CN114315979A

CN114315979A - Light-cleavable protein mutant with high light-cleavage efficiency and application thereof

Info

Publication number: CN114315979A
Application number: CN202111467326.1A
Authority: CN
Inventors: 周娜娜; 安泰; 王小艳; 王磊; 张媛; 赵国淼; 王靖; 李凡; 杨凯; 王满意; 牛兴和; 郝小明
Original assignee: Cofco Nutrition and Health Research Institute Co Ltd
Current assignee: Cofco Nutrition and Health Research Institute Co Ltd
Priority date: 2021-12-03
Filing date: 2021-12-03
Publication date: 2022-04-12
Anticipated expiration: 2041-12-03
Also published as: CN114315979B

Abstract

The invention relates to the technical field of genetic engineering, and discloses a photocleavable protein mutant with high photocleavage efficiency and application thereof. The mutant of the photo-cleavable protein is a mutant of photo-cleavable protein phoCl, the amino acid sequence of the photo-cleavable protein phoCl is shown as SEQ ID NO:1, and the mutant of the photo-cleavable protein has mutation in at least one of the 8 th to 232 th amino acid residues of the photo-cleavable protein phoCl. The photocleavable protein mutant provided by the invention can obtain higher cleavage efficiency in shorter UV illumination time. The photocleavable protein mutant provided by the invention can be fused and expressed with different target proteins, the obtained fusion protein has high purification efficiency, and the high-efficiency recovery of the target protein is realized.

Description

Light-cleavable protein mutant with high light-cleavage efficiency and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a photocleavable protein mutant with high photocleavage efficiency and application thereof.

Background

With the development of biotechnology, mature high-efficiency prokaryotic and eukaryotic cell protein expression systems have been developed at present, and the production of target proteins by adopting a microbial expression system has gradually formed a large-scale industrial production system. However, the expression of the target protein is often accompanied by the expression of other proteins in the expression system, so that the obtained expression product is often a mixture of the target protein and other proteins. Therefore, protein purification becomes a key link in genetic engineering and protein engineering, the efficiency of protein purification determines the yield of recombinant protein, and the protein purification technology also determines the production cost of recombinant protein. The purification methods mainly employed at present include chromatographic techniques (e.g., ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, etc.) and electrophoretic techniques. Although the chromatographic technique has high precision, the universality and the purification efficiency are insufficient, the cost is high, and although the electrophoretic technique has low cost, the separation precision and the purification efficiency are often difficult to meet the production requirement.

The recent development of optogenetic technology has enabled peptide cleavage to occur in response to light stimuli by expression in vivoThe split fluorescent protein realizes the regulation and control of functional protein positioning or protein purification, and is a novel protein purification technology. Such as Floyd, N.et al., 2009, Photoinduced, family-specific, site-selective cleavage ofTIM-barrel proteins. J Am ChemSoc131, 12518-12519 discloses the attachment of a GH-1 fragment which cleaves in response to UV light to an affinity chromatography tag (e.g.a hexahistidine tag) to avoid folding errors in elution due to changes in buffer pH or the addition of protein denaturants. Also for example, US10370420B2 discloses a photocleavable protein that can be used for intracellular protein localization, enzyme activation, bio-based surface materials, etc., by breaking peptide bonds under irradiation of UV light; CN111154119A discloses a method for preparing hydrogel with controllable mechanical properties by utilizing the photo-cleavable protein; CN109971776A also developed a protein purification method based on this photo-cleavable protein. However, the photo-cleavage efficiency of the photo-cleavable protein is only about 60%, which has the defect of low cleavage efficiency, and the defect can cause the problems of low recovery rate of the target protein, waste of the target protein and the like, and is not favorable for scientific research and production work.

Disclosure of Invention

The invention aims to solve the problems of long UV light irradiation time, low cutting efficiency and the like in the process of carrying out light cutting on light-cleavable protein in the prior art, and provides a light-cleavable protein mutant with high light cutting efficiency and application thereof. The UV light irradiation time of the photo-cleavable protein mutant is short, the cleavage efficiency is high, and the high-efficiency recovery of the target protein can be realized.

In order to achieve the above object, the present invention provides a photo-cleavable protein mutant, wherein the mutant is a mutant of photo-cleavable protein phoCl, wherein the amino acid sequence of the photo-cleavable protein phoCl is shown as SEQ ID NO:1, and the mutant has a mutation in at least one of the amino acid residues 8-232 of the photo-cleavable protein phoCl.

In a second aspect, the invention provides the use of a photocleavable protein mutant as described above in protein purification.

In a third aspect, the invention provides a fusion protein comprising a protein of interest and a photocleavable protein mutant as described above.

The fourth aspect of the invention provides a protein purification method, which comprises the steps of fusion expression of fusion protein containing target protein and a light-cleavable protein mutant, purification of the fusion protein, and light cleavage of the purified fusion protein to obtain the purified target protein, wherein the fusion protein is the fusion protein.

Through the technical scheme, the invention can obtain the following beneficial effects:

(1) the photo-cleavable protein mutant provided by the invention can complete photo-cleavage in a short time, and has high photo-cleavage efficiency, so that the purification efficiency of protein purification by using the photo-cleavable protein is effectively improved.

(2) The photocleavable protein mutant provided by the invention can be subjected to fusion expression with various target proteins, and the obtained fusion protein has high purification efficiency and high recovery rate of the target protein.

(3) Compared with the original photocleavable protein phoCl, the photocleavable protein mutant provided by the invention has the advantages that the photocleavage efficiency is obviously improved, the purification yield of the target protein is improved, and the purification cost is reduced.

