CN115298204A

CN115298204A - proNGF mutants and uses thereof

Info

Publication number: CN115298204A
Application number: CN202280003074.3A
Authority: CN
Inventors: 裴秀芝; 卢世香; 王帅; 刘忠凯; 王海; 王琪; 孙秀明; 张晓苹; 梁千惠; 费东雪; 刁树青
Original assignee: Qingdao Wanming Saibo Pharmaceutical Co ltd
Current assignee: Qingdao Wanming Saibo Pharmaceutical Co ltd
Priority date: 2022-01-25
Filing date: 2022-01-25
Publication date: 2022-11-04
Anticipated expiration: 2042-01-25
Also published as: WO2023141754A1; CN115298204B

Abstract

The present invention relates to proNGF mutants and uses thereof. According to the invention, the cleavage efficiency of Furin (Furin) on the obtained proNGF mutant is changed by mutating a Furin cleavage site in proNGF, so that the expression amount of beta-NGF is increased while the proNGF proportion is reduced in the cell expression process. The proNGF mutant provided by the invention is beneficial to reducing the pressure of downstream purification for removing proNGF and increasing the space for improving the expression quantity of beta-NGF by an upstream process.

Description

proNGF mutants and uses thereof

Technical Field

The invention relates to the field of protein engineering, in particular to a proNGF mutant with substitution at a protease cleavage site, a preparation method thereof, a polynucleotide encoding the proNGF mutant, a corresponding recombinant expression vector and a cell, and use of the proNGF mutant in preparing beta-nerve growth factor.

Background

Nerve Growth Factor (NGF) promotes neuronal differentiation and maintains neuronal survival, and clinical trial indications have been reported for acute or degenerative neurological diseases such as neurotrophic keratitis, retinal pigment degeneration, alzheimer's disease, and the like. The entire human NGF exon encodes a 241 amino acid protein, commonly referred to as preproNGF. The signal peptide of preproNGF is cleaved in the endoplasmic reticulum to form the nerve growth factor precursor (proNGF, 223 amino acids). proNGF exists as a homodimer in the endoplasmic reticulum, is then transferred to the golgi apparatus and is cleaved enzymatically by proteases to form the mature β -NGF dimer (monomer contains 120 amino acids).

proNGF is the major form of NGF in the brain, and increased amounts of proNGF have been detected in animal models of neurodegenerative diseases and neurotrauma, suggesting that the ratio of proNGF to NGF may have a correlation in the development and progression of neurological diseases. proNGF is able to activate the p75NTR-sortilin receptor complex (Nykjaer et al, 2004), causing apoptosis of nerve cells, and is a key impurity affecting the quality of β -NGF. There are also two major splice variants of proNGF, long proNGF-Sub>A and short proNGF-B, which further increase the complexity of the NGF-related protein product.

At present, all the rat nerve growth factors are on the market in China, are extracted from the submaxillary gland of a mouse, have the risk of murine virus pollution in production and have large difficulty in batch quality control. The first recombinant human beta-NGF medicine sold in the world in the company Domp é Italy in 2017 is produced by expressing a recombinant human beta-nerve growth factor precursor protein (rhpro-NGF) inclusion body by using a prokaryotic expression system (E.coli), diluting and renaturing the inclusion body, cutting off a leader peptide by using trypsin, and purifying to obtain the beta-NGF protein. However, following inclusion body renaturation, the activity of β -NGF is affected.

Eukaryotic cells express NGF products with a high number of precursors, including intact proNGF and variants of precursors that are not cleaved completely. The use of trypsin to cleave proNGF in vitro to produce beta-NGF is inefficient, requiring a large amount of enzyme, which undoubtedly increases downstream purification procedures to remove large amounts of enzyme. If in vitro enzyme digestion is not used, various precursor variants are directly purified and removed, which brings great challenges to the purification process. Meanwhile, the precursor variant contains rhNGF sequence, has similar physicochemical properties with rhNGF products, and increases the purification complexity due to the generation of different splice variants.

Disclosure of Invention

The invention provides a mutant of a nerve growth factor precursor and application thereof in producing beta-nerve growth factor, aiming at improving the expression quantity of mature peptide beta-NGF and reducing the proportion of pronNGF so as to relieve the purification pressure downstream of the beta-NGF production.

In a first aspect, the present invention provides a proNGF mutant comprising:

(i) 1-12 as shown in SEQ ID NO;

(ii) An amino acid sequence having at least 90% sequence identity to the amino acid sequences shown in SEQ ID NO 1-12; or

(iii) An amino acid sequence having one or more site substitutions, deletions or insertions compared to the amino acid sequence shown in SEQ ID Nos. 1 to 12.

In a second aspect, the present invention provides a polynucleotide encoding said proNGF mutant provided herein.

In a third aspect, the invention provides a recombinant expression vector comprising the polynucleotide provided by the invention.

In some embodiments, the recombinant expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

In some embodiments, the eukaryotic expression vector is pcDNA3.1, pEGFP/pEGFT, pCHO1.0, pCMV, pSV2, or pGN.

In a fourth aspect, the invention provides a cell comprising the recombinant expression vector provided by the invention.

In some embodiments, the cell is a prokaryotic cell or a eukaryotic cell.

In some embodiments, the eukaryotic cell is a CHO cell or 293 cell.

In a fifth aspect, the present invention provides the use of said proNGF mutant, said polynucleotide, said recombinant expression vector and/or said cell in the preparation of β -NGF.

