RU2801248C2 - Hybrid protein containing fragments of fluorescent proteins and its application - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to hybrid proteins based on fragments of fluorescent proteins and target therapeutic polypeptides, and can be used to express and obtain the target polypeptide. A fusion protein construct is proposed that contains fragments of fluorescent proteins and a target therapeutic polypeptide selected from human insulin precursor protein, insulin lispro precursor protein, insulin glargine precursor protein, glucagon-like peptide and its derivatives exenatide and liraglutide, insulin, calcitonin, teduglutide.

EFFECT: invention provides an increase in the efficiency of obtaining said target therapeutic polypeptides.

10 cl, 5 dwg, 1 tbl, 6 ex

Description

Technical field

The present invention relates to biotechnology, in particular, to hybrid proteins containing fragments of fluorescent proteins, and to the field of their application.

State of the art

Peptides are a class of biomolecules widely used as reagents in many areas of biomedical research, as well as therapeutic agents in the treatment of diseases, as diagnostic agents in the detection of pathogenic bacteria, and as biomarkers. Two methods for synthesizing peptides are widely used; one is chemical synthesis and the other is recombinant expression. Chemical synthesis is used to prepare a variety of therapeutic peptides, including corticorelin, parathyroid hormone (PTH), glucagon-like peptide (GLP-1) and its derivatives exenatide and liraglutide, as well as enfuvirtide, calcitonin, bivalirudin, ziconotide, semorelin, somatorelin, secretin, teduglutide and insulin. This method requires multi-step condensation of amino acid fragments to form peptides, and also requires inconvenient protection, deprotection and purification steps. To date, most commercial peptides with amino acid residues less than 50 are produced in this manner. As the demand for peptides in the pharmaceutical and biomedical research industries increases, so does the price of amino acid fragments used in chemical synthesis. As a consequence, maintaining affordable prices for peptide-based therapeutics (eg, GLP-1) used for daily use may be problematic in the future. Although from a technical point of view there is the possibility of chemical synthesis of peptides with the number of amino acid residues less than 50, however, the low useful productivity of this process and the large amount of organic waste generated in this process make such a synthesis process uneconomical. Currently, most peptides with more than 50 amino acid residues are produced by recombinant expression in host cells of organisms such as bacteria, yeast, insects and mammals. For many years, the usual way to ensure the expression of polypeptides was the use of hybrid proteins. However, the use of currently available methods of using hybrid proteins to provide expression of peptides is associated with many technical problems, especially when obtaining peptides producing less than 50 amino acid residues. For example, the fusion protein known from the prior art is characterized by high molecular weight, strong hydrophobicity, and difficulty in separation. The target protein is characterized by low specific gravity, low fusion ratio, stable structure, difficult digestion, and significant dead volume in the loading ion column and hydrophobic column.

Thus, there is an urgent need to develop a new fusion protein to provide expression of the target peptide, to overcome the limitations associated with the use of existing fusion proteins and, at the same time, to increase the useful productivity of the inserted amino acid proteins, for example, increase the production of artificial amino acid proteins. such as tert-butoxycarbonyl-lysine proteins.

The essence of the invention

The aim of the present invention is to provide a novel fusion protein to allow expression of the target peptide and at the same time increase the beneficial productivity of the inserted artificial amino acid proteins, for example, increase the production of artificial amino acid proteins such as t-butoxycarbonyl-lysine proteins.

The first object of the present invention relates to a hybrid protein having the structure shown in Formula I:

Where

"-" denotes a peptide bond or linker peptide,

each P1 is independently the first target peptide;

each P2 is independently the second target peptide;

each L1 is independently the missing or first linker peptide;

each L2 is independently the missing or second linker peptide;

A1 is a missing or signal peptide;

A2 is a missing or signal peptide;

each X is independently a single β-pleated block of a fluorescent protein;

n is a positive integer from 1 to 8;

s can take the values 0, 1 or 2;

t can take the values 0, 1 or 2; And

an additional condition is the inequality s and t to zero simultaneously.

In yet another preferred embodiment, s = 0 and t = 1.

In yet another preferred embodiment, s = 1 and t = 0.

In yet another preferred embodiment, the value of n is in the range 1-6, preferably in the range 2-4.

In yet another preferred embodiment, A1 is the signal peptide and A2 is the missing peptide.

In yet another preferred embodiment, the β-fold block is selected from the group consisting of u1, u2, u3, u4, u5, u6, u7, u8, u9, u10 and u11 fluorescent protein β-sheet blocks.

In yet another preferred embodiment, each X is 10-14 amino acids in length.

In another preferred embodiment, each X is different.

In yet another preferred embodiment, each X is the same.

In yet another preferred embodiment, the fluorescent protein is selected from the group consisting of green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein (GBP), blue fluorescent protein (GFP) gene, and any combination of these elements.

In yet another preferred embodiment, green fluorescent protein (GFP) is used as the fluorescent protein.

In another preferred embodiment, the GFP has the amino acid sequence shown in SEQ ID NO.: 13.

In yet another preferred embodiment, there is no flexible connection between any two (3-fold) blocks.

In yet another preferred embodiment, (X)n acts as a protein expression facilitator.

In another preferred embodiment, the total length Ln of element (X)n is 3.7-30% of the total length L0 of the fluorescent protein, preferably 7.5-20.5%, more preferably 7.5-15.5%) .

In another preferred embodiment, X is selected from the group consisting of

element uj, which has the amino acid sequence shown in SEQ ID NO.: j;

where j can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

In another preferred embodiment, X is selected from the group consisting of:

In yet another preferred embodiment, X further comprises an amino acid sequence formed by mutual substitution of the elements R, K and H in any of the sequences as shown in SEQIDNO.: 1-11; and/or

X further includes an amino acid sequence formed by mutual substitution of P and Q elements in the sequence as shown in any one of SEQ ID NO.: 1-11; and/or

X further includes an amino acid sequence formed by mutual substitution of elements T and S in the sequence as shown in any one of SEQ ID NO.: 1-11.

In another preferred embodiment, X is selected from the group consisting of

(A) a polypeptide having the amino acid sequence shown in any of SEQ ID NO.: 1-11;

(B) a polypeptide having ≥80% homology (preferably ≥85% homology, more preferably ≥90%, more preferably ≥95%, most preferably ≥97%) with the amino acid sequence shown in any of SEQ ID NO : 1-11, with preservation of characteristics;

(C) a polypeptide derivative formed by substitution, deletion or addition of amino acid residues 1-5 to the amino acid sequence shown in any of SEQ ID NOs: 1-11, while maintaining characteristics.

In yet another preferred embodiment, (X)n may be u8, u9, u2-u3, u4-u5, u8-u9, u1-u2-u3, u2-u3-u4, u3-u4-u5, u5-u6- u7, u8-u9-u 10, u9-u10-u11, u3-u5-u7, u3-u4-u6, u4-u7-u10, u6-u8-u10, u1-u2-u3-u4, u2-u3 -u4-u5, u3-u4-u3-u4, u3-u5-u7-u9, u5-u6-u7-u8, u1-u3-u7-u9, u2-u2-u7-u8, u7-u2-u5 -u11, u3-u4-u7-u 10, u1-I-u2, u1-I-u5, u2-I-u4, u3-I-u8, u5-I-u6 or u 10-I-u11.

In yet another preferred embodiment, A1 or A2 has the amino acid sequence shown in SEQ ID NO.: 12 (MVSKGEELFTGV).