(4) The photocleavable protein mutant provided by the invention can realize high-flux protein purification, has a wide application range, can be used for laboratory protein purification, and is also suitable for industrial scale popularization and application.

Drawings

FIG. 1 is a graph of the fluorescence intensity of the different concentrations of the original protein phoCl detected in example 1.

FIGS. 2-4 are graphs of the fluorescence transitions of the proto-protein phoCl in example 1.

FIG. 5 shows the results of the mutant high-throughput activity screening in example 1.

FIG. 6 is a schematic diagram of a reference protein junction in the construction of a multiple mutation site-combining mutant in example 1.

FIG. 7 is a graph showing the change in fluorescence intensity of mutant 2 obtained in example 1.

FIG. 8 is a graph comparing the results of photocleavage efficiency of the original protein phoCl, mutant 1 and mutant 2 obtained in example 1.

FIG. 9 is a graph showing the comparison of the photocleavage efficiencies of mutant 1 and mutant 2 in example 1 at different irradiation wavelengths.

FIG. 10 is a graph showing the comparison of the photocleavage efficiency of mutant 1 in example 1 under different illumination times.

FIG. 11 is a graph showing the comparison of the photocleavage efficiency of mutant 2 in example 1 under different illumination times.

FIG. 12 is an SDS-PAGE of the product of the fusion protein phoCl-Histatin1 after different periods of light exposure in example 2.

FIG. 13 is an SDS-PAGE electrophoresis of the products of the fusion protein mutant 1-Histatin1 after being exposed to light for different periods in example 2.

FIG. 14 is an SDS-PAGE electrophoresis of the products of the fusion protein mutant 2-Histatin1 after being exposed to light for different periods in example 2.

FIG. 15 is a graph showing the results of measurement of the minimum inhibitory concentration of Histatin1 obtained by photocleavage in example 2.

FIG. 16 is an SDS-PAGE electrophoresis of the products of the fusion protein mutant 2-Rm (fungal detoxification enzyme) in example 2 after exposure to light at different times.

FIG. 17 is an SDS-PAGE electrophoresis of the products of the fusion protein mutant 2-Cel12a (Endocellulose) in example 2 after being subjected to different times of light irradiation.

FIG. 18 is a graph showing the ZEN degradation rate results of the fungal detoxification enzyme obtained by photocleavage in example 2.

FIG. 19 is a graph showing the results of measuring the protein activity of the endonuclease obtained by photocleavage in example 2.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

The invention provides a (high light cutting efficiency) photo-cleavable protein mutant, which is a mutant of photo-cleavable protein phoCl, wherein the amino acid sequence of the phoCl is shown as SEQ ID NO:1, and the photo-cleavable protein mutant has mutation in at least one of the amino acid residues from 8 to 232 of the phoCl (namely the photo-cleavable protein mutant is a mutant with mutation in at least one of the amino acid residues from 8 to 232 of the photo-cleavable protein phoCl).

MVIPDYFKQSFPEGYSWERSMTYEDGGICIATNDITMEGDSFINKIHFKGTNFPPNGPVMQKRTVGWEASTEKMYERDGVLKGDVKMKLLLKGGGHYRCDYRTTYKVKQKPVKLPDYHFVDHRIEILSHDKDYNKVKLYEHAVARNSTDSMDELYKGGSGGMVSKGEETITSVIKPDMKNKLRMEGNVNGHAFVIEGEGSGKPFEGIQTIDLEVKEGAPLPFAYDILTTAFHYGNRVFTKYPR（SEQ ID NO:1）

Any mutant of phoCl having the above characteristics is within the scope of the present invention. In order to obtain higher photocleavage efficiency, according to a preferred embodiment of the invention, said mutant photocleavable protein has a mutation at least one of

amino acid residues

8, 9, 10, 18, 20, 28, 34, 43, 49, 52, 64, 73, 76, 80, 82, 84, 86, 90, 92, 98, 100, 108, 112, 113, 119, 125, 128, 135, 147, 148, 150, 151, 162, 165, 168, 179, 202, 207, 215, 232 of the photocleavable protein phoCl.

In order to obtain higher photocleavage efficiency, preferably, the photocleavable protein mutant is a multi-point mutein. Preferably, the mutant photo-cleavable protein has 10-15 mutation sites (at amino acid residue positions as described above).

According to a particularly preferred embodiment of the present invention, the mutant photo-cleavable protein comprises a protein having an amino acid sequence shown as SEQ ID NO. 2 or SEQ ID NO. 3.

MVIPDYFKQSFPEGYSWGRSMTYEDGGICIATNDITMEGDSFINKIHFEGTHFPPNGPVMQKRTVGWEASTEEMYGRDGELKGYVKMKLQLKGGGHYRCDYRTTYKVKQKPVKLPDYHCVDHRIEILSHDKDYNRVKLYEHAVARNSTDGMDELYKGGSGGVVSKGEETITSVIKPDMRNKLRMEGNVNGHAFVIEGEGSGKPFEGIQTIDLEVKEGAPLPFAYDILTTAFHYGNRVFTKYPR（SEQ ID NO:2）

MVIPDYFKQSFPEGYSWGRSMTYEDGGICIATNDITMEGDSFINKIHFEGTHFPPNGPVMQKRTVGWEASTEEMYGRDGELKGYVKMKLQLKGGGHYRCDYRTTYKVKQKPVKLPDYHCVDHRIEILSHDKDYNRVKLYEHAVARNSTDGMDELYKGGSGGVVSKGEETITSVIKPDMRNKLRMEGNVNGHAFVIEGEGSGMPFEGIQTIDLEVKEGAPLPFAYDILTTAFHYGNRVFTKYPR（SEQ ID NO:3）

To facilitate protein purification, according to a preferred embodiment of the present invention, wherein the photo-cleavable protein mutant further comprises an amino acid tag for purification.