In a sixth aspect, the present invention provides a method for the preparation of β -NGF, comprising:

inserting a polynucleotide encoding the proNGF mutant into an expression vector to obtain a recombinant expression vector;

transfecting the recombinant expression vector into a cell;

the cells are cultured to obtain the beta-NGF.

In some embodiments, the recombinant expression vector is a eukaryotic expression vector and the cell is a eukaryotic cell.

In a seventh aspect, the present invention provides another method for preparing β -NGF, comprising:

providing said proNGF mutant;

cleaving said proNGF mutant in vitro using a protease to obtain β -NGF.

In some embodiments, the protease is a precursor protein converting enzyme.

In some embodiments, the protease is selected from at least one of furin, trypsin, plasmin.

The proNGF mutants provided by the invention all change the cleavage efficiency of Furin (Furin) on the proNGF mutant by mutating Furin cleavage sites in the proNGF, so that the proNGF ratio is reduced and the expression quantity of beta-NGF is increased in the cell expression process, and the N-terminal sequence of the obtained beta-NGF is normal and has normal biological activity. The proNGF mutant provided by the invention is beneficial to reducing the pressure of downstream purification for removing proNGF and increasing the space for improving the expression quantity of beta-NGF by an upstream process.

Drawings

FIG. 1 is the full-length amino acid sequence of wild-type prepro NGF (including the signal peptide);

FIG. 2 is a SDS-PAGE analysis of a prototype of the culture supernatant from day 8 of Fed Batch culture (Fed-Batch), in which 0 represents wild-type NGF and 1-12 in turn represent NGF mutants 1-12;

FIG. 3 is a Western Blot analysis of culture supernatants at day 8 of fed-batch culture (primary antibody is. Beta. -NGF antibody), in which 0 represents wild-type NGF, and 1-12 sequentially represent NGF mutants 1-12;

FIG. 4 is a Western Blot analysis of culture supernatants at day 8 of fed-batch culture (primary antibody is pronGF antibody), wherein 0 represents wild-type pronGF, and 1-12 in turn represent pronGF mutants 1-12;

FIG. 5 is a fed batch culture harvest supernatant reduction prototype SDS-PAGE analysis in which 0 is wild-type NGF,1-12 are NGF mutants 1-12 in sequence;

FIG. 6 is a Western Blot analysis of the supernatant of a fed batch culture harvest (primary antibody is. Beta. -NGF antibody), in which 0 represents wild-type NGF, and 1-12, in turn, represent NGF mutants 1-12;

FIG. 7 is a Western Blot analysis of the supernatant of a batch culture harvest (primary antibody is a pronGF antibody), wherein 0 represents wild-type pronGF, and 1-12 in turn represent pronGF mutants 1-12;

FIG. 8 is a fed-batch culture harvest supernatant SEC chromatogram;

FIG. 9 is a graph of biological activity of purified samples, in which 0 is wild-type NGF and 1-11 are NGF mutants 1-11 in that order;

FIG. 10 is a LC-MS deconvolution map of the purified sample (wild type 0);

FIG. 11 is a LC-MS deconvolution map of the purified sample (mutant 1);

FIG. 12 is a LC-MS deconvolution map of the purified sample (mutant 5);

FIG. 13 is the LC-MS deconvolution map of the purified sample (mutant 11).

Detailed Description

The present invention will be further illustrated with reference to the following examples, which are not intended to limit the invention in any way.

The present invention provides a proNGF mutant comprising:

(i) 1-12 as shown in SEQ ID NO;

In particular, proNGF or proNGF mutants according to the invention refers to human proNGF or human proNGF mutants. To obtain mature β -NGF, proNGF must be cleaved by a protease. The proNGF mutant is obtained by mutating the furin cleavage site of wild-type proNGF. Wherein, the amino acid sequence of proNGF is shown in SEQ ID NO. 13.

A significant feature of furin substrates is the requirement that the cleavage sequence must be arginine at positions P1 and P4 (

amino acids

1 and 4 from the amino terminus of the cleavage site). -R-X-X-X-R ↓ - (X: arbitrary amino acids; ↓: cleavage site) is the shortest cleavage sequence of furin. The inventors of the present invention found that mutations not limited to a single site can be made to the P1-P7 site of proNGF, and that mutations in a specific combination contribute to an increase in the cleavage efficiency of furin and, in turn, an increase in the expression level of β -NGF.

The amino acid sequences of the furin cleavage sites of wild-type proNGF (positions 115 to 121) and of the furin cleavage sites of proNGF mutants 1 to 12 according to the invention are shown in table 1. The number of amino acids at positions P1 to P7 after mutation is not limited to 7, and some of the amino acids are 6 amino acids, and the amino acids are 8 amino acids.