In another preferred embodiment, A1-(X)n has the amino acid sequence shown in SEQ ID NO.: 14, 15, 16, 17, 22, 23, 24, 26, 27, 28, 29, or 30.

In yet another preferred embodiment, the first linker peptide contains a first restriction site (eg, a TEV restriction site).

In another preferred embodiment, the second linker peptide contains a second restriction site.

In yet another preferred embodiment, flexible linker I contains a third restriction site.

In yet another preferred embodiment, the third restriction site is a EK restriction site (as shown in the sequence DDDDK, SEQ ID NO.: 25).

In yet another preferred embodiment, (X)n may be one of the following: u1-EK-u2, u1-EK-u5, u2-EK-u4, u3-EK-u8, u5-EK-u6, or u10-EK -u11, where EK is the restriction site of the EK enzyme.

In another preferred embodiment, the first, second and third restriction sites are different from each other.

In another preferred embodiment, two or three of the composition of the first, second and third restriction sites are the same.

In another preferred embodiment, the first, second and third restriction sites are absent in P1 and P2.

In another preferred embodiment, the first linker peptide and/or the second linker peptide further comprise restriction sites other than the first, second and third restriction sites.

In yet another preferred embodiment, the first linker peptide and/or the second linker peptide comprise a trypsin restriction site, preferably the first linker peptide and/or the second linker peptide comprise at least either arginine (R) or lysine (K).

In another preferred embodiment, the N-terminal amino acid of the first linker peptide is Arg or Lys.

In another preferred embodiment, the C-terminal amino acid of the second linker peptide is Arg or Lys.

In yet another preferred embodiment, the first linker peptide and/or the second linker peptide further comprise a tobacco etch virus protease recognition sequence.

In yet another preferred embodiment, the first linker peptide and/or the second linker peptide comprise the amino acid sequence shown in SEQ ID NO.: 18.

In yet another preferred embodiment, the first linker peptide and/or the second linker peptide further comprise a tag sequence to aid in expression and/or purification.

In yet another preferred embodiment, the tag sequence is a histidine tag, preferably a 6xHIS tag.

In another preferred embodiment, both P1 and P2 are independently selected from the following: human insulin precursor protein, insulin lispro precursor protein, insulin glargine precursor protein, parathyroid hormone, corticorelin, calcitonin, bivalirudin, glucagon-like peptide, and exenatide derivatives thereof. and liraglutide, as well as ziconotide, sermorelin, somatorelin, secretin, teduglutide, hirudin, growth hormone, growth factor, growth hormone releasing factor, adrenocorticotropic hormone, releasing factor, deslorelin, desmopressin, elcatonin, glucagon, leuprorelin, lutropin-releasing- hormone, somatostatin, thyrotropin-releasing hormone, triptorelin, intestinal vasoactive peptide, interferon, parathyroid hormone, BH3 peptide, amyloid peptide or fragments of the above peptides, or one of the combinations (preferably, but not limited to the above polypeptides).

In yet another preferred embodiment, both P1 and P2 are independently an artificial amino acid protein.

In another preferred embodiment, both P1 and P2 independently have a sequence of 10-200 amino acids, preferably a sequence of 10-80 amino acids.

In another preferred embodiment, both P1 and P2 are independently proinsulin or insulin, preferably human proinsulin or human insulin.

In yet another preferred embodiment, the insulin includes slow acting insulin or rapid acting insulin.

In another preferred embodiment, the lysine at position 29 of proinsulin is an alkynyloxycarbonyl lysine derivative, a tert-butoxycarbonyl-lysine protein derivative (tert-butoxycarbonyl-L-lysine), an acylated aliphatic lysine, or a combination of these elements.

In yet another preferred embodiment, proinsulin has the amino acid sequence shown in SEQ ID NO: 19 or 20.

In yet another preferred embodiment, the target P2 peptide is located at the C-terminus of (X)n (or connected to it).

In yet another preferred embodiment, the fusion protein is processed to form a first target peptide and/or a second target peptide.

In another preferred embodiment, the first target peptide and the second target peptide may be the same or different.

In yet another preferred embodiment, the fusion protein is processed to form a first target peptide and/or a second target peptide, and (X)n is separated to form a short peptide of length Lx, much shorter than the length Lp of the first target peptide and/or second target peptide.

In yet another preferred embodiment, each Lx is amino acid 1-25.

In yet another preferred embodiment, the ratio of length Lx to length Lp is 1/2 to 1/10, preferably 1/3 to 1/8.

In yet another preferred embodiment, the difference between the length Lp and the length L is greater than 1.3KD.

In yet another preferred embodiment, the fusion protein has a structure selected from the group consisting of: A1-u8-L2-P2, A1-u9-L2-P2, A1-u2-u3-L2-P2, A1-u4-u5- L2-P2, A1-u8-u9-L2-P2, A1-u3-u5-u7-L2-P2, A1-u1-u2-u3-L2-P2, A1-u1-u2-u3-u4-L2- P2, A1-u3-u4-u6-L2-P2, A1-u4-u7-u10-L2-P2, A1-u3-u4-u7-u10-L2-P2 or A1-u5-EK-u6-L2- P2.

In another preferred embodiment, the fusion protein has the amino acid sequence shown in SEQ ID NO.: 21.

The second aspect of the present invention relates to the isolated polynucleotide encoding of a fusion protein according to the first aspect of the present invention.

The third object of the present invention relates to a vector that includes a polynucleotide in accordance with the second object of the present invention.

In yet another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenovirus vector, retroviral vector, transposon, or a combination of the above.

In another preferred embodiment, the vector is a plasmid; preferably, the vector is a pBAD-HisA vector and/or a pEvol-pBpF vector.

The fourth object of the present invention relates to a host cell containing a vector according to the third object of the present invention, or a polynucleotide according to the second object of the present invention integrated into a chromosome or allowing the expression of a fusion protein according to the first object of the present invention.

In another preferred embodiment, the host cell is E. coli, hay bacillus, yeast cell, insect cell, mammalian cell, or a combination of the above elements.

In another preferred embodiment, none of the host cells contains proteases corresponding to the first, second and third restriction sites.

The fifth object of the present invention relates to a method for preparing a protein, comprising the following steps:

(a) culture the host cell according to the fourth aspect of the present invention, thereby obtaining a fusion protein according to the first aspect of the present invention.

In yet another preferred embodiment, the method further comprises the following step: (b) the fusion protein obtained in step (a) is purified.

In yet another preferred embodiment, the method further comprises a procedure for proteolytically processing the fusion protein according to the first aspect of the present invention to release the target peptide from the fusion protein.

In yet another preferred embodiment, in step (c), the fusion protein according to the first aspect of the present invention is processed using a protease to obtain a processed product; and optionally

(d) isolate or purify the target peptide from the processed product.

In yet another preferred embodiment, the purification step includes further purification of the target peptide by gel filtration or the use of a high performance liquid chromatography process.

The sixth aspect of the present invention relates to the use of a fusion protein according to the first aspect of the present invention, or a polynucleotide according to the second aspect of the present invention, or a vector according to the third aspect of the present invention, or a host cell according to the fourth aspect of the present invention, to ensure the expression and preparation of the target peptide.

It should be understood that within the scope of the present invention, all of the aforementioned technical features of the present invention and all of the technical features detailed below (eg, embodiments) may be combined with each other to form a new or preferred technical solution. Due to space limitations, this document does not repeat.