Any amino acid tag commonly used in the art for protein purification may be suitable for use in the present invention. Preferably, the amino acid tag comprises at least one of a poly-histidine tag, a glutathione-S-transferase tag (GST), a hemagglutinin tag, a FLAG tag, a myc tag, a maltose binding protein tag (MBP), and a fluorescent tag (e.g., EGFP, etc.). The present invention is not particularly limited with respect to the specific sequence of the amino acid tag, for example, the amino acid tag may include the following amino acid sequence:

poly-histidine tag: HHHHHHHHHH (SEQ ID NO: 4)

glutathione-S-transferase tag (GST): MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSPEFPGRLERPH (SEQ ID NO: 5)

Hemagglutinin labeling: YPYDVPDYA (SEQ ID NO: 6)

And (3) FLAG label: DYKDDDDK (SEQ ID NO: 7)

myc tag: EQKLISEEDL (SEQ ID NO: 8)

Maltose binding protein tag (MBP): MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRISEFGSSRVDLQASLALAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWQLGCFGG (SEQ ID NO: 9)

Fluorescent tag (EGFP): MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO: 10)

The invention also relates to genes of the light-cleavable protein mutant. From the amino acid sequences disclosed in the present invention, the nucleotide sequences encoding the genes can be completely deduced by those skilled in the art from well-known codon tables, and the genes can be obtained by biological methods (e.g., PCR method, mutation method) or chemical synthesis method. The gene can contain other elements besides the coding gene of the photo-cleavable protein mutant, and the elements are used for assisting the expression of the coding gene of the photo-cleavable protein mutant or improving the expression efficiency and the expression quantity. The skilled person can design and select corresponding elements, such as promoters, terminators, enhancers, regulatory sequences, inducible elements, etc., according to well-known biological knowledge.

In the invention, the application may include connecting the photocleavable protein mutant provided by the invention with a target protein and a purification tag (or other amino acid sequences or groups convenient for protein purification, etc.), so as to prepare a fusion protein, and separating and purifying the fusion protein. And separating the photocleavable protein mutant from the target protein by utilizing the characteristic that the photocleavable protein mutant can be cracked under specific photocleavage conditions (including irradiation light wavelength, irradiation time and the like), thereby obtaining the purified target protein.

The third aspect of the invention provides a fusion protein, which is characterized in that the fusion protein comprises a target protein and the photocleavable protein mutant.

The fusion protein provided by the invention can be obtained by any existing bioengineering technical means. According to a preferred embodiment of the present invention, wherein the fusion protein is obtained by fusion expression of the photocleavable protein mutant as described above with the target protein. The characteristics of the mutant of the photo-cleavable protein are as described above and will not be described herein.

In the invention, the fusion protein can be obtained by any method existing in the field for carrying out fusion expression on the photocleavable protein mutant provided by the invention and the target protein. Preferably, the fusion protein may be obtained in a manner that: constructing a fusion gene by the gene expressing the photocleavable protein mutant and the gene expressing the target protein, and expressing the fusion gene by an expression vector to obtain the expression vector (host cell) containing the fusion protein. Then, cracking or crushing an expression vector (host cell) containing the fusion protein, centrifuging lysate or homogenate of the host cell and performing affinity chromatography, and performing UV light irradiation on the fusion protein after the affinity chromatography so as to separate the fusion protein from the host cell; and then performing affinity chromatography on the irradiated mixed solution again, and collecting the affinity column permeate to obtain the target protein. In the above method, the fusion gene can be constructed by any method existing in the art, and the present invention is not particularly limited to the specific method and procedure thereof as long as the target protein can be obtained. The choice of the expression vector in the present invention is also not particularly limited, and any expression vector that is currently available in the art for expression of fusion proteins can be applied to the present invention, for example, yeast, Escherichia coli, Bacillus subtilis, (mammalian) cells, and the like.

The photocleavable protein mutant provided by the invention can be used for purifying any target protein. In order to obtain a better purification effect, according to a preferred embodiment of the present invention, wherein the molecular weight of the target protein is 5-50 kDa.

Preferably, the molecular weight of the target protein is 5-30 kDa. For example, 5kDa, 7 kDa, 10 kDa, 12 kDa, 15 kDa, 18 kDa, 20 kDa, 22 kDa, 25 kDa, 28 kDa, 30kDa, or any intermediate value between any two of the above values.

The fourth aspect of the present invention provides a protein purification (recovery) method, comprising fusion expression of a fusion protein comprising a target protein and a photocleavable protein mutant, purification of the fusion protein, and photocleavage of the purified fusion protein to obtain a purified target protein, wherein the fusion protein is the fusion protein as described above.

In order to improve the photocleavage efficiency, according to a preferred embodiment of the present invention, the photocleavage conditions include a wavelength of 365-405nm and a time of 20-90 min. Preferably, the photocleavage time is 20-60 min.

Preferably, the light cutting conditions include a wavelength of 380-385nm and a time of 25-35 min.

The present invention will be described in detail below by way of examples. It should be understood that the following examples are only intended to further illustrate and explain the contents of the present invention by way of example, and are not intended to limit the present invention.

In the following examples, the amino acid sequence of the original photocleavable protein phoCl is shown in SEQ ID NO 1. Unless otherwise specified, all biological/chemical reagents used were purchased from the normal biological/chemical suppliers and were analytically pure.

Example 1

This example serves to illustrate the obtaining of the photo-cleavable proteins provided by the present invention.