TABLE 1 furin cleavage site of pronGF mutants and wild-type pronGF

Source	Amino acid sequence	Sequence numbering
			proNGF mutant 1	RTHRRKR	14
proNGF mutant 2	RTHRRRR	15
			proNGF mutant 3	RTRRRRR	16
proNGF mutant 4	RTHRRRRR	17
			proNGF mutant 5	RRRVRR	18
proNGF mutant 6	RRRRVRR	19
			proNGF mutant 7	RRRRRR	20
proNGF mutant 8	RRRRRRR	21
			proNGF mutant 9	RRRSKR	22
proNGF mutant 10	RRRRSKR	23
			proNGF mutant 11	RGIRRKR	24
proNGF mutant 12	QTKRSKR	25
			Wild-type proNGF	RTHRSKR	26

Accordingly, the amino acid sequences of proNGF mutants 1 to 12 and of wild-type proNGF are as follows (without signal peptide):

amino acid sequence of proNGF mutant 1 (SEQ ID NO: 1):

EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRRKRSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA*

amino acid sequence of proNGF mutant 2 (SEQ ID NO: 2):

EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRRRRSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA*

amino acid sequence of proNGF mutant 3 (SEQ ID NO: 3):

EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTRRRRRSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA*

amino acid sequence of proNGF mutant 4 (SEQ ID NO: 4):

EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRRRRRSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA*

amino acid sequence of proNGF mutant 5 (SEQ ID NO: 5):

EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRRRVRRSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA*

amino acid sequence of proNGF mutant 6 (SEQ ID NO: 6):

EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRRRRVRRSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA*

amino acid sequence of proNGF mutant 7 (SEQ ID NO: 7):

EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRRRRRRSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA*

amino acid sequence of proNGF mutant 8 (SEQ ID NO: 8):

EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRRRRRRRSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA*

amino acid sequence of proNGF mutant 9 (SEQ ID NO: 9):

EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRRRSKRSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA*

amino acid sequence of proNGF mutant 10 (SEQ ID NO: 10):

EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRRRRSKRSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA*

amino acid sequence of proNGF mutant 11 (SEQ ID NO: 11):

EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRGIRRKRSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA*

amino acid sequence of proNGF mutant 12 (SEQ ID NO: 12):

EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNQTKRSKRSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA*

amino acid sequence of human wild-type proNGF (SEQ ID NO: 13):

EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSKRSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA*

in the present invention, having at least 90% sequence identity to the amino acid sequences depicted in SEQ ID NOs 1-12 means that, when aligned, this percentage of amino acids is identical in a comparison of the two sequences. Specifically, the invention includes those portions of the amino acid sequence that have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the amino acid sequences set forth in SEQ ID Nos 1-12.

The proNGF mutant also comprises an amino acid sequence with one or more site substitutions, deletions or insertions compared with the amino acid sequence shown in SEQ ID NO. 1-12, and the function and/or activity of the proNGF mutant corresponding to the proNGF mutant are not obviously changed compared with the amino acid sequence shown in SEQ ID NO. 1-12, so that the proNGF mutant cleavage efficiency by furin can be improved.

The present invention provides a polynucleotide encoding said proNGF mutant provided herein.

As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications, if present, may confer modifications to the nucleotide structure before or after polynucleotide assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by coupling to a labeling component. The term also refers to double-stranded molecules and single-stranded molecules. Unless otherwise specified or required, a polynucleotide in any embodiment of the invention includes both the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

The term "encoding" as applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide if it can be transcribed and/or translated in its natural state or when manipulated by methods well known to those skilled in the art to produce mRNA for the polypeptide and/or fragments thereof. The antisense strand is the complement of such a nucleic acid, from which the coding sequence can be deduced.

In the present invention, the furin cleavage site of wild-type proNGF can be mutated by genetic engineering methods. Nucleic acid sequences encoding wild-type proNGF are known in the art. In some embodiments, the nucleic acid sequence encoding wild-type proNGF is subjected to gene-level mutagenesis to encode an amino acid other than the furin cleavage site of wild-type proNGF. Methods for mutagenesis at the gene level of a nucleic acid sequence encoding wild-type proNGF are routine in the art. Furthermore, in the case of known amino acid sequences of proNGF mutants, the synthesis of the corresponding coding nucleotide sequences is also a routine procedure known in the art.

The invention also provides a recombinant expression vector, which comprises the polynucleotide provided by the invention.

Any suitable vector known in the art can be used for the construction of the recombinant expression vector. The method of ligating the polynucleotide to an expression vector to construct the recombinant expression vector is a routine method in the art.

In some embodiments, the recombinant expression vector is a prokaryotic expression vector or a eukaryotic expression vector. The eukaryotic expression vector may be selected from eukaryotic expression vectors known in the art or modified vectors thereof, including but not limited to pcDNA3.1, pEGFP/pEGFT, pCHO1.0, pCMV, pSV2, pGN. Preferably, the eukaryotic expression vector is pcDNA3.1.

The invention also provides a cell comprising the recombinant expression vector provided by the invention.

Any suitable cell known in the art for expressing β -NGF can be used. Methods for introducing the polynucleotide into the cell to express β -NGF, or transfecting the recombinant expression vector into the cell to express β -NGF, are all conventional in the art.

In some embodiments, the cell is a prokaryotic cell or a eukaryotic cell. The eukaryotic cell may be selected from eukaryotic host cells known in the art or engineered cells thereof, including but not limited to CHO cells or 293 cells. Preferably, the eukaryotic cell is a CHO K1 cell. By using eukaryotic cells for expression, the enzyme digestion efficiency of furin of host cells can be directly improved, the proportion of beta-NGF is further improved, uniform beta-NGF products can be obtained, and the problems of non-uniform enzyme digestion and protease digestion after prokaryotic cells are expressed in the form of inclusion bodies and need in vitro renaturation are solved.

The invention also provides the use of the proNGF mutant, the polynucleotide, the recombinant expression vector and/or the cell for the preparation of beta-NGF.

The proNGF mutant provided by the invention can change the cleavage efficiency of the proNGF mutant of furin pairs, obviously reduce the secretion of proNGF during expression and improve the expression quantity of beta-NGF. Meanwhile, the N-terminal sequence of the obtained beta-NGF is normal and has normal biological activity.