Description of drawings

Figure 1 shows the structure of mature insulin correctly forming disulfide bonds.

Figure 2 shows a plasmid expression map of the recombinant human insulin fusion protein.

Figure 3 shows a schematic representation of a fusion protein of recombinant human insulin.

Figure 4 shows the structure of tert-butoxycarbonyl-lysine.

Figure 5 shows the expression of the recombinant fusion protein. Lane 1, Expression of the 11.2 kD A1-u4-u5-TEV-R-MinilNS fusion protein; Lane 2, Expression of the 8.7 kD A1-u4-u5-TEV-R-GLP1 fusion protein; Lane 3, Expression of the 10.0 kD A1-u8-TEV-R-MiniINS fusion protein; lane 4, expression of the 12.2 kD A1-u3-u5-u7-TEV-R-MiniINS fusion protein; line 5, Expression of the hybrid protein A1-u5-EK-u6-TEV-R-MiniINS with a molecular weight of 12.0 kD; M is the protein molecular weight standard.

Detailed description

After extensive and comprehensive research, the inventors for the first time have received a new hybrid protein suitable for providing expression of the target peptide. The presence of (X)n or A1-(X)n in the fusion protein of the present invention provides improved parameters for the folding process and expression of the target fusion peptide; this increases the useful productivity and solubility of the hybrid protein and reduces the degree of its intermolecular interaction, so that the target hybrid peptide can be folded at high concentrations, which ensures the industrial significance of the process. In addition, the (X)n or A1-(X)n elements of the fusion protein can be cleaved into many short peptides of much smaller length compared to the target peptide, which provides a more efficient separation of it from the target peptide, increasing the convenience of the target peptide purification procedure. . Based on the foregoing, the inventors have presented the present invention.

Terms

Before presenting the description of the present invention, it is necessary to point out that the present invention is not limited to the specific methods and experimental conditions described; such methods and conditions may vary. It should also be understood that the terms used herein are only intended to describe specific embodiments, and not to limit the scope of the present invention, which is limited only by the appended claims.

Unless otherwise indicated, all technical and scientific terms used in this document have the meaning consistent with the usual interpretation of its specialists in the field of technology to which the present invention pertains.

In the context of this document, when used in relation to a specifically recited value, the term "about" means that the value may deviate from the recited value by no more than 1%. For example, in the context of this document, the expression "about 100" includes all values from 99 to 101 (eg, 99.1; 99.2; 99.3; 99.4, etc.).

In the context of this document, the term "comprising" or "including" may be non-limiting, partially limiting and limiting. In other words, such a term also includes the meanings "substantially consisting of" or "consisting of".

The three-letter and one-letter amino acid codes used in the present invention are as described in J. biool. chem, 243, p. 3558 (1968).

In the context of this document, the use of the term "optional" or "optional" means that the event or situation described below may occur or occur, but does not have to occur or occur.

The term "sequence identity" as used in the present invention refers to the degree of identity between two nucleic acids or two amino acid sequences when optimally aligned and compared to allowable mutations such as substitution, insertion or deletion. The degree of identity when comparing the sequence described in the present invention, with its identical sequence can reach at least 85%, 90% or 95%; preferably at least up to 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

hybrid protein

In the context of this document, the terms "fusion protein of the present invention", "recombinant fusion protein" and "polypeptide" refer to a fusion protein according to the first aspect of the present invention.

Thus, the fusion protein of the present invention includes the structure shown in Formula I:

Where

"-" denotes a peptide bond or linker peptide,

each P1 is independently the first target peptide;

each P2 is independently the second target peptide;

each L1 is independently the missing or first linker peptide;

each L2 is independently the missing or second linker peptide;

A1 is a missing or signal peptide;

A2 is a missing or signal peptide;

each X is independently a single β-pleated block of a fluorescent protein;

n is a positive integer from 1 to 8;

s can take the values 0, 1 or 2;

t can take the values 0, 1 or 2; And

an additional condition is the inequality s and t to zero simultaneously.

In yet another preferred embodiment, the fluorescent protein used is GFB, and preferably the amino acid sequence of GFB includes the amino acid sequence (241 AA) as shown in SEQ ID NO.: 13.

Among them, in the above sequence, the underlined portions are the β-pleated blocks u1, u2, u3, u4, u5, u6, u7, u8, u9, u10 and u11 of the fluorescent protein sequence.

In yet another preferred embodiment, (X)n may be one of the following: u8, u9, u2-u3, u4-u5, u8-u9, u1-u2-u3, u2-u3-u4, u3-u4-u5 , u5-u6-u7, u8-u9-u10, u9-u10-u11, u3-u5-u7, u3-u4-u6, u4-u7-10 u10, u6-u8-u10, u1-u2-u3- u4, u2-u3-u4-u5, u3-u4-u3-u4, u3-u5-u7-u9, u5-u6-u7-u8, u1-u3-u7-u9, u2-u2-u7-u8, u7-u2-u5-u11, u3-u4-u7-u10, u1-I-u2, u1-I-u5, u2-I-u4, u3-I-u8, u5-I-u6 or u10-I- u11.

In yet another preferred embodiment, each X(uj) of composition (X)n has or does not have a flexible compound I, preferably a flexible linker I.

In yet another preferred embodiment, flexible linker I contains a third restriction site.

In yet another preferred embodiment, flexible linker I contains a EK restriction enzyme site (DDDDK).

In yet another preferred embodiment, the fusion protein

is processed to form a first target peptide and/or a second target peptide, and (X)n is cut to form a short peptide with a length Lx that is much shorter than the length Lp of the first target peptide and/or the second target peptide.

In another preferred embodiment, A1-(X)n is A1-u8, A1-u9, A1-u2-u3, A1-u4-u5, A1-u8-u9, A1-u3-u5-u7, A1- u1-u2-u3, A1-u1-u2-u3-u4, A1-u3-u4-u6, A1-u4-u7-u10, A1-u3-u4-u7-u10 or A1-u5-EK-u6.

In yet another preferred embodiment, A1-(X)n has the 30 amino acid sequence shown in SEQ ID NO.: 14, 15, 16, 17, 22, 23, 24, 26, 27, 28, 29, or 30.

Sequence A1-u8:

MVSKGEELFTGVGIKANFKIRHNVED (SEQ ID NO.: 23)

Sequence A1-u9:

MVSKGEELFTGVVQLADHYQQNTPIG (SEQ ID NO.: 17)

Sequence A1-u2-u3:

MVSKGEELFTGVHKFSVRGEGEGDATKLTLKFICTT (SEQ ID NO.: 24)

Sequence A1-u4-u5:

MVSKGEELFTGVYVQERTISFKDTYKTRAEVKFEGD (SEQ ID NO.: 16)

Sequence A1-u3-u5-u7:

MVSKGEELFTGVKLTLKFICTTTYKTRAEVKFEGDHNVYITADKQ (SEQ ID NO.: 15)

Sequence u8-u9 (A1 missing):

GIKANFKIRHNVEDVQLADHYQQNTPIG (SEQ ID NO.: 14)

Sequence A1-u5-EK-u6:

MVSKGEELFTGVTYKTRAEVKFEGDDDDDKTLVNRIELKGIDF (SEQ ID NO.: 22)

Sequence A1-u1-u2-u3:

MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATKLTLKFICTT (SEQ ID NO.: 26)

Sequence A1-u1-u2-u3-u4:

MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATKLTLKFICTTT

YVQERTISFKD (SEQ ID NO.: 27)

Sequence A1-u3-u4-u6:

MVSKGEELFTGVKLTLKFICTTYVQERTISFKDTLVNRIELKGIDF (SEQ ID NO.: 28)

Sequence A1-u4-u7-u10:

MVSKGEELFTGVYVQERTISFKDHNVYITADKQHYLSTQSVLSKD (SEQ ID NO.: 29)

Sequence A1-u3-u4-u7-u10:

MVSKGEELFTGVKLTLKFICTTYVQERTISFKDHNVYITADKQHYLSTQS VLSKD (SEQ ID NO.: 30)

In yet another preferred embodiment, the first linker peptide and/or the second linker peptide comprise the amino acid sequence shown in SEQ ID NO.: 18 (ENLYFQGR).