The pBAD-phoCl plasmid adopted in the embodiment is an expression system which takes the pBAD plasmid as a vector and is inserted with phoCl expression genes, and the preparation method can refer to Zhang Sai, 2012, the preparation of recombinant bifidobacterium longum engineering bacteria for expressing interferon-alpha 2b, and China journal of pathogenic biology, 007(003), 190-191 and 194.

In this example, primers F1 (ATGGTGATCCCTGACTACTTCAAGCAG, SEQ ID NO: 11) and R1 (TTACCGTGGGTACTTGGTGAACACG, SEQ ID NO: 12) were synthesized by Kui Rui Boxing Ke Biotechnology, Inc.

(one) construction of a library of photocleavable protein mutants

The method comprises the steps of taking pBAD-phoCl plasmid as a template, taking primers F1 and R1 as a forward primer and a reverse primer respectively, carrying out directed evolution of error-prone PCR according to a reaction system in a table 1 and an error-prone PCR program in a table 2, and constructing a mutant library.

The PCR product was subjected to 1% agarose gel electrophoresis using an agarose gel recovery kit (purchased from Tiangen Biochemical technology, Beijing, Ltd.) according to the procedures described in the kit instructions, and the fragment of 732bp was recovered by cutting the gel. The recovered fragments were subjected to a golden gate reaction with pBAD vector (purchased from biowind corporation) (reaction system shown in table 3). After completion of the reaction, the cells were transferred to Trans10 competent cells (purchased from Kyoto Kogyo Co., Ltd.), plated on LB solid culture plates (containing 150. mu.g/mL ampicillin), and cultured overnight (about 16 to 18 hours) at 37 ℃ by inversion to obtain a phoCl mutant library.

(II) mutant library activation and inducible expression

The obtained phoCl mutant library was subjected to QPix series high-throughput microbial clone screening system, and a total of 6000 single clones were picked in batches. The picked monoclonals were inoculated into 96-well plates containing 800. mu.L of LB liquid medium (containing 150. mu.g/mL ampicillin) per well, and cultured overnight (about 16-18 hours) at 37 ℃ with shaking at 1000 rpm.

mu.L of the overnight-cultured culture broth was aspirated, added to a 24-well plate containing 1.8mL of fresh LB liquid medium (containing 150. mu.g/mL of ampicillin) per well, and after shaking-culturing at 500rpm at 37 ℃ for 4 hours, arabinose (final concentration: 0.02%) was added and the culture was induced at 25 ℃ for 20 hours.

And (3) centrifuging the bacterial liquid subjected to induction culture at 4 ℃ and 4000rpm for 15min, and removing the supernatant. The pellet was suspended in 100. mu.L of sodium phosphate buffer (50mM, pH7), lysozyme (purchased from Sigma-Aldrich Co., Ltd., final concentration: 10 mg/L) was added, and the mixture was treated at 37 ℃ for 1 hour. Then, the mixture was centrifuged at 4000rpm for 30min at 4 ℃ to transfer 200. mu.L of the supernatant to a new 96-well microplate for use as a template for activity screening.

(III) high throughput screening of mutants

The calculated cleavage efficiency (fluorescence quenching ratio) is used as the standard for screening protein mutants. The specific method comprises the following steps:

the original photocleavable protein phoCl was added to a 96-well microplate in a concentration gradient (200 μ L per well) and the fluorescence intensity was read with a microplate reader (results are shown in fig. 1). When phoCl is irradiated with 365-405nm wavelength light, fluorescence transition occurs (as shown in FIG. 2, the green fluorescence transition is on the left side, and the red fluorescence transition is on the right side). With the increase of the irradiation time, the intensity of the green fluorescence gradually weakens (as shown in fig. 3), and the red fluorescence is enhanced and then weakened (as shown in fig. 4). According to the change of the fluorescence curve, the green fluorescence is found to be in negative correlation with the irradiation time, and the intensity of the green fluorescence is stabilized at a certain value when the irradiation time is 90-120min, which indicates that the maximum fluorescence quenching proportion is reached at the moment. Based on previous studies, the fluorescence quenching ratio was equivalent to the photocleavage efficiency. The fluorescence intensity before and after photocleavage was measured by a microplate reader, and the photocleavage efficiency was calculated according to the following formula.

Photocleavage efficiency (%) = (green fluorescence intensity of protein before photocleavage-green fluorescence intensity of protein after photocleavage)/green fluorescence intensity of protein before photocleavage × 100%

Placing the 96-well enzyme label plate containing 200uL of mutant supernatant in each well obtained in the test (II) in an enzyme label instrument, and performing fluorescence measurement (excitation wavelength is 470nm, emission wavelength is 510 nm) and marking as Fl 1; the ELISA plate was irradiated under a 405nm UV lamp for 90min, and the fluorescence was measured again and recorded as Fl 2. The light cutting efficiency was calculated as follows.

Photocleavage efficiency (%) = (Fl 1-Fl 2)/Fl 1X 100%

The photocleavage efficiency of the original protein phoCl (60%) was used as a control. FIG. 5 shows the photocleavage efficiency of the selected mutants, based on the cleavage efficiency of 100% at "1", where the numbers represent the specific photocleavage efficiency of each mutant. Analysis shows that the cutting efficiency of the 115 mutants is obviously higher than that of the original protein, and the mutants are subjected to amplification culture to verify whether the cutting efficiency is obviously improved.