The invention also provides a preparation method of the beta-NGF, which comprises the following steps:

(11) Inserting a polynucleotide encoding the proNGF mutant into an expression vector to obtain a recombinant expression vector;

(12) Transfecting the recombinant expression vector into a cell;

(13) The cells are cultured to obtain the beta-NGF.

Specifically, in step (11), the method of mutating the furin cleavage site (positions 115-121) of proNGF is a method conventional in the art. The method of constructing the recombinant expression vector by inserting a polynucleotide encoding the proNGF mutant into an expression vector is a routine method in the art. In some embodiments, the recombinant expression vector is a prokaryotic expression vector or a eukaryotic expression vector. Preferably, the eukaryotic expression vector is pcDNA3.1.

In step (12), the method for transfecting the recombinant expression vector into the cell is a conventional method in the art. In some embodiments, the cell is a prokaryotic cell or a eukaryotic cell. Preferably, the eukaryotic cell is a CHO K1 cell.

In step (13), the method for culturing the cells is a method conventional in the art. In some embodiments, the culture method is Fed Batch culture (Fed-Batch).

It will be appreciated that the proNGF mutants provided herein, when used to produce β -NGF, not only directly increase furin cleavage efficiency during eukaryotic expression to obtain β -NGF, but are also suitable for in vitro cleavage to obtain β -NGF. Correspondingly, the invention also provides another preparation method of the beta-NGF, which comprises the following steps:

(21) Providing said proNGF mutant;

(22) Cleaving said proNGF mutant in vitro using a protease to obtain β -NGF.

Specifically, in step (22), the protease having a protease substrate specificity cleaves the protein substrate but does not digest the active portion of the protein. There are a number of proprotein convertases that can be used to cleave proNGF to obtain β -NGF. In some embodiments, the protease is preferably at least one of furin, trypsin, plasmin, more preferably furin.

By appropriately adjusting the ratio of the protease to the proNGF mutant and the cleavage time, the proNGF mutant can be completely cleaved. The method of adjusting the ratio and the cutting time is a conventional method in the art.

In order to make the details and operation of the above-mentioned embodiments of the present invention clearly understandable to those skilled in the art and to make the progress of the proNGF mutants and their uses apparent, the above-mentioned technical solutions are illustrated below by way of examples.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 construction of proNGF mutant expression vectors

The whole length of prepro NGF is 241 amino acids, after a signal peptide with 18 amino acids at the N end is cut off, pronNGF is formed, and simultaneously, 2 enzyme cutting sites are arranged, and incomplete pronNGF, namely proA and proB, is formed by enzyme cutting respectively. Furin recognizes the RSKR at positions 118-121 and cleaves after the last R to form a 120 amino acid mature β -NGF protein. The invention mutates 115-121 RTHRSKR of wild type proNGF to form 12 mutants. Synthesizing the full-length nucleotide sequences of 12 mutants by gene, connecting to eukaryotic expression vector pcDNA3.1 after enzyme digestion to construct 12 recombinant expression vectors (1-12), connecting the full-length nucleotide sequences of proNGF of wild type and mutants 1-12 to eukaryotic expression vector pcDNA3.1 after enzyme digestion of HindIII and BamHI to construct a control recombinant expression vector. The sequence is verified to be correct through sequencing.

Example 2 evaluation of the expression of different proNGF mutants

The constructed 13 expression vectors were transfected into CHO K1 cells, respectively, and pressure-screened using L-amino sulfoxide Methionine (MSX), and maintained in pressure culture. And (3) when the cell viability is recovered to be more than 90%, performing fed batch culture on the obtained cell pool, and harvesting the cells when the cell viability is close to 80%. On day 8 of culture (D8), 1ml of each sample was centrifuged to collect the supernatant, which was frozen at-80 ℃. The D8 culture supernatant and final harvest were subjected to SDS-PAGE analysis, western Blot analysis for β -NGF and proNGF antibodies, respectively.

The reduced SDS-PAGE results of the supernatants from the fed batch culture on day 8 are shown in FIG. 2. Western Blot analysis gray-scale scanning (shown in FIG. 3 and FIG. 4) was performed on SDS-PAGE mature peptide beta-NGF and proNGF at day 8, and the expression level of the protein and the ratio of mature peptide to propeptide were quantitatively analyzed. Due to the complexity of proNGF, proNGF was detected with two different molecular weights, each scanned in gray scale.

The gray values and ratios of proNGF and β -NGF in day 8 reduced SDS-PAGE are shown in table 2.

TABLE 2 reduction SDS-PAGE Gray Scale Scan at day 8 of fed batch culture

As can be seen from Table 2, mutant 5, mutant 4, mutant 3 and mutant 1 exhibited high β -NGF expression levels and low proNGF ratios, and mutant 8 and mutant 9 exhibited the lowest β -NGF expression levels, although the proNGF ratios were low; wherein 0 represents wild-type proNGF, and samples 1 to 12, in turn, represent proNGF mutants 1 to 12.

Western Blot grayscale scan data for samples from day 8 of fed-batch culture are shown in Table 3.

TABLE 3 Western Blot (pronGF and. Beta. -NGF antibodies) grayscale scan at day 8 of fed-batch culture

In Table 3, the ratio of β -NGF/proNGF is relative, due to the differences in sensitivity and exposure of different antibodies. As can be seen from Table 3 (column 5-. Beta. -NGF),

mutants

0, 1, 3, 2, 4, 12 and 5 were found in the order of higher expression of β -NGF.

Mutants

6, 7, 10 and 11 expressed a very low proNGF ratio (column 6 for NGF/proNGF, a high value indicates a low proNGF ratio), but their beta-NGF expression was too low, possibly causing a lower proNGF expression than in the test line, which highlights a high NGF ratio.

Mutants

2, 12 and 0, however, had high NGF expression and high proNGF ratios. Wherein 0 represents wild-type proNGF, and samples 1 to 12, in turn, represent proNGF mutants 1 to 12.

The final harvest supernatants from the fed cultures were also subjected to SDS-PAGE (as shown in FIG. 5) and Western Blot analysis of both antibodies (as shown in FIGS. 6-7). As can be seen from FIGS. 5 to 7, the expression levels in the following days are remarkably accumulated, and according to the experience, the expression levels in the last days are almost doubled every day, and the influence of the harvest time on the expression levels is large. Due to the inconsistency of 12-14 days of final harvest time (12 days of culture for mutants 2-5, 13 days of culture for

mutants

1, 6, 7, 11, and 14 days of culture for control 0 and mutants 8-10), the expression level of different mutants is not completely consistent with the detection result of D8. Therefore, the detection results of SDS-PAGE and Western Blot at the 8 th day have more reference significance. Except that mutant 12 had a higher proNGF ratio than the control wild type, all other mutants had a lower proNGF ratio than the control wild type. Therefore, the proNGF mutants 1-11 can achieve the purpose of reducing proNGF expression, and the expression quantity can be improved in later-stage clone screening and process development optimization.

Mutants

1, 5 and 11 and a control wild type 0 are selected, the supernatant of the final harvest solution is purified, and whether the mutation sites affect the restriction enzyme of furin or not is further analyzed, i.e., whether the N-terminal of the mature beta-NGF is consistent with the wild type or not is further analyzed.

EXAMPLE 3 purification of supernatants preparation of Small samples

The final harvest of the fed-batch culture of the

mutants

1, 5 and 11 selected in example 2 and the wild-type control 0 was subjected to purification analysis, and samples with a purity of 90% or more were prepared. The final harvest was centrifuged at the fed batch culture and the supernatant was purified by cation chromatography using Hitrp SPFF 5ml (Cytiva, 29401108) and molecular sieve gel filtration Chromedex 75pg (Booglong, ezload,16/60, 120 ml) to recover samples of higher purity. Molecular sieve gel filtration chromatography elutes 3 major uv absorption peaks, a macromolecular protein elution peak, then proNGF, again beta-NGF. The SEC elution peaks for the 4 samples are shown in FIG. 8, and analyzed for proNGF and β -NGF elution peaks, as shown in Table 4.

TABLE 4 molecular Sieve purification chromatogram analysis

As can be seen from FIG. 8 and Table 4, the proNGF ratios expressed in sample No. 1 (mutant 1) and control 0 (wild type) were high, and those in sample No. 5 (mutant 5) and sample No. 11 (mutant 11) were low. The sample treatment mode before the SEC chromatography of the sample No. 1 is different from that of other samples, the SP elution sample of the sample No. 1 is concentrated by a 10kD ultrafiltration tube, the precipitation appears, and the sample is filtered by 0.22 mu m and then is subjected to SEC chromatography. It is possible that part of the NGF forms aggregates/precipitates, resulting in a high ProNGF content. Thus the other samples were not ultrafiltered, and the highest concentration fraction of the SP eluate was directly subjected to SEC chromatography, and the lower concentration fraction (20-30%) was subjected to SEC chromatography again, as shown in Table 4 for comparison with the data for the higher concentration fraction.

Since the molecular weight of proNGF is close to the size of the β -NGF dimer, variants formed by different splice forms of proNGF are complex and proNGF could be detected as containing β -NGF dimer.

EXAMPLE 4 analysis of biological Activity of purified samples (TF-1 cell proliferation method)

Human erythrocytic leukemia cells (TF-1 cells, GM-CSF dependent, derived from the recombinant protein Chamber of Chinese institute for food and drug assay) in good growth state were plated into 96-well plates at an amount of 10000 cells per well using a basal medium (1640 +10% FBS), with a volume of 100. Mu.L per well; then 100 mu L of sample solution to be tested diluted by 3 times of the basic culture medium in a gradient way is added into each hole, the concentration is set to be 1000, 333.33, 111.11, 37.03, 12.34, 4.12, 1.37, 0.46, 0.15 and 0.05ng/ml, and each concentration is two multiple holes; mixing, adding into 37 deg.C, 5% CO ₂ Culturing for 72h in an incubator; adding 20 mu L of MTS into each well, mixing uniformly, and incubating for 4h at 37 ℃; detecting the OD value of each hole at 490nm of an enzyme-labeling instrument; fitting the absorbance-concentration relation curve of each group by Graphpad Prism 7.0 software (selecting four-parameter nonlinear regression equation for fitting); EC50 values for stimulating the proliferation of TF-1 cells were calculated for each sample, and the results are shown in FIG. 9 and Table 5. Wherein 0 represents wild-type proNGF, and 1 to 11 in turn represent proNGF mutants 1 to 11.

TABLE 5 biological Activity assay results

Sample(s)	Concentration (mg/ml)	EC50(ng/ml)
			0	0.533	10.77
1	0.756	6.896
			2	1.396	28.31
3	0.443	30.20
			4	0.854	8.48
5	1.013	10.16
			6	0.332	16.73
7	0.674	20.49
			8	0.568	12.73
9	0.377	9.38
			10	0.81	7.61
11	0.673	11.48

The biological activity is a cell method, the coefficient of variation is higher, and the EC50 range of 50-200% of the activity of the control 0 can be considered to be similar. The EC50 of the other samples was not much different from the EC50 of control sample 0, except for slightly lower activity of mutant 2 and mutant 3. Thus, mutation of the cleavage site does not affect the biological activity of β -NGF protein.

EXAMPLE 5 sequencing of N-terminal amino acids of purified samples

Purified mutants

1, 5, 11 and wild type control 0 were subjected to amino acid sequencing (as shown in FIGS. 10-13). The sequencing results of FIGS. 10-13 show that: the beta-NGF expressed by the three proNGF mutants and the wild-type proNGF in the

samples

1, 5 and 11 contains a complete sequence of 120 amino acids, and also contains two amino acid deletion variants, namely 5 amino acids (6-120 aa) deleted from the N terminal and 2 amino acids (1-118 aa) deleted from the C terminal. According to research reports, the phenomenon of deletion of amino acids at the N terminal and the C terminal is more common in the natural expression of beta-NGF. As can be seen from Table 6, the proNGF mutant corresponds to the beta-NGF amino acid sequence expressed by wild-type proNGF, only slightly in proportion. Therefore, the mutation of the cleavage sites of mutant 1, mutant 5 and mutant 11 does not affect the cleavage and maturation of the target protein. In addition, possible modifications of the full-length mature peptide NGF were also detected: oxidation and saccharification, both of mutant 1, mutant 5, mutant 11 and wild type, were modified independently of the mutation.

TABLE 6ProNGF mutant and wild-type ProNGF and the contents of 2 deletion fragments (%)

Sample (I)	Complete molecule (120 aa)	Fragment (6-120 aa)	Fragment (1-118 aa)
				0	85.02	6.10	8.88
1	81.44	5.57	12.99
				5	86.91	5.10	7.99
11	86.79	4.55	8.66

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Islands Wanming Saibei pharmaceutical Co., ltd

<120> proNGF mutants and uses thereof

<130> DSP1V224220ZJ

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<170> PatentIn version 3.5

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Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg Arg Leu

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Arg Ser Pro Arg Val Leu Phe Ser Thr Gln Pro Pro Arg Glu Ala Ala

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Asp Thr Gln Asp Leu Asp Phe Glu Val Gly Gly Ala Ala Pro Phe Asn

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Arg Thr His Arg Arg Lys Arg Ser Ser Ser His Pro Ile Phe His Arg

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Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp Lys

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Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu

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Val Asn Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu Thr Lys

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Cys Arg Asp Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile Asp Ser

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Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala

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Leu Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp

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Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg Arg Ala

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Glu Pro His Ser Glu Ser Asn Val Pro Ala Gly His Thr Ile Pro Gln

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Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu Arg Arg

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Ala Arg Ser Ala Pro Ala Ala Ala Ile Ala Ala Arg Val Ala Gly Gln

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Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg Arg Leu

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Arg Ser Pro Arg Val Leu Phe Ser Thr Gln Pro Pro Arg Glu Ala Ala

65 70 75 80

Asp Thr Gln Asp Leu Asp Phe Glu Val Gly Gly Ala Ala Pro Phe Asn

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Arg Thr His Arg Arg Arg Arg Ser Ser Ser His Pro Ile Phe His Arg

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Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp Lys

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Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu

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Val Asn Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu Thr Lys

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Cys Arg Asp Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile Asp Ser

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Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala

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Leu Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp

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Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg Arg Ala

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Glu Pro His Ser Glu Ser Asn Val Pro Ala Gly His Thr Ile Pro Gln

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Ala Arg Ser Ala Pro Ala Ala Ala Ile Ala Ala Arg Val Ala Gly Gln

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Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg Arg Leu

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Arg Ser Pro Arg Val Leu Phe Ser Thr Gln Pro Pro Arg Glu Ala Ala

65 70 75 80

Asp Thr Gln Asp Leu Asp Phe Glu Val Gly Gly Ala Ala Pro Phe Asn

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Arg Thr Arg Arg Arg Arg Arg Ser Ser Ser His Pro Ile Phe His Arg

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Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp Lys

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Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu

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Val Asn Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu Thr Lys

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Cys Arg Asp Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile Asp Ser

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Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala

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Leu Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp

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Glu Pro His Ser Glu Ser Asn Val Pro Ala Gly His Thr Ile Pro Gln

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Ala Arg Ser Ala Pro Ala Ala Ala Ile Ala Ala Arg Val Ala Gly Gln

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Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg Arg Leu

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Arg Ser Pro Arg Val Leu Phe Ser Thr Gln Pro Pro Arg Glu Ala Ala

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Asp Thr Gln Asp Leu Asp Phe Glu Val Gly Gly Ala Ala Pro Phe Asn

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Arg Thr His Arg Arg Arg Arg Arg Ser Ser Ser His Pro Ile Phe His

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Arg Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp

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Lys Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu Gly

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Glu Val Asn Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu Thr

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Lys Cys Arg Asp Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile Asp

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Ser Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys

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Ala Leu Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile

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Asp Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg Arg Ala

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Glu Pro His Ser Glu Ser Asn Val Pro Ala Gly His Thr Ile Pro Gln

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Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu Arg Arg

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Ala Arg Ser Ala Pro Ala Ala Ala Ile Ala Ala Arg Val Ala Gly Gln

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Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg Arg Leu

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Arg Ser Pro Arg Val Leu Phe Ser Thr Gln Pro Pro Arg Glu Ala Ala

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Asp Thr Gln Asp Leu Asp Phe Glu Val Gly Gly Ala Ala Pro Phe Asn

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Arg Arg Arg Val Arg Arg Ser Ser Ser His Pro Ile Phe His Arg Gly

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Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp Lys Thr

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Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu Val

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

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Arg Asp Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys

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His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala Leu

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Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr

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Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg Arg Ala

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Glu Pro His Ser Glu Ser Asn Val Pro Ala Gly His Thr Ile Pro Gln

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Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu Arg Arg

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Ala Arg Ser Ala Pro Ala Ala Ala Ile Ala Ala Arg Val Ala Gly Gln

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Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg Arg Leu

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Arg Ser Pro Arg Val Leu Phe Ser Thr Gln Pro Pro Arg Glu Ala Ala

65 70 75 80

Asp Thr Gln Asp Leu Asp Phe Glu Val Gly Gly Ala Ala Pro Phe Asn

85 90 95

Arg Arg Arg Arg Val Arg Arg Ser Ser Ser His Pro Ile Phe His Arg

100 105 110

Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp Lys

115 120 125

Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu

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Val Asn Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu Thr Lys

145 150 155 160

Cys Arg Asp Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile Asp Ser

165 170 175

Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala

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Leu Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp

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Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg Arg Ala

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Glu Pro His Ser Glu Ser Asn Val Pro Ala Gly His Thr Ile Pro Gln

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Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu Arg Arg

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Ala Arg Ser Ala Pro Ala Ala Ala Ile Ala Ala Arg Val Ala Gly Gln

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Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg Arg Leu

50 55 60

Arg Ser Pro Arg Val Leu Phe Ser Thr Gln Pro Pro Arg Glu Ala Ala

65 70 75 80

Asp Thr Gln Asp Leu Asp Phe Glu Val Gly Gly Ala Ala Pro Phe Asn

85 90 95

Arg Arg Arg Arg Arg Arg Ser Ser Ser His Pro Ile Phe His Arg Gly

100 105 110

Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp Lys Thr

115 120 125

Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu Val

130 135 140

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

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Arg Asp Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys

165 170 175

His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala Leu

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Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr

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Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg Arg Ala

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Glu Pro His Ser Glu Ser Asn Val Pro Ala Gly His Thr Ile Pro Gln

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Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu Arg Arg

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Ala Arg Ser Ala Pro Ala Ala Ala Ile Ala Ala Arg Val Ala Gly Gln

35 40 45

Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg Arg Leu

50 55 60

Arg Ser Pro Arg Val Leu Phe Ser Thr Gln Pro Pro Arg Glu Ala Ala

65 70 75 80

Asp Thr Gln Asp Leu Asp Phe Glu Val Gly Gly Ala Ala Pro Phe Asn

85 90 95

Arg Arg Arg Arg Arg Arg Arg Ser Ser Ser His Pro Ile Phe His Arg

100 105 110

Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp Lys

115 120 125

Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu

130 135 140

Val Asn Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu Thr Lys

145 150 155 160

Cys Arg Asp Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile Asp Ser

165 170 175

Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala

180 185 190

Leu Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp

195 200 205

Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg Arg Ala

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Glu Pro His Ser Glu Ser Asn Val Pro Ala Gly His Thr Ile Pro Gln

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Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu Arg Arg

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Ala Arg Ser Ala Pro Ala Ala Ala Ile Ala Ala Arg Val Ala Gly Gln

35 40 45

Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg Arg Leu

50 55 60

Arg Ser Pro Arg Val Leu Phe Ser Thr Gln Pro Pro Arg Glu Ala Ala

65 70 75 80

Asp Thr Gln Asp Leu Asp Phe Glu Val Gly Gly Ala Ala Pro Phe Asn

85 90 95

Arg Arg Arg Ser Lys Arg Ser Ser Ser His Pro Ile Phe His Arg Gly

100 105 110

Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp Lys Thr

115 120 125

Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu Val

130 135 140

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

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Arg Asp Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys

165 170 175

His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala Leu

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Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr

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Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg Arg Ala

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Glu Pro His Ser Glu Ser Asn Val Pro Ala Gly His Thr Ile Pro Gln

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Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu Arg Arg

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Ala Arg Ser Ala Pro Ala Ala Ala Ile Ala Ala Arg Val Ala Gly Gln

35 40 45

Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg Arg Leu

50 55 60

Arg Ser Pro Arg Val Leu Phe Ser Thr Gln Pro Pro Arg Glu Ala Ala

65 70 75 80

Asp Thr Gln Asp Leu Asp Phe Glu Val Gly Gly Ala Ala Pro Phe Asn

85 90 95

Arg Arg Arg Arg Ser Lys Arg Ser Ser Ser His Pro Ile Phe His Arg

100 105 110

Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp Lys

115 120 125

Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu

130 135 140

Val Asn Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu Thr Lys

145 150 155 160

Cys Arg Asp Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile Asp Ser

165 170 175

Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala

180 185 190

Leu Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp

195 200 205

Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg Arg Ala

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Glu Pro His Ser Glu Ser Asn Val Pro Ala Gly His Thr Ile Pro Gln

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Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu Arg Arg

20 25 30

Ala Arg Ser Ala Pro Ala Ala Ala Ile Ala Ala Arg Val Ala Gly Gln

35 40 45

Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg Arg Leu

50 55 60

Arg Ser Pro Arg Val Leu Phe Ser Thr Gln Pro Pro Arg Glu Ala Ala

65 70 75 80

Asp Thr Gln Asp Leu Asp Phe Glu Val Gly Gly Ala Ala Pro Phe Asn

85 90 95

Arg Gly Ile Arg Arg Lys Arg Ser Ser Ser His Pro Ile Phe His Arg

100 105 110

Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp Lys

115 120 125

Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu

130 135 140

Val Asn Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu Thr Lys

145 150 155 160

Cys Arg Asp Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile Asp Ser

165 170 175

Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala

180 185 190

Leu Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp

195 200 205

Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg Arg Ala

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Glu Pro His Ser Glu Ser Asn Val Pro Ala Gly His Thr Ile Pro Gln

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Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu Arg Arg

20 25 30

Ala Arg Ser Ala Pro Ala Ala Ala Ile Ala Ala Arg Val Ala Gly Gln

35 40 45

Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg Arg Leu

50 55 60

Arg Ser Pro Arg Val Leu Phe Ser Thr Gln Pro Pro Arg Glu Ala Ala

65 70 75 80

Asp Thr Gln Asp Leu Asp Phe Glu Val Gly Gly Ala Ala Pro Phe Asn

85 90 95

Gln Thr Lys Arg Ser Lys Arg Ser Ser Ser His Pro Ile Phe His Arg

100 105 110

Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp Lys

115 120 125

Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu

130 135 140

Val Asn Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu Thr Lys

145 150 155 160

Cys Arg Asp Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile Asp Ser

165 170 175

Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala

180 185 190

Leu Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp

195 200 205

Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg Arg Ala

210 215 220

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Glu Pro His Ser Glu Ser Asn Val Pro Ala Gly His Thr Ile Pro Gln

1 5 10 15

Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu Arg Arg

20 25 30

Ala Arg Ser Ala Pro Ala Ala Ala Ile Ala Ala Arg Val Ala Gly Gln

35 40 45

Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg Arg Leu

50 55 60

Arg Ser Pro Arg Val Leu Phe Ser Thr Gln Pro Pro Arg Glu Ala Ala

65 70 75 80

Asp Thr Gln Asp Leu Asp Phe Glu Val Gly Gly Ala Ala Pro Phe Asn

85 90 95

Arg Thr His Arg Ser Lys Arg Ser Ser Ser His Pro Ile Phe His Arg

100 105 110

Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp Lys

115 120 125

Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu

130 135 140

Val Asn Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu Thr Lys

145 150 155 160

Cys Arg Asp Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile Asp Ser

165 170 175

Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala

180 185 190

Leu Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp

195 200 205

Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg Arg Ala

210 215 220

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Arg Thr His Arg Arg Lys Arg

1 5

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Arg Thr His Arg Arg Arg Arg

1 5

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Arg Thr Arg Arg Arg Arg Arg

1 5

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Arg Thr His Arg Arg Arg Arg Arg

1 5

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Arg Arg Arg Val Arg Arg

1 5

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Arg Arg Arg Arg Val Arg Arg

1 5

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Arg Arg Arg Arg Arg Arg

1 5

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Arg Arg Arg Arg Arg Arg Arg

1 5

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Arg Arg Arg Ser Lys Arg

1 5

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Arg Arg Arg Arg Ser Lys Arg

1 5

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Arg Gly Ile Arg Arg Lys Arg

1 5

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Gln Thr Lys Arg Ser Lys Arg

1 5

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Arg Thr His Arg Ser Lys Arg

1 5

Claims

A proNGF mutant, comprising:

(i) An amino acid sequence as shown in SEQ ID NO. 1-12;

(ii) An amino acid sequence having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID Nos. 1-12; or

(iii) An amino acid sequence having one or more site substitutions, deletions or insertions compared to the amino acid sequence shown in SEQ ID NO. 1-12.
2. A polynucleotide encoding a proNGF mutant as claimed in claim 1.
3. A recombinant expression vector comprising the polynucleotide of claim 2.
4. The recombinant expression vector according to claim 3, wherein the recombinant expression vector is a prokaryotic expression vector or a eukaryotic expression vector.
5. The recombinant expression vector of claim 4, wherein the eukaryotic expression vector is pcDNA3.1, pEGFP/pEGFT, pCHO1.0, pCMV, pSV2 or pGN.
6. A cell comprising the polynucleotide of claim 2 or the recombinant expression vector of claim 3.
7. The cell of claim 6, wherein the cell is a prokaryotic cell or a eukaryotic cell.
8. The cell of claim 7, wherein the eukaryotic cell is a CHO cell or 293 cell.
9. Use of a proNGF mutant as claimed in claim 1, a polynucleotide as claimed in claim 2, a recombinant expression vector as claimed in any one of claims 3 to 5 and/or a cell as claimed in any one of claims 6 to 8 for the preparation of β -NGF.
10. A method for producing beta-NGF, comprising:

inserting a polynucleotide encoding a proNGF mutant as claimed in claim 1 into an expression vector to obtain a recombinant expression vector;

transfecting the recombinant expression vector into a cell;

the cells are cultured to obtain the beta-NGF.
11. The method of claim 10, wherein the recombinant expression vector is a eukaryotic expression vector and the cell is a eukaryotic cell.
12. A method for producing beta-NGF, comprising:

providing a proNGF mutant as defined in claim 1;

the proNGF mutants were cleaved in vitro using protease to give β -NGF.
13. The method of claim 12, wherein the protease is a precursor protein converting enzyme, preferably at least one of furin, trypsin, and plasmin.