In yet another preferred embodiment, proinsulin has the amino acid sequence shown in SEQ ID NO: 19 or 20.

In another preferred embodiment, the fusion protein has the amino acid sequence shown in SEQ ID NO.: 21.

In the context of this document, the term "fusion protein" also includes options that exhibit the above activity. These options include the following (however, the list is not exhaustive): 1-3 (usually 1-2, more preferably 1) amino acid deletions, insertions and / or substitutions, as well as the addition or removal of one or more (usually 3 or less, preferably 2 or less, more preferably 1 or less) amino acids at the C-terminus and/or N-terminus. As an example, in this region, when amino acids with similar or identical properties are substituted, the function of the protein is usually not altered. As another example, the addition or removal of one or more amino acids at the C-terminus and/or N-terminus usually does not change the structure and function of the protein. In addition, this term also includes the polypeptide of the present invention in monomeric and multimeric forms. The term also includes linear and non-linear polypeptides (eg, cyclic peptides).

The present invention also includes active fragments, derivatives and analogues of the fusion protein mentioned above. As used herein, the terms "fragment", "derivative", and "analogue" refer to a polypeptide that substantially retains the function or activity of the fusion protein of the present invention. Fragments, derivatives or analogs of a polypeptide of the present invention may be (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) a polypeptide with a substituent group on one or more amino acid residues, or (iii) a polypeptide formed by fusing a polypeptide with another compound (e.g., a compound that extends the half-life of a polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusing an additional amino acid sequence with that of the polypeptide (a fusion protein formed by fusion with a leader sequence, a secretory sequence, or a 6His tag sequence). According to the principles contained in this document, these fragments, derivatives and analogs fall within the scope, well known to specialists in this field.

The use of the term "preferred type" when applied to an active derivative means that, compared to the amino acid sequence of the present invention, a maximum of 3, preferably a maximum of 2, and more preferably a maximum of 1 amino acid is replaced by amino acids with similar or similar properties during the formation of the polypeptide. The best way to produce these conserved variant polypeptides, according to Table A, is amino acid substitution.

The present invention also provides analogues of the fusion protein of the present invention. The difference between these analogs and the polypeptide of the present invention may be the presence of a different amino acid sequence, may also be the presence of a different modified form that does not affect the sequence, or both of these differences. Analogues also include analogs having residues other than naturally occurring L-amino acids (e.g. D-amino acids) and analogs having amino acids not naturally occurring or synthetic amino acids (e.g. β- and γ-amino acids) . It is to be understood that the polypeptide of the present invention may be other than one of the representative polypeptides exemplified above.

In addition, the fusion protein of the present invention can also be modified. Modified (as a rule, without changing the primary structure) forms include: chemically obtained (for example, in the process of acetylation or carboxylation) in vivo or in vitro forms of polypeptides. Modification procedures also include glycosylation used, for example, to obtain polypeptides during glycosylation modification during polypeptide synthesis and processing, or during further processing steps. Such modification can be achieved by exposing the polypeptide to an enzyme that performs a glycosylating role (eg, a mammalian glycosylase or deglycosylase). Modified forms also include sequences with amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). They also include polypeptides that have been modified to improve their antiproteolytic properties or optimize their solubility properties.

The term "polynucleotide encoding a fusion protein of the present invention" may include a polynucleotide encoding a fusion protein of the present invention, or a polynucleotide also including additional coding and/or non-coding sequences.

The present invention also relates to variants of the aforementioned polynucleotides that encode fragments, analogs and derivatives of polypeptides or fusion proteins having an amino acid sequence similar to that discussed in the present invention. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide. It may be a substitution, deletion, or insertion of one or more nucleotides, but does not significantly alter the function of the encoded fusion protein.

The present invention also relates to polynucleotides hybridizing to the above sequences and having at least 50%, preferably at least 70% and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to a polynucleotide of the present invention under stringent conditions (or under stringent conditions). In the present invention, the term "stringent conditions" refers to the following: (1) hybridization and elution at low ionic strengths and high temperatures, for example, 0.2×SSC, 0.1%) SDS, 60°C; or (2) adding a denaturant during hybridization, eg 50% (v/v) formamide, 0.1% calf serum/0.1% ficoll, 42°C, etc.; or (3) hybridization occurring only when the degree of identity between the two sequences reaches 90% or more, and more preferably 95% or more.

Preferably, the fusion protein and polynucleotides of the present invention are provided in isolated form and, more preferably, purified to homogeneity.

The full-length sequence of a polynucleotide of the present invention can generally be obtained using a PCR amplification method, a recombination method, or an artificial synthesis method. When using the PCR amplification method, primers can be designed based on the corresponding nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, as well as using a commercially available cDNA library, or a cDNA library prepared using a traditional method known to those skilled in the art. of the art, as a template for amplifying the corresponding sequence. If the sequence is long, it is often necessary to perform two or more PCR amplifications; the amplified fragments are then spliced in the correct order.

Once the corresponding sequence has been obtained, a recombination method can be used to obtain this corresponding sequence in large quantities. Typically, this is achieved by cloning the sequence into a vector and then transferring it into a cell and isolating it from proliferated host cells using conventional methods.

In addition, artificial synthesis methods can also be used to synthesize related sequences, especially in the case of a small fragment length. (Typically by synthesizing multiple small fragments, followed by very long sequence fragments.)

At this point in time, the procedure for encoding a protein (or a fragment or derivative thereof) with a DNA sequence, according to the present invention, is implemented entirely using chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or, for example, vectors) and cells known in the art.

Preferably, to obtain a polynucleotide of the present invention, a method consisting in the use of PCR technology to amplify DNA/RNA should be used. Preferably, a rapid cDNA end amplification (RACE-cDNA) method should be used, especially in cases where it is difficult to obtain full-length cDNA from a library, and proper selection of primers used for PCR can be carried out in accordance with the sequence information disclosed in the present invention. ; these primers can be synthesized using conventional methods. Amplified DNA/RNA fragments can be separated and purified using traditional methods such as gel electrophoresis.

expression vector

The present invention also relates to a vector containing a polynucleotide of the present invention, a host cell obtained by a genetic engineering method using a vector of the present invention, or a fusion protein coding sequence of the present invention, as well as a method for producing a polypeptide of the present invention using recombinant technology.

In the case of conventional recombinant DNA technology, the polynucleotide sequence of the present invention may be used to provide expression or production of a recombinant fusion protein. In general, the following steps are present:

(1) using a polynucleotide (or variant) of the present invention to encode a fusion protein of the present invention, or using a recombinant expression vector containing the polynucleotide to transform or convert a suitable host cell;

(2) culture the host cell in a suitable medium;

(3) isolation and purification of the protein from the culture medium or cells.

In the present invention, a polynucleotide sequence encoding a fusion protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors known in the art. Any plasmid and any vector can be used, as long as they can be reproduced and stabilized in the host. An important characteristic of an expression vector is that it typically contains an origin of replication, a promoter, a marker gene, and translational controls.

Methods well known to those skilled in the art can be used to construct an expression vector containing a DNA sequence encoding a fusion protein of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology and in vivo recombinant technology. The DNA sequence can be efficiently linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Typical examples of these promoters are the following: Lactose promoter or tryptophan promoter of Escherichia coli; lambda phage PL promoter; eukaryotic promoters, including the CMV immediate early promoter, the HSV thymidine kinase promoter, the SV40 virus early and late promoter, retroviral LTR sequences, and certain other known promoters capable of controlling gene expression in prokaryotic or eukaryotic cells or viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance gene, and green fluorescent protein (GFP) or resistance gene. E. coli. to tetracycline or ampicillin.

A vector containing the aforementioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell in such a way that it is capable of expressing the protein.

The host cell may be a prokaryotic cell, eg a bacterial cell, or a eukaryotic cell, eg a yeast cell, or a higher eukaryotic cell, eg a mammalian cell. Typical examples are the following: Escherichia coli, streptomycetes; bacterial cells of mouse typhoid fever; fungal cells such as yeast; and plant cells (eg ginseng cells).

If the expression of the polynucleotide of the present invention occurs in higher eukaryotic cells, then when the enhancer sequence is inserted into the vector, transcription will be increased. Enhancers are DNA factors typically consisting of 10-300 base pairs that act cis on promoters to enhance gene transcription. Examples include the SV40 enhancer (consisting of 100-270 base pairs) on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and adenoviral enhancers (including the like).

Those of ordinary skill in the art are familiar with the procedure for selecting appropriate vectors, promoters, enhancers, and host cells.

Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. If the host is a prokaryote (eg, Escherichia coli), then the collection of competent cells capable of absorbing DNA can be carried out after the exponential growth phase, and processing them - by the method of CaCl ₂ . The steps involved are well known in the art. Another way is to use MgCl ₂ . If necessary, the transformation process can be carried out using electroporation. If the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, traditional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The resulting transformants can be cultured using conventional methods to ensure expression of the polypeptide encoded by the gene of the present invention. Depending on the host cell used, one of the conventional culture media may be used. The culture process is carried out under conditions suitable for growing host cells. Once the desired cell density has been reached during growth of the host cells, the selected promoter is induced using an appropriate method (eg, temperature conversion or chemical induction) and the cells are cultured for a specified period of time.

Using the above method, the recombinant polypeptide can be expressed in the cell or on the cell membrane, or secreted from the cell. If necessary, physical, chemical and other characteristics of the recombinant protein can be used to separate and purify it using various separation methods. These methods are well known to those skilled in the art. Examples of these methods include the following (however, the list is not exhaustive): conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, destruction of bacteria by osmosis, ultraprocessing, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography methods, as well as combinations of these methods.

Insulin synthesis

Insulin is a well-defined peptide with a known amino acid sequence and structural characteristics. It is a 51 amino acid protein with two amino acid chains. The hormone contains two independent peptide chains, the A chain (21 amino acids) and the B chain (30 amino acids). There are 6 cysteine residues in two amino acid chains, and each of the chains has two cysteine residues connected to each other through disulfide bonds. In terms of statistics, there are 15 possible variants of the formation of disulfide bonds in the human insulin molecule. However, in biologically active human insulin, only one of these 15 possible variants is present, and these disulfide bonds are as follows: 1) A6-A11; 2) A7-B7; 3) A20-B19. Proinsulin is the biological precursor of insulin. It is a single chain peptide formed by linking chain A and chain B with peptide C. Two peptide chains of insulin are connected by disulfide bonds (Figure 1).

Insulin is a protein hormone secreted by pancreatic insular cells stimulated by endogenous substances such as glucose. The first secretion of the pancreatic island cells is proinsulin, a long chain polypeptide of 84 amino acids. Proinsulin is cleaved from the middle part (chain C) of proinsulin by the action of special enzymes PC1 and PC2, which convert the protease into proinsulin, as well as by the action of carboxypeptidase E (CPE), while the carboxyl part (chain A) and the amine part (chain B ) of proinsulin are connected by disulfide bonds to form insulin. Mature insulin is contained in the secretory vesicles of the pancreatic islet cells and is coordinated by zinc ions to form hexamers. With external stimulation, insulin is released into the blood along with secretory vesicles and exerts its physiological effects. The blood of patients with type 1 diabetes loses the ability to regulate glucose levels due to the loss of the ability of pancreatic island cells to form insulin.

Currently, there are two methods for producing different types of commercial recombinant human insulin - the "chain combination" method and the "pro-insulin" method. When using the "chain combination" method, the two peptide chains that make up insulin - chain A and chain B - are synthesized separately using biological recombination, and then chain A and chain B are mixed to form disulfide bonds to form biologically active human insulin. However, the efficiency of the process of direct mixing of two peptide chains to form biologically active human insulin is relatively low, and the final yield of the substance is only about 7%. This method is gradually being replaced by the second, "pro-insulin" method. When using the "proinsulin" method, proinsulin, consisting of insulin chains B, C and A, is first expressed in E. coli or yeast and then, after purification, is renatured in vitro. Renatured proinsulin is then hydrolyzed and processed using trypsin and carboxypeptidase B to produce naturally active human insulin. When using the "pro-insulin" method, trypsin specifically recognizes lysine and arginine in the protein and cleaves the peptide bond at the C-terminus of lysine and arginine. For conformational reasons, the arginine at position B22 of proinsulin is not hydrolyzed by trypsin. However, trypsin recognizes and hydrolyzes the lysine at the B29 position of the insulin, resulting in insulin as a by-product (insulin DesB30), which eliminates the threonine at the B30 position. To reduce insulin production DesB30, the amount of trypsin and reaction time must be strictly controlled. However, a certain amount of DesB30 insulin will still be produced. Because insulin DesB30 and regular insulin differ only in the presence or absence of threonine, their separation is a very difficult task. For the industrial separation of insulin, high-performance liquid chromatography technology is used, carried out on a large scale; however, the separation of conventional insulin and DesB30 insulin using this method generates a large amount of industrial waste, which significantly increases the cost of production of existing recombinant human insulin.

The present invention provides a process for the production of human insulin with properly connected cysteine bridges. In the course of its implementation, this method requires fewer steps, due to which it is possible to achieve greater productivity in the process of producing human insulin.

A DNA fragment was synthesized chemically; wherein the coding structure of the 2β-TEV-R-MiniINS DNA fragment consisted of the amino acid sequence shown in SEQ ID NO.: 21. This DNA fragment was cloned into a bacterial expression vector regulated by the araBAD promoter. The expression vector containing 2β-TEV-R-MiniINS was converted into E. coli Top10 strain, and the recombinant cells were cultured in Luria-Bertani medium containing contaminants. The 2β-TEV-R-MiniINS fusion protein was extracted from intracellular inclusions and folded under certain conditions, resulting in the formed disulfide bond in the folded 2β-TEV-R-MiniINS fusion protein being identical to the disulfide bond in folded human proinsulin, i.e. e., disulfide bonds A6-A11, A7-B7 and A20-B19 were formed. The separated, correctly folded 2β-TEV-R-MiniINS fusion protein was processed using trypsin and carboxypeptidase B to form correctly folded tert-butoxycarbonyl human insulin. Tert-butoxycarbonyl human insulin was purified to 90% purity using a hydrophobic chromatography process. The tert-butoxycarbonyl was then deprotected with acid to form correctly folded human insulin, which was then purified by reverse phase HPLC. N-terminal sequence analysis, molecular weight determination, and peptide mapping processes were used to determine the properties of pure human insulin thus obtained.

Compared with the prior art, the main advantages of the present invention are as follows:

1) An element of the fusion protein of the present invention that promotes protein expression may also contribute to the folding of the fusion protein.

2) The protein expression promoting element of the fusion protein of the present invention can increase the solubility of the soluble fusion protein and reduce the degree of intermolecular interaction thereof, so that the fusion protein can be folded at high concentrations, which makes the process industrially useful.

3) In the process of preparing the target peptide, there is no need to carry out cyanogen bromide cleavage, oxidative hydrolysis of sulfites and associated purification steps.

4) In the process of preparing the target peptides, there is no need to use thiols or hydrophobic adsorbent resins in high concentrations.

5) The target peptides are protected from intracellular destruction of microbial hosts.

6) The target protein is characterized by high values of specific gravity (increased fusion coefficient). The (X)n or A1-(X)n elements of the fusion protein can be processed into small fragments by the protease. Compared to the target protein, a wide variation in molecular weight values and significant ease of separation are provided.

7) The fusion protein of the present invention can promote the expression of the target peptide; the level of expression and the yield of the target peptide are significantly increased.

8) The fusion protein of the present invention is highly suitable for expression of artificial amino acid target peptides, and can obviously assist in the folding of artificial amino acid target peptides.

The present invention will be further explained below with demonstration of specific embodiments. It should be understood that these embodiments are used only to illustrate the present invention and not to limit the scope of the present invention. Experimental methods without specifying the specific conditions for their implementation in the following examples are usually carried out under conventional conditions, for example, under the conditions described in Sambrook (Sambrook) with co-authors "Molecular Cloning": Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989 ), or in accordance with the conditions offered by the manufacturer. Unless otherwise indicated, percentages and parts are weight percent and weight parts.

These embodiments use human insulin as an example of the target peptide, however, this example is not limiting.

Example 1 Construction of the A1-u4-u5-TEV-R-MiniINS Expression Vector

The A1-u4-u5-TEV-R-MiniINS expression structure contains the gene encoding the human insulin protein, which is fused to the C-terminus of the A1-u4-u5 structure. The linker peptide connecting A1-u4-u5 and MiniINS insulin protein is Glu-Asn-Leu-Tyr-Phe-Gln-Gly-Arg octapeptide. This octapeptide can be hydrolysed by trypsin at the carboxyl terminus of Arg, and can also be hydrolyzed by TEV protease between Gin and Gly. The DNA sequence of the octapeptide is codon-optimized, which allows high-level expression of a functional protein in E. coli.

All fragments of the hybrid recombinant protein used were synthesized using Gen Script and loaded into the pUC57 vector. The A1-u4-u5-TEV-R-MiniINS structure was removed from the synthetic vector pUC57-A1-u4-u5-TEV-R-MiniINS using restriction enzymes NcoI and XhoI; at the same time, the expression vector pBAD/His A(KanaR) was excised using the same restriction enzymes NcoI and XhoI. The processed products were separated first using an agarose gel electrophoresis procedure and then an agarose gel DNA repair kit for extraction; finally, the two DNA fragments were joined using T4 DNA ligase. The ligation product was transformed into E. coli Top 10 cells, and then the transformed cells were cultured on a Luria-Bertani agar medium containing kanamycin at a concentration of 50 μg/ml (yeast peptone 10 g/l, yeast extract powder 5 g/l, NaCl 10 g /l, agar 1.5%), overnight. 3 living colonies were selected; overnight, they were cultured in 5 ml of Luria-Bertani liquid medium (yeast peptone - 10 g/l, yeast extract powder - 5 g/l, NaCl - 10 t/l) containing 50 μg/ml of kanamycin, and using for plasmid extraction with the Plasmid Mini-Kit. The extracted plasmid was then subjected to sequencing. The plasmid obtained at the end of all these manipulations was named pBAD-A1-u4-u5-TEV-R-MiniINS. The plasmid map and fusion protein scheme are shown in FIG. 2 and 3, respectively.

Example 2 Construction of the A1-u4-u5-TEV-R-GLP1 Expression Structure

The procedure for constructing and synthesizing the expression structure pUC57-A1-u4-u5-TEV-R-GLPl is described in Example 1. At the same time, restriction enzymes NcoI and XhoI were used to excise the pBAD/His A(KanaR) expression vector, and processed products separated using an agarose gel electrophoresis procedure and then using an agarose DNA recovery gel extraction kit; finally, the two DNA fragments were joined using T4 DNA ligase. The ligation product was chemically transformed into E. coli Top10 cells, and then the transformed cells were cultured on a Luria-Bertani agar medium containing kanamycin at a concentration of 50 μg/ml (yeast peptone - 10 g/l, yeast extract powder - 5 g/l, NaCl - 10 g/l, agar - 1.5%) during the night. 3 living colonies were selected; overnight, they were cultured in 5 ml of liquid Luria-Bertani medium (yeast peptone - 10 g/l, yeast extract powder - 5 g/l, NaCl - 10 g/l) containing 50 μg/ml of kanamycin, and using for plasmid extraction with the Plasmid Mini-Kit. The extracted plasmid was then subjected to sequencing using the oligonucleotide sequencing primer 5'-ATGCCATAGCATTTTTATCC-3' to confirm the correct insertion. The resulting plasmid was named pBAD-A 1-u4-u5-TEV-R-GLP1.

Example 3 Expression, separation and purification of the A1-u4-u5-TEV-R-MiniINS fusion protein

To allow expression of the A1-u4-u5-TEV-R-MiniINS fusion fragment containing the amino acid sequence of SEQ ID NO.: 21. Plasmid pBAD-A1-u4-u5-TEV-R-MiniINS, verified by

sequencing, and the pyrrolysin aminoacyl-tRNA synthetase plasmid pEvol-pylRs-pylT (wherein the pyrrolysin aminoacyl-tRNA synthetase plasmid pEvol-pylRs-pylT was used to allow the expression of aminoacyl-tRNA synthetase and tRNA as shown in Example 1 of the Application for patent No. 2011103886241) were co-transformed into E. coli strain Top10. The transformation solution was placed overnight on a Luria-Bertani agar medium containing kanamycin at a concentration of 25 μg/ml and chloramphenicol at a concentration of 17 μg/ml. A single colony was chosen; overnight, it was cultured in a liquid Luria-Bertani medium containing kanamycin at a concentration of 25 μg/ml and chloramphenicol at a concentration of 17 μg/ml. Then the overnight culture was added to 100 ml of Luria-Bertani medium (Luria-Bertani liquid medium: yeast peptone - 12 g/l, yeast extract powder - 24 g/l, glycerol - 4 ml/l, kinase partner protein 4%, antifoam agent 0.3% kinase partner protein solution: KH ₂ PO ₄ 23.1 g/l, anhydrous K ₂ HPO ₄ 125.4 g/l), containing kanamycin at a concentration of 25 μg/ml and chloramphenicol at a concentration of 17 μg/l ml; incubation was carried out at 37°C until reaching the level of values of 2~4 optical density OD ₆₀₀ . Then a 25% solution of arabinose was added to the medium until a final concentration of 0.25% was reached, and 0.1 M tert-butoxycarbonyl-lysine solution (tert-butoxycarbonyl-lysine, where the structure of tert-butoxycarbonyl is shown in Fig. 4) was added until the final concentration was reached. concentration of 5 nm to induce the expression of the hybrid protein. The culture solution was continuously cultured for 16-20 hours, followed by a centrifugation collection procedure (10,000 rpm, 5 min, 4°C).

The expression of the hybrid protein A1-u4-u5-TEV-R-MiniINS was carried out in the form of insoluble "intracellular inclusions". To release intracellular inclusions, E. coli cells were destroyed using a high pressure homogenizer. Nucleic acids, cell debris and soluble proteins were removed by centrifugation at 10,000g. Intracellular inclusions containing the A1-u4-u5-TEV-R-MiniINS fusion protein were washed out with pure water; the resulting precipitate of intracellular inclusions was used as the raw material for folding. The final level of expression of the hybrid protein was 14 g/l in terms of fermentation broth. A scheme of SDS-PAGE fusion protein electrophoresis is shown in FIG. 5. From FIG. 5, it can be seen that the target protein expressed by the fusion protein can be expressed completely without break, and the fusion protein inserted into the target artificial amino acid protein can also be expressed intact without break.

To refold the fusion protein, intracellular inclusions are dissolved in a 7.5 M urea solution (pH 10.5) containing 2-10 mM mercaptoethanol; so that the total protein concentration after dissolution was 10-25 mg/ml. The sample was diluted 5-10 times; the traditional folding process was carried out for 16-30 hours at a temperature of 4-8°C and a pH value of 10.5 to 11.7. At a temperature of 18-25°C, the pH value was maintained at the level of 8.0-9.5; trypsin and carboxypeptidase B were used to process the fusion protein for 10-20 hours; then 0.45 M ammonium sulfate solution was added to terminate the enzymatic hydrolysis reaction. The results of HPLC analysis in reverse phase mode show that the yield of the product at this stage of enzymatic hydrolysis is more than 90%. The insulin analogue obtained after processing with the participation of trypsin and carboxypeptidase B is called "tert-butoxycarbonyl human insulin". The procedure for enzymatic hydrolysis of tert-butoxycarbonyl human insulin under the above conditions is not possible. The sample was clarified by membrane filtration using 0.45 mM ammonium sulfate as a buffer; pre-purification of tert-butoxycarbonyl human insulin was carried out using a hydrophobic chromatography process. The degree of purity of polyacrylamide gel electrophoresis using sodium dodecyl sulfate reaches 90%, and the final yield of tert-butoxycarbonyl human insulin per 1 liter of fermentation broth is about 2.1 g; the resulting tert-butoxycarbonyl human insulin was analyzed using a matrix-assisted laser desorption/ionization (MALDI-TOF) mass spectrometry method; it was found that the actual value of the molecular weight corresponded to the theoretical value of 5907.7 Da. The collection of samples was carried out using the method of hydrophobic chromatography; hydrochloric acid was added to allow the deprotection of tert-butoxycarbonyl human insulin to proceed. Sodium hydroxide solution was added to bring the pH to 2.8 ~ 3.2 (to stop the reaction). After two steps of reverse phase high pressure chromatography, the yield of recombinant human insulin was over 85%. The final yield of recombinant human insulin per liter of fermentation broth is about 700 mg.

Example 4 Construction of the pBAD-A1-u8-TEV-R-MiniINS Expression Structure and Expression, Separation and Purification of the A1-u8-TEV-R-MiniINS Fusion Protein

The same experimental method as in Example 1-3 is used; A1-u4-u5 (SEQ ID NO.: 16) is replaced in A1-u4-u5-TEV-R-MiniINS with the sequence A1-u8 (SEQ ID NO.: 23). A recombinant plasmid was constructed and named pBAD-A1-u8-TEV-R-MiniINS; the expression of the hybrid protein A1-u8-TEV-R-MiniINS was provided according to the specified method (Fig. 5). The final yield of tert-butoxycarbonyl human insulin is about 1.9 g/L; the resulting tert-butoxycarbonyl human insulin was analyzed using a matrix-assisted laser desorption/ionization (MALDI-TOF) mass spectrometry method. The results show that the actual value of the molecular weight corresponds to the theoretical one.

Example 5 Construction of the pBAD-A1-u3-u5-u7-TEV-R-MiniINS Expression Structure and Expression, Separation and Purification of the A1-u3-u5-u7-TEV-R-MiniINS Fusion Protein

The same experimental method as in Example 1-3 is used; A1-u4-u5 (SEQ ID NO.: 16) is replaced in A1-u4-u5-TEV-R-MiniINS with the sequence A1-u3-u5-u7 (SEQ ID NO.: 15). A recombinant plasmid was constructed and named pBAD-A1-u3-u5-u7-TEV-R-MiniINS; the expression of the hybrid protein A1-u3-u5-u7-TEV-R-MiniINS was provided according to the specified method. The final yield of tert-butoxycarbonyl human insulin is about 2.0 g/l; the resulting tert-butoxycarbonyl human insulin was analyzed using a matrix-assisted laser desorption/ionization (MALDI-TOF) mass spectrometry method. The results show that the actual value of the molecular weight corresponds to the theoretical one.

Example 6 Construction of the pBAD-A1-u5-EK-u6-TEV-R-MiniINS Expression Structure and Expression, Separation and Purification of the A1-u5-EK-u6-TEV-R-MiniINS Fusion Protein

The same experimental method as in Example 1-3 is used; A1-u4-u5 (SEQ ID NO.: 16) is replaced in A1-u4-u5-TEV-R-MiniINS with the sequence A1-u5-EK-u6 (SEQ ID NO.: 22), where EK is a restriction site enzyme EK DDDDK (SEQ ID NO.: 25). A recombinant plasmid was constructed and named pBAD-A1-u5-EK-u6-TEV-R-MiniINS; the expression of the hybrid protein A1-u5-EK-u6-TEV-R-MiniINS was provided according to the specified method. The final yield of tert-butoxycarbonyl human insulin is about 1.9 g/L; the resulting tert-butoxycarbonyl human insulin was analyzed using a matrix-assisted laser desorption/ionization (MALDI-TOF) mass spectrometry method. The results show that the actual value of the molecular weight corresponds to the theoretical one.

All documents referred to in the present invention are incorporated herein by reference as if they were incorporated by reference individually. In addition, it should be understood that after becoming familiar with the above ideas of the present invention, various changes and modifications can be made to the invention by specialists in this field of technology, and also that these equivalents equally fall within the scope of the claims appended to this application.

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SEQUENCE LIST

<110> NINGBO KUNPENG BIOTECH CO., LTD.

<120> FUSION PROTEIN CONTAINING FLUORESCENT PROTEIN FRAGMENTS AND ITS APPLICATIONS

<130> P2020-0086

<150> CN201910210102.9

<151> 2019-03-19

<160> 31

<170>PatentIn version 3.5

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Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln

65 70 75 80

Leu Glu Asn Tyr Cys Asn

85

<210> 20

<211> 52

<212> PRT

<213> artificial sequence

<220>

<223> proinsulin

<400> 20

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Gly

20 25 30

Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu

35 40 45

Asn Tyr Cys Asn

50

<210> 21

<211> 96

<212> PRT

<213> artificial sequence

<220>

<223> fusion protein

<400> 21

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Tyr Val Gln Glu

1 5 10 15

Arg Thr Ile Ser Phe Lys Asp Thr Tyr Lys Thr Arg Ala Glu Val Lys

20 25 30

Phe Glu Gly Asp Glu Asn Leu Tyr Phe Gln Gly Arg Phe Val Asn Gln

35 40 45

His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly

50 55 60

Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Gly Ile Val Glu Gln

65 70 75 80

Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn

85 90 95

<210> 22

<211> 43

<212> PRT

<213> artificial sequence

<220>

<223> A1-u5-EK-u6

<400> 22

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Thr Tyr Lys Thr

1 5 10 15

Arg Ala Glu Val Lys Phe Glu Gly Asp Asp Asp Asp Asp Lys Thr Leu

20 25 30

Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe

35 40

<210> 23

<211> 26

<212> PRT

<213> artificial sequence

<220>

<223> A1-u8

<400> 23

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Gly Ile Lys Ala

1 5 10 15

Asn Phe Lys Ile Arg His Asn Val Glu Asp

20 25

<210> 24

<211> 36

<212> PRT

<213> artificial sequence

<220>

<223> A1-u2-u3

<400> 24

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val His Lys Phe Ser

1 5 10 15

Val Arg Gly Glu Gly Glu Gly Asp Ala Thr Lys Leu Thr Leu Lys Phe

20 25 30

Ile Cys Thr Thr

35

<210> 25

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> EK restriction site

<400> 25

Asp Asp Asp Asp Lys

15

<210> 26

<211> 49

<212> PRT

<213> artificial sequence

<220>

<223> A1-u1-u2-u3

<400> 26

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu

1 5 10 15

Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly

20 25 30

Glu Gly Glu Gly Asp Ala Thr Lys Leu Thr Leu Lys Phe Ile Cys Thr

35 40 45

Thr

<210> 27

<211> 60

<212> PRT

<213> artificial sequence

<220>

<223> A1-u1-u2-u3-u4

<400> 27

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu

1 5 10 15

Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly

20 25 30

Glu Gly Glu Gly Asp Ala Thr Lys Leu Thr Leu Lys Phe Ile Cys Thr

35 40 45

Thr Tyr Val Gln Glu Arg Thr Ile Ser Phe Lys Asp

50 55 60

<210> 28

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> A1-u3-u4-u6

<400> 28

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Lys Leu Thr Leu

1 5 10 15

Lys Phe Ile Cys Thr Thr Tyr Val Gln Glu Arg Thr Ile Ser Phe Lys

20 25 30

Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe

35 40 45

<210> 29

<211> 45

<212> PRT

<213> artificial sequence

<220>

<223> A1-u4-u7-u10

<400> 29

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Tyr Val Gln Glu

1 5 10 15

Arg Thr Ile Ser Phe Lys Asp His Asn Val Tyr Ile Thr Ala Asp Lys

20 25 30

Gln His Tyr Leu Ser Thr Gln Ser Val Leu Ser Lys Asp

35 40 45

<210> 30

<211> 55

<212> PRT

<213> artificial sequence

<220>

<223> A1-u3-u4-u7-u10

<400> 30

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Lys Leu Thr Leu

1 5 10 15

Lys Phe Ile Cys Thr Thr Tyr Val Gln Glu Arg Thr Ile Ser Phe Lys

20 25 30

Asp His Asn Val Tyr Ile Thr Ala Asp Lys Gln His Tyr Leu Ser Thr

35 40 45

Gln Ser Val Leu Ser Lys Asp

50 55

<210> 31

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Oligonucleosides

<400> 31

atgccatagc atttttatcc 20

<---

Claims (30)

1. Fusion protein for expression and production of the target polypeptide containing the structure shown in Formula I: (P1-L1)s-A1-(X)n-A2-(L2-P2)t (I) Where "-" denotes a peptide bond or linker peptide, each P1 is independently the first target peptide; each P2 is independently the second target peptide; each L1 is independently the missing or first linker peptide; each L2 is independently the missing or second linker peptide; A1 is a missing or signal peptide; A2 is a missing or signal peptide; each X is independently a single β-pleated block of a fluorescent protein; n is a positive integer from 1 to 8; s can take the values 0, 1 or 2; t can take the values 0, 1 or 2; And an additional condition is the inequality of s and t to zero simultaneously; and (X)n is u8, u9, u2-u3, u4-u5, u8-u9, u1-u2-u3, u2-u3-u4, u3-u4-u5, u5-u6-u7, u8-u9- u10, u9-u10-u11, u3-u5-u7, u3-u4-u6, u4-u7-u10, u6-u8-u10, u1-u2-u3-u4, u2-u3-u4-u5, u3- u4-u3-u4, u3-u5-u7-u9, u5-u6-u7-u8, u1-u3-u7-u9, u2-u2-u7-u8, u7-u2-u5-u11, u3-u4- u7-u10, u1-I-u2, u1-I-u5, u2-I-u4, u3-I-u8, u5-I-u6 or u10-I-u11; I - flexible linker containing a restriction site; where the amino acid sequences u1-u11 are shown below: u1 VPILVELDGDVNG (SEQ ID NO.: 1) u2 HKFSVRGEGEGDAT (SEQ ID NO.: 2) u3 KLTLKFICTTT (SEQ ID NO.: 3) u4 YVQERTISFKD (SEQ ID NO.: 4) u5 TYKTRAEVKFEGD (SEQ ID NO.: 5) u6 TLVNRIELKGIDF (SEQ ID NO.: 6) u7 HNVYITADKQ (SEQ ID NO.: 7) u8 GIKANFKIRHNVED (SEQ ID NO.: 8) u9 VQLADHYQQNTPIG (SEQ ID NO.: 9) u10 HYLSTQSVLSKD (SEQ ID NO.: 10) u11 HMVLLEFVTAAGI (SEQ ID NO.: 11); P1 and P2 are each independently selected from: human insulin precursor protein, insulin lispro precursor protein, insulin glargine precursor protein, glucagon-like peptide and its derivatives exenatide and liraglutide, insulin, calcitonin, teduglutide. 2. The fusion protein of claim 1, wherein the first target peptide and/or the second target peptide is a protein with an artificial amino acid. 3. The fusion protein of claim 1, wherein A1-(X)n has the amino acid sequence shown in SEQ ID NO.: 14, 15, 16, 17, 22, 23, 24, 26, 27, 28, 29, or 30 . 4. The fusion protein of claim 1, wherein a flexible linker I is present between any two β-fold blocks and the flexible linker I contains a restriction site. 5. An isolated polynucleotide encoding a fusion protein according to claim 1. 6. A vector for expressing a fusion protein according to claim 1, which includes a polynucleotide according to claim 5. 7. Host cell for expression of the fusion protein according to claim 1, containing the vector according to claim 6. 8. A host cell for expressing the fusion protein of claim 1, wherein the exogenous polynucleotide of claim 5 is integrated into the chromosome of the host cell. 9. A method for producing a fusion protein according to claim 1, comprising the following steps: (a) under conditions suitable for obtaining a fusion protein according to claim 1, a host cell according to any one of claims 7 to 8 is cultured, thereby obtaining a fusion protein according to claim 1. 10. Use of a host cell according to any one of claims 7-8 for expression and production of the target polypeptide according to claim 1.