(IV) mutant amplification culture and Activity verification

The mutants selected in test (III) and the original protein strain were inoculated into a test tube containing 5mL of LB liquid medium (containing 150. mu.g/mL of ampicillin), and cultured overnight (about 16 to 18 hours) at 37 ℃ with shaking at 220 rpm. The next day, the cells were inoculated at 1% inoculum size into 100mL of LB liquid medium (containing 150. mu.g/mL ampicillin) in a Erlenmeyer flask, and cultured at 37 ℃ for 3 to 4 hours with shaking at 220rpm, followed by addition of arabinose (final concentration: 0.02%), followed by induction at 25 ℃ for 200rpm overnight (about 20 hours).

The bacterial suspension obtained by the induction culture was centrifuged at 8000rpm for 10min at 4 ℃ and the cells were washed twice with 20mL of sodium phosphate buffer (50mM, pH7), and then 10mL of sodium phosphate buffer (50mM, pH7) was added to the washed cells to suspend the cells. Subsequently, ultrasonication was carried out under ice bath conditions (ultrasonic power 200W, ultrasonic 3 s/intermittent 5s, treatment time 10min in total). Centrifuging the sample after ultrasonic treatment at 12000rpm for 20min at 4 deg.C, collecting the supernatant to detect fluorescence, performing optical cutting at 405nm for 90min, detecting fluorescence again, and calculating the optical cutting efficiency according to the method of test (III).

The results show that the fluorescence quenching ratio of the 115 mutant proteins screened in the test (III) is obviously higher than that of the original protein. The 115 mutant proteins were subsequently sequenced and aligned with the original protein PhoCl amino acid sequence (consigned to Beijing Rui Boxing Biotech, Inc.), and the mutation sites of the 115 mutant proteins were found to include 40 amino acid positions: k8, Q9, S10, E18, S20, I28, D34, I43, K49, N52, T64, K73, E76, V80, K82, D84, K86, L90, K92, R98, D100, K108, V112, K113, F119, E125, S128, K135, S147, T148, S150, M151, M162, K165, E168, K179, K202, I207, K215, H232.

(V) construction of mutants with combined multiple mutation sites

The 40 mutation sites were combined using Site-Directed Mutagenesis using the QuikChange Site-Directed Mutagenesis Kit (FIG. 6) from Stratagene. Iterative mutation experiments prove that 2 mutant proteins with obviously improved cutting efficiency are screened out finally (mutant 1 and mutant 2, the mutation sites are shown in table 4 in detail, and the corresponding coding genes are respectively shown in SEQ ID NO. 13 and SEQ ID NO. 14).

ATGGTTATCCCGGACTACTTCAAACAGTCTTTCCCGGAAGGTTACTCTTGGGGTCGTTCTATGACCTACGAAGACGGTGGTATCTGCATCGCTACCAACGACATCACCATGGAAGGTGACTCTTTCATCAACAAAATCCACTTCGAAGGTACCCACTTCCCGCCGAACGGTCCGGTTATGCAGAAACGTACCGTTGGTTGGGAAGCTTCTACCGAAGAAATGTACGGTCGTGACGGTGAACTGAAAGGTTACGTTAAAATGAAACTGCAGCTGAAAGGTGGTGGTCACTACCGTTGCGACTACCGTACCACCTACAAAGTTAAACAGAAACCGGTTAAACTGCCGGACTACCACTGCGTTGACCACCGTATCGAAATCCTGTCTCACGACAAAGACTACAACCGTGTTAAACTGTACGAACACGCTGTTGCTCGTAACTCTACCGACGGTATGGACGAACTGTACAAAGGTGGTTCTGGTGGTGTTGTTTCTAAAGGTGAAGAAACCATCACCTCTGTTATCAAACCGGACATGCGTAACAAACTGCGTATGGAAGGTAACGTTAACGGTCACGCTTTCGTTATCGAAGGTGAAGGTTCTGGTAAACCGTTCGAAGGTATCCAGACCATCGACCTGGAAGTTAAAGAAGGTGCTCCGCTGCCGTTCGCTTACGACATCCTGACCACCGCTTTCCACTACGGTAACCGTGTTTTCACCAAATACCCGCGT（SEQ ID NO:13）

ATGGTTATCCCGGACTACTTCAAACAGTCTTTCCCGGAAGGTTACTCTTGGGGTCGTTCTATGACCTACGAAGACGGTGGTATCTGCATCGCTACCAACGACATCACCATGGAAGGTGACTCTTTCATCAACAAAATCCACTTCGAAGGTACCCACTTCCCGCCGAACGGTCCGGTTATGCAGAAACGTACCGTTGGTTGGGAAGCTTCTACCGAAGAAATGTACGGTCGTGACGGTGAACTGAAAGGTTACGTTAAAATGAAACTGCAGCTGAAAGGTGGTGGTCACTACCGTTGCGACTACCGTACCACCTACAAAGTTAAACAGAAACCGGTTAAACTGCCGGACTACCACTGCGTTGACCACCGTATCGAAATCCTGTCTCACGACAAAGACTACAACCGTGTTAAACTGTACGAACACGCTGTTGCTCGTAACTCTACCGACGGTATGGACGAACTGTACAAAGGTGGTTCTGGTGGTGTTGTTTCTAAAGGTGAAGAAACCATCACCTCTGTTATCAAACCGGACATGCGTAACAAACTGCGTATGGAAGGTAACGTTAACGGTCACGCTTTCGTTATCGAAGGTGAAGGTTCTGGTATGCCGTTCGAAGGTATCCAGACCATCGACCTGGAAGTTAAAGAAGGTGCTCCGCTGCCGTTCGCTTACGACATCCTGACCACCGCTTTCCACTACGGTAACCGTGTTTTCACCAAATACCCGCGT（SEQ ID NO:14）

The change in fluorescence intensity with photocleavage time (shown in FIG. 7) was observed for mutant 2 at different protein concentrations, and was significantly different from the original protein phoCl (shown in FIG. 1). Specifically, as shown in fig. 1, as the cleavage time of the original protein phoCl is prolonged, the green fluorescence intensities corresponding to different protein concentrations are gradually reduced, and a stronger green fluorescence intensity is displayed until 120 min; as shown in FIG. 7, in mutant 2, the green fluorescence intensity corresponding to different protein concentrations was gradually reduced with the increase of the cleavage time, and no green fluorescence was detected at 30 min.

The results of the photocleavage efficiency calculations for the original protein phoCl, mutant 1 and mutant 2 are shown in detail in fig. 8. Through calculation, compared with the original protein, the photocleavage efficiency of the mutant 1 is improved by 34.6%, and the photocleavage efficiency of the mutant 2 is improved by 35%.

(VI) optimization of mutant photocleavage conditions with combination of multiple mutant sites

The two mutant proteins obtained in the test (five) were subjected to photocleavage under the conditions of a protein concentration of 0.2mg/mL and an irradiation time (i.e., photocleavage time) of 90min, and the cleavage efficiencies of the mutant proteins were measured under the conditions of different irradiation wavelengths (the results are shown in FIG. 9), and it was found that the cleavage efficiencies of the two mutant proteins were the highest at an irradiation wavelength of 385 nm.

The two mutant proteins obtained in the test (IV) were subjected to photo-cleavage under the conditions that the protein concentration was 0.2mg/mL and the irradiation wavelength was 385nm, and the photo-cleavage efficiencies of the mutant proteins at different irradiation times were measured (the results are shown in FIGS. 10 and 11), and it was found that the two mutant proteins substantially reached the maximum cleavage efficiency at the irradiation time of 30 min.

Example 2

This example serves to illustrate the expression and purification of the fusion proteins provided by the present invention.

(I) detection of fusion protein Activity

Based on molecular biological operation,

mutant proteins

1 and 2 screened in the test (five) in example 1 are subjected to fusion expression with the antibacterial peptide Hisatatin 1.

Original protein phoCl connected with a purification tag (poly-histidine tag, amino acid sequence is shown as SEQ ID NO: 4), mutant 1 and mutant 2 are respectively fused and expressed with antimicrobial peptide Histatin1 (synthesized by general biological company), after purification by using the purification tag, the purified protein is cut for 0min, 1min, 5min, 10min, 30min, 60min, 90min and 120min at the wavelength of 385nm, and the cutting condition is detected by electrophoresis.

SDS-PAGE electrophoresis images of the antimicrobial peptide (phoCl-Histatin 1) fusion expressed with the original protein, the antimicrobial peptide (mutant 1-Histatin 1) fusion expressed with mutant 1, and the antimicrobial peptide (mutant 2-Histatin 1) fusion expressed with mutant 2 are shown in FIGS. 12-14, respectively. As can be seen from the figure, the cleavage of mutant 1-Histatin1 and mutant 2-Histatin1 was essentially complete at 30min, while the cleavage of phoCl-Histatin1 was followed by 90min for the remainder of the fusion protein.

The Minimal Inhibitory Concentration (MIC) of Histatin1 against Candida albicans (ATCC 10231, purchased from ATCC) obtained by photocleavage and purification of three fusion proteins (phoCl-Histatin 1, mutant 1-Histatin1, mutant 2-Histatin 1) was determined by broth dilution (cf. Chenxiu et al, 1994. evaluation of MIC determination by broth dilution, journal of Chinese medical examination, 17(2): 95-98). Candida albicans was cultured overnight (about 16 h) to OD at 37 ℃ in LB medium₆₀₀And = 1. After diluting Candida albicans 1000 times with LB medium, adding into 96-well cell culture plate (50. mu.L per well), adding Histatin1 after photocleavage, so that Histatin1 in experimental group is in 100. mu.

L reaction systemWere

50, 40, 30, 20, 10 and 5. mu.g/mL, respectively, as blanks in the reaction system without Histatin 1. Placing a 96-hole cell culture plate in a 30 ℃ constant temperature incubator, performing shake culture at 180rpm for 16h, and measuring OD (optical density) by using a microplate reader₆₀₀And calculating the inhibition rate of Histatin1 on Candida albicans at different concentrations.

Inhibition (%) = (control OD)₆₀₀Experimental group OD₆₀₀) Control group OD₆₀₀×100%

The results of the bacteriostatic activity of Histatin1 obtained after photocleavage of the three fusion proteins are consistent, and are shown in FIG. 15, and it can be seen from the figure that the Minimum Inhibitory Concentration (MIC) of Histatin1 is 30 μ g/mL, and the results are in the same way as the literature (e.g. Xing L)et al., 2013, Facile Expression and Purification of Antimicrobial Peptide histatin 1 with Cleavable Self-Aggregating Tag (cSAT) Fusion in Escherichia coliProtein Expression and Purification, 88: 248-253.) reported values close.

(II) protein purification Using the mutant

Based on molecular biological operation, mutant 2 screened in test (five) in example 1 was fusion expressed with target proteins of different sizes.

Mutant 2 connected with a purification tag (poly-histidine tag, amino acid sequence shown as SEQ ID NO: 4) is respectively fused and expressed with fungus detoxification enzyme (Rm, 29.6 KDa) and endocellulase (Cel 12a, 23.5 KDa), purified by using the purification tag, purified protein is cut for 0min, 1min, 5min, 10min, 30min, 60min, 90min and 120min at 385nm wavelength, and the cutting condition is detected by electrophoresis.

FIGS. 16 and 17 show SDS-PAGE electrophoresis of mutant 2-Rm and mutant 2-Cel12a, respectively. As can be seen from the figure, the mutant 2-Rm fusion protein was weaker in cleavage efficiency than the mutant 2-Cel12a, indicating that different target proteins slightly affect the cleavage efficiency.

Respectively detecting the enzyme activities of the cut fungus detoxification enzyme (Rm) and the cut fibrous incision enzyme (Cel 12 a), wherein the specific detection method comprises the following steps:

fungal detoxification enzyme (Rm): mixing the cut fungus detoxification enzyme with Zearalenone (ZEN), and detecting the degradation rate of the fungus detoxification enzyme on zearalenone by a liquid chromatography (Zorbax SB C18 column, wherein the mobile phase is acetonitrile + water + methanol, the flow rate is 1 mL/min, the column temperature is 25 ℃, and a fluorescence detector). The results are shown in FIG. 18. As can be seen from the figure, the degradation rate of ZEN reaches more than 70%, which indicates that the activity of the fungus detoxification enzyme is higher.

Endocellulase (Cel 12 a): mixing the cut endo-cellulase with sodium carboxymethyl cellulose, and detecting the degradation of the endo-cellulase to the sodium carboxymethyl cellulose by using a DNS (domain name system) color development method so as to determine the protein activity of the endo-cellulase. As a result, as shown in FIG. 19, it can be seen that the activity of the cleaved Cel12a reached about 4U/mg.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

SEQUENCE LISTING

<110> Zhongliang Nutrition and health research institute Co., Ltd

<120> photo-cleavable protein mutant with high photo-cleavage efficiency and application thereof

<130> YSI70944COF

<160> 14

<170> PatentIn version 3.5

<210> 1

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<400> 1

Met Val Ile Pro Asp Tyr Phe Lys Gln Ser Phe Pro Glu Gly Tyr Ser

1 5 10 15

Trp Glu Arg Ser Met Thr Tyr Glu Asp Gly Gly Ile Cys Ile Ala Thr

20 25 30

Asn Asp Ile Thr Met Glu Gly Asp Ser Phe Ile Asn Lys Ile His Phe

35 40 45

Lys Gly Thr Asn Phe Pro Pro Asn Gly Pro Val Met Gln Lys Arg Thr

50 55 60

Val Gly Trp Glu Ala Ser Thr Glu Lys Met Tyr Glu Arg Asp Gly Val

65 70 75 80

Leu Lys Gly Asp Val Lys Met Lys Leu Leu Leu Lys Gly Gly Gly His

85 90 95

Tyr Arg Cys Asp Tyr Arg Thr Thr Tyr Lys Val Lys Gln Lys Pro Val

100 105 110

Lys Leu Pro Asp Tyr His Phe Val Asp His Arg Ile Glu Ile Leu Ser

115 120 125

His Asp Lys Asp Tyr Asn Lys Val Lys Leu Tyr Glu His Ala Val Ala

130 135 140

Arg Asn Ser Thr Asp Ser Met Asp Glu Leu Tyr Lys Gly Gly Ser Gly

145 150 155 160

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

165 170 175

Asp Met Lys Asn Lys Leu Arg Met Glu Gly Asn Val Asn Gly His Ala

180 185 190

Phe Val Ile Glu Gly Glu Gly Ser Gly Lys Pro Phe Glu Gly Ile Gln

195 200 205

Thr Ile Asp Leu Glu Val Lys Glu Gly Ala Pro Leu Pro Phe Ala Tyr

210 215 220

Asp Ile Leu Thr Thr Ala Phe His Tyr Gly Asn Arg Val Phe Thr Lys

225 230 235 240

Tyr Pro Arg

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<212> PRT

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Met Val Ile Pro Asp Tyr Phe Lys Gln Ser Phe Pro Glu Gly Tyr Ser

1 5 10 15

Trp Gly Arg Ser Met Thr Tyr Glu Asp Gly Gly Ile Cys Ile Ala Thr

20 25 30

Asn Asp Ile Thr Met Glu Gly Asp Ser Phe Ile Asn Lys Ile His Phe

35 40 45

Glu Gly Thr His Phe Pro Pro Asn Gly Pro Val Met Gln Lys Arg Thr

50 55 60

Val Gly Trp Glu Ala Ser Thr Glu Glu Met Tyr Gly Arg Asp Gly Glu

65 70 75 80

Leu Lys Gly Tyr Val Lys Met Lys Leu Gln Leu Lys Gly Gly Gly His

85 90 95

Tyr Arg Cys Asp Tyr Arg Thr Thr Tyr Lys Val Lys Gln Lys Pro Val

100 105 110

Lys Leu Pro Asp Tyr His Cys Val Asp His Arg Ile Glu Ile Leu Ser

115 120 125

His Asp Lys Asp Tyr Asn Arg Val Lys Leu Tyr Glu His Ala Val Ala

130 135 140

Arg Asn Ser Thr Asp Gly Met Asp Glu Leu Tyr Lys Gly Gly Ser Gly

145 150 155 160

Gly Val Val Ser Lys Gly Glu Glu Thr Ile Thr Ser Val Ile Lys Pro

165 170 175

Asp Met Arg Asn Lys Leu Arg Met Glu Gly Asn Val Asn Gly His Ala

180 185 190

Phe Val Ile Glu Gly Glu Gly Ser Gly Lys Pro Phe Glu Gly Ile Gln

195 200 205

Thr Ile Asp Leu Glu Val Lys Glu Gly Ala Pro Leu Pro Phe Ala Tyr

210 215 220

Asp Ile Leu Thr Thr Ala Phe His Tyr Gly Asn Arg Val Phe Thr Lys

225 230 235 240

Tyr Pro Arg

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Met Val Ile Pro Asp Tyr Phe Lys Gln Ser Phe Pro Glu Gly Tyr Ser

1 5 10 15

Trp Gly Arg Ser Met Thr Tyr Glu Asp Gly Gly Ile Cys Ile Ala Thr

20 25 30

Asn Asp Ile Thr Met Glu Gly Asp Ser Phe Ile Asn Lys Ile His Phe

35 40 45

Glu Gly Thr His Phe Pro Pro Asn Gly Pro Val Met Gln Lys Arg Thr

50 55 60

Val Gly Trp Glu Ala Ser Thr Glu Glu Met Tyr Gly Arg Asp Gly Glu

65 70 75 80

Leu Lys Gly Tyr Val Lys Met Lys Leu Gln Leu Lys Gly Gly Gly His

85 90 95

Tyr Arg Cys Asp Tyr Arg Thr Thr Tyr Lys Val Lys Gln Lys Pro Val

100 105 110

Lys Leu Pro Asp Tyr His Cys Val Asp His Arg Ile Glu Ile Leu Ser

115 120 125

His Asp Lys Asp Tyr Asn Arg Val Lys Leu Tyr Glu His Ala Val Ala

130 135 140

Arg Asn Ser Thr Asp Gly Met Asp Glu Leu Tyr Lys Gly Gly Ser Gly

145 150 155 160

Gly Val Val Ser Lys Gly Glu Glu Thr Ile Thr Ser Val Ile Lys Pro

165 170 175

Asp Met Arg Asn Lys Leu Arg Met Glu Gly Asn Val Asn Gly His Ala

180 185 190

Phe Val Ile Glu Gly Glu Gly Ser Gly Met Pro Phe Glu Gly Ile Gln

195 200 205

Thr Ile Asp Leu Glu Val Lys Glu Gly Ala Pro Leu Pro Phe Ala Tyr

210 215 220

Asp Ile Leu Thr Thr Ala Phe His Tyr Gly Asn Arg Val Phe Thr Lys

225 230 235 240

Tyr Pro Arg

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His His His His His His

1 5

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Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro

1 5 10 15

Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu

20 25 30

Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu

35 40 45

Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys

50 55 60

Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn

65 70 75 80

Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu

85 90 95

Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser

100 105 110

Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu

115 120 125

Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn

130 135 140

Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp

145 150 155 160

Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu

165 170 175

Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr

180 185 190

Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala

195 200 205

Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Glu Val Leu

210 215 220

Phe Gln Gly Pro Leu Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg

225 230 235 240

Pro His

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<211> 9

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Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

1 5

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Asp Tyr Lys Asp Asp Asp Asp Lys

1 5

<210> 8

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Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

1 5 10

<210> 9

<211> 462

<212> PRT

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<400> 9

Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys

1 5 10 15

Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr

20 25 30

Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe

35 40 45

Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala

50 55 60

His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile

65 70 75 80

Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp

85 90 95

Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu

100 105 110

Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys

115 120 125

Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly

130 135 140

Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro

145 150 155 160

Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys

165 170 175

Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly

180 185 190

Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp

195 200 205

Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala

210 215 220

Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys

225 230 235 240

Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser

245 250 255

Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro

260 265 270

Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp

275 280 285

Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala

290 295 300

Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala

305 310 315 320

Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln

325 330 335

Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala

340 345 350

Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn

355 360 365

Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Ile

370 375 380

Glu Gly Arg Ile Ser Glu Phe Gly Ser Ser Arg Val Asp Leu Gln Ala

385 390 395 400

Ser Leu Ala Leu Ala Val Val Leu Gln Arg Arg Asp Trp Glu Asn Pro

405 410 415

Gly Val Thr Gln Leu Asn Arg Leu Ala Ala His Pro Pro Phe Ala Ser

420 425 430

Trp Arg Asn Ser Glu Glu Ala Arg Thr Asp Arg Pro Ser Gln Gln Leu

435 440 445

Arg Ser Leu Asn Gly Glu Trp Gln Leu Gly Cys Phe Gly Gly

450 455 460

<210> 10

<211> 239

<212> PRT

<213> Artificial sequence

<400> 10

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 Ser Gly

20 25 30

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

35 40 45

Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr

50 55 60

Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys

65 70 75 80

Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu

85 90 95

Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu

100 105 110

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

115 120 125

Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr

130 135 140

Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn

145 150 155 160

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

165 170 175

Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly

180 185 190

Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu

195 200 205

Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe

210 215 220

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

225 230 235

<210> 11

<211> 27

<212> DNA

<213> Artificial sequence

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atggtgatcc ctgactactt caagcag 27

<210> 12

<211> 25

<212> DNA

<213> Artificial sequence

<400> 12

ttaccgtggg tacttggtga acacg 25

<210> 13

<211> 729

<212> DNA

<213> Artificial sequence

<400> 13

atggttatcc cggactactt caaacagtct ttcccggaag gttactcttg gggtcgttct 60

atgacctacg aagacggtgg tatctgcatc gctaccaacg acatcaccat ggaaggtgac 120

tctttcatca acaaaatcca cttcgaaggt acccacttcc cgccgaacgg tccggttatg 180

cagaaacgta ccgttggttg ggaagcttct accgaagaaa tgtacggtcg tgacggtgaa 240

ctgaaaggtt acgttaaaat gaaactgcag ctgaaaggtg gtggtcacta ccgttgcgac 300

taccgtacca cctacaaagt taaacagaaa ccggttaaac tgccggacta ccactgcgtt 360

gaccaccgta tcgaaatcct gtctcacgac aaagactaca accgtgttaa actgtacgaa 420

cacgctgttg ctcgtaactc taccgacggt atggacgaac tