, and the mixture was separated into a culture supernatant and cells by centrifugation (8,000 Xg, 4 ℃ C., 10 minutes), and 50. mu.l of the culture supernatant and the total amount of cells were stored at-80 ℃.

(3) Extraction of proteins from Bacillus pumilus

50. mu.l of 2 Xsample buffer (manufactured by EZ Apply, ATTO) was added to 50. mu.l of the frozen culture supernatant, and the mixture was stirred by a vortex mixer and then heated in boiling water for 10 minutes to SDS.

On the other hand, the cells were disrupted with 100. mu.l of an xTractor buffer (TAKARABio) containing 0.01% SEM-nuclease (Wako), and then centrifuged at 10,000x g for 10 minutes at 4 ℃. Then, 50. mu.l of 2 Xsample buffer was added to 50. mu.l of the supernatant fraction (intracellular soluble fraction), and SDS was performed by the same procedure.

(4) Western analysis

An Esterase (ESTA) standard was used as a standard substance for protein quantification. Dilution series were prepared by repeating 2-fold dilution with a sample buffer, and these were used as standards.

Protein electrophoresis (SDS-PAGE) was performed using a Criterion cell (BIO RAD) and a Criterion TGX-gel (BIO RAD). Electrophoresis was performed for 40 minutes at a constant voltage of 200V by adding a migration buffer (Tris/Glycine/SDS buffer, BIO RAD) to the electrophoresis chamber, dropping 4. mu.l of the SDS-treated sample to the well, and performing electrophoresis.

The gel after electrophoresis was blotted using a Trans-Blot transfer system (BIO RAD) using a Trans-Blot Turbo (BIO RAD).

The blotted membrane was immersed in a blocking solution (TBS system, pH 7.2, Nacalai Tesque), shaken at room temperature for 1 hour or left to stand at 4 ℃ for 16 hours, and then washed with TBS-T (137mM sodium chloride, 2.68mM potassium chloride, 1% polyoxyethylene sorbitan monolaurate, 25mM Tris-HCl, pH 7.4) by shaking at room temperature for 3 times for 5 minutes.

For detection of esterase, an antiserum mouse monoclonal anti-6 XHis antibody (Abcam) was diluted 6,000-fold with TBS-T and used. The membrane was immersed in the diluent and shaken at room temperature for 2 hours to perform antigen-antibody reaction, and washed in TBS-T at room temperature for 3 times and 5 minutes. As a secondary antibody, 3,000-fold dilution of anti-mouse IgG, AP-linked antibody (Cell Signaling TECHNOLOGY) with TBS-T was used.

The membrane was immersed in this diluted solution, shaken at room temperature for 1 hour to perform antigen-antibody reaction, and washed in TBS-T at room temperature for 3 times for 5 minutes. The color reaction with alkaline phosphatase was carried out by immersing the membrane in a color-developing solution (0.1M sodium chloride, 5mM magnesium chloride, 0.33mg/ml nitrotetrazolium blue, 0.33mg/ml 5-bromo-4-chloro-3-indolyl-phosphoric acid, 0.1M Tris-HCl, pH 9.5), shaking at room temperature for 15 minutes, washing the membrane with distilled water, and drying at room temperature.

The developed membrane was imaged at a resolution of 600dpi using a scanner (PM-A900, EPSON), and the amount of esterase in the culture supernatant and in the cell-solubilized fraction was determined using image analysis software (CS Analyzer ver.3.0, ATTO), and the total amount of the enzyme in the culture supernatant and in the cell-solubilized fraction was compared as the total amount of solubilized esterase.

(5) Preparation of culture samples for enzyme purification

The constructed Bacillus pumilus strain for esterase expression, which was stored at-80 ℃, was spread on an MTNm plate using a sterile loop and cultured at 37 ℃ for 16 to 20 hours. Subsequently, 5ml of TMNm was pipetted into a 50ml conical tube made of polypropylene, and the plate sample was inoculated with a sterile ring. After culturing at 30 ℃ for 16 to 20 hours with shaking at 120rpm, the whole amount of the above culture was added to a 500ml baffled Erlenmeyer flask into which 150ml of TMNm medium was pipetted, and culturing was carried out at 30 ℃ for 48 hours with shaking at 120 rpm. 50ml of the culture was dispensed into 50ml conical tubes made of polypropylene, centrifuged at 8,000 Xg at 4 ℃ for 10 minutes, the cells were precipitated, 40ml of the supernatant was transferred to another tube, the remaining supernatant was completely aspirated out by a pipette, the tubes containing the supernatant and the cells (precipitates) were frozen with liquid nitrogen, and the frozen cells were stored at-80 ℃.

(6) Purification of His-tag fusion enzyme protein from supernatant fraction

After thawing 40ml of the supernatant fraction frozen at-80 ℃, 13.3ml of pH 7.4, 0.4M sodium phosphate buffer containing 1.2M NaCl and 0.04M imidazole was added and mixed. After 5ml of TALON His-tag fusion protein purification resin (Clontech) was packed into an Econo-Pac column (BIO RAD), and equilibrated with 25ml of an equilibration buffer (pH 7.4 containing 0.3M NaCl and 0.01M imidazole, 0.1M sodium phosphate buffer), about 10ml of the supernatant sample prepared with a phosphate buffer was packed into the column, the resin was suspended, and the whole amount of the resin was washed with the remaining sample and transferred to a 100ml polypropylene centrifuge tube (manufactured by IWAKI). The centrifuge tube containing the sample was set in a rotor, and the His-tag fusion enzyme protein was adsorbed to the resin for 2 hours in a low-temperature chamber (4 ℃).

Next, the sample buffer in which the resin having the sample adsorbed thereon is suspended is transferred to an empty column, and only the buffer is eluted. Further, 50ml of equilibration buffer was passed through the column to wash the resin.

After completion of the washing, the resin-adsorbed His-tag fusion enzyme protein was eluted with 20ml of an elution buffer (pH 7.4, 0.1M sodium phosphate buffer containing 0.3M NaCl and 0.15M imidazole). For the elution, a method of dividing each 2ml fraction into 2ml Eppendorf tubes ice-cooled with an aluminum block and dividing the fractions into 10 tubes by repeating this operation 10 times was employed. Each 20. mu.l fraction was dispensed into a 1.5ml Eppendorf tube, 20. mu.l of 2 Xsample buffer (EZ Apply, manufactured by ATTO) was added thereto, and the mixture was stirred by a vortex mixer and then heated in boiling water for 10 minutes to SDS-transform the sample.

(7) Purification of His-tag fusion enzyme protein from cell fractions

After thawing the cell fractions frozen at-80 ℃, 50ml of an xTracer buffer (Clonthech) containing 5. mu.l of SEM nuclease (manufactured by Wako) was added, and the cells were dissolved by shaking at room temperature for about 20 minutes. 5ml of TALON His-tag fusion protein purification resin (Clontech) was packed into an Econo-Pac column (BIO RAD), and a supernatant sample was purified by the same method as described below, and 20. mu.l of each fraction was dispensed into a 1.5ml Eppendorf tube, and 20. mu.l of 2 Xsample buffer (EZ Apply, manufactured by ATTO) was added thereto, and the mixture was stirred by a vortex mixer and then heated in boiling water for 10 minutes to SDS-form the sample.

(8) Western analysis of purified fractions

For the standard substance for protein quantification, the purified esterase was used as a standard substance in the same manner as for the quantification of the crude extract fraction. Dilution series were prepared by repeating 2-fold dilution with a sample buffer, and these were used as standards. Protein electrophoresis (SDS-PAGE) was performed using a Criterion cell (BIO RAD) and a Criterion TGX-gel (BIO RAD). Electrophoresis buffer (Tris/Glycine/SDS buffer, BIO RAD) was added to the electrophoresis chamber, and 4. mu.l of the SDS-fractionated sample was dispensed into the well, followed by electrophoresis at a constant voltage of 200V for 40 minutes.

The gel after electrophoresis was blotted using a Trans-Blot transfer system (BIO RAD) using a Trans-Blot Turbo (BIO RAD).

The post-blotting procedures were carried out in the same manner as the Western analysis of the crude extract described above.

(9) Ultrafiltration and concentration of purified His-tag fusion enzyme protein

The fractions in which the presence of the target enzyme protein was confirmed by the Western analysis were pooled and transferred to a centrifugal filtration unit (Amicon Ultra-10K, Merck) and subjected to ultrafiltration at 5,000 Xg at 4 ℃ for 30 minutes. Subsequently, the filtrate was discarded, and 10ml of 1 XPBS buffer, pH 7.4, was added to the filtrate to carry out ultrafiltration by the same method as described above. The same operation was further repeated 3 times, and the sample was concentrated to about 1.0ml to remove imidazole from the sample. The enzyme protein concentration of the concentrate was determined according to the Bradford method using BSA as a standard.

(10) Determination of esterase Activity

The esterase activity was measured by the following method using the Lipase Kit S from DS Pharma Biomedical.

First, 2.4ml of the buffer solution was added to the developer container to dissolve the buffer solution completely, and 22ml of distilled water was added to prepare a color developing stock solution. 1.1ml of the prepared color developing stock solution was pipetted into a 1.5ml Eppendorf tube and frozen at-20 ℃.

Then, the reaction-terminated raw solution was incubated at 30 ℃ for 5 to 10 minutes to dissolve the reaction-terminated raw solution, and the total amount was diluted with distilled water to 500ml, and then stored in a refrigerator at 4 ℃. Immediately before the activity measurement, 1.1ml of the above-mentioned color developing stock solution was dissolved, and 1.1ml of the buffer solution and 8.8ml of distilled water were added thereto to prepare a color developing solution.

Subsequently, 5. mu.l of the esterase sample for measurement was pipetted into a 96-well microtiter plate relative to 100. mu.l of the developing solution, and mixed by a vortex mixer, 10. mu.l of the attached substrate solution (BALB) was added to each well, and further mixed. Subsequently, the plate was covered with aluminum foil, and the enzyme reaction was carried out at 30 ℃ for 30 minutes under a dark condition. After the reaction, 150. mu.l of a reaction-stopping solution incubated at 30 ℃ in advance was added to stop the reaction. As a blank, a sample series in which a substrate was added after the reaction was prepared, and the absorbance (412nm) of both was measured by a microplate reader, and the value obtained by subtracting the blank value was multiplied by 1,000 to obtain a BALB unit value, which was used as an indicator of the enzyme activity.

(11) Evaluation in s.cerevisiae

Transformed yeasts into each of which a plasmid encoding a fusion protein in which a peptide tag is attached to the N-terminus of human growth hormone protein (hGH) was introduced were cultured by the method described in FIG. 2 of WO2017/115853, 200. mu.l of the culture was dispensed into a 1.5ml Eppendorf tube, centrifuged at 2,000Xg at 4 ℃ for 5 minutes, the supernatant was discarded, and the pellet (cell) was frozen with liquid nitrogen and stored at-80 ℃. Next, after thawing the pellet on ice, the cell pellet was dispersed by vortexing. Further, 0.5ml of Yeast Prelysine buffer (ATTO) containing cOmplete ULTRA cocktail (Merck) and 0.1. mu.g/ml SEM nuclease (Fuji film and Wako pure chemical industries, Ltd.) was added thereto and the mixture was allowed to stand at room temperature for 10 minutes. Then, the cell lysate was centrifuged at 10,000Xg at 4 ℃ for 10 minutes, and the centrifuged supernatant and the pellet were made into a soluble fraction and an insoluble fraction, respectively, and SDS-fractionated by a predetermined method, followed by electrophoresis by SDS-PAGE, and the hGH content of each fraction was measured by Western analysis.

< results >

(1) Increased expression of proteins in soluble fractions of Bacillus pumilus by N-terminal attachment of various peptide tags

The prepared recombinant Bacillus pumilus was cultured under given conditions, and the esterase was extracted under given conditions, and the expression level of the enzyme in the soluble fraction was determined by Western analysis. As a result, according to FIG. 3, when PG12 (SEQ ID NO: 1) was added to the N-terminus as comparative example 2, the expression of esterase in the soluble fraction was not observed to be improved as compared with that in the case where no label was added. In addition, in comparative example 3 in which PX12-20 was added to the N-terminus, the expression level was 3.6 times higher than that in the case where no tag was added. On the other hand, addition of PX12-32, PX12-33, PX12-34, PX12-35, PX12-36, PX12-90, PX12-89, and PX12-83 to the N-terminus of esterase gave an expression level 6 times or more higher than that without the tag, and was clearly superior to the comparative examples. In particular, when PX12-32 was added to the N-terminus, high productivity was obtained.

(2) Increased expression of proteins in the soluble fraction of Bacillus pumilus by C-terminal attachment of various peptide tags

The prepared recombinant Bacillus pumilus was cultured under given conditions, and the esterase was extracted under given conditions, and the expression level of the enzyme in the soluble fraction was determined by Western analysis. As a result, according to FIG. 4, in the case of comparative example 5 in which PG12 (SEQ ID NO: 1) was added to the C-terminal, the expression of esterase in the soluble fraction was not observed to be improved as compared with the case where no label was added. In comparative example 6, in which PX12-20 was added to the C-terminal, the expression level was about 4 times that in the case where no tag was added. On the other hand, addition of PX12-32 and PX12-32 to the C-terminus of the esterase gave higher expression levels than the unlabeled one by 8 times or more, and the esterase yield in the soluble fraction was clearly superior to that in comparative example 6 (addition of PX 12-20).

(3) Effect of various peptide tag additions on esterase Activity

The prepared recombinant Bacillus pumilus was cultured under predetermined conditions, and the esterase secreted into the medium and the esterase in the soluble fraction in the bacterial body were purified under predetermined conditions, whereby the influence of the addition of the tag on the enzymatic activity was confirmed. As a result, as shown in FIG. 5, it was confirmed that the N-terminally tagged esterase (PX12-32-S, PX12-34-S) secreted into the medium had activity equivalent to the unlabeled (tag (-)) esterase. In addition, it was confirmed that the esterase (PXl2-32-P, PX12-34-P) having a tag attached to the N-terminal end obtained by disrupting the bacterial cells had an activity equivalent to that of the unlabeled (tagged (-) esterase).

(4) Increase in expression level of protein in soluble fraction in Yeast due to addition of various peptide tags to N-terminus

The prepared recombinant yeast was cultured, and the expression amount of hGH in each fraction was measured by Western analysis. As a result, according to FIG. 6, when PX12-20-N was added to the N-terminus, the amount of hGH in the soluble fraction was smaller than that in the insoluble fraction, and when PX12-32 and PXl2-34 were added to the N-terminus of hGH, the expression amount of hGH in the soluble fraction was significantly increased. From this fact, it was found that the effect of increasing the expression level of the protein in the soluble fraction was also observed in the eukaryotic cells.

The effect of inhibiting the degradation of the fusion protein by linking a peptide tag containing a specific sequence in eukaryote can be expected.

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Protein soluble expression using peptide tags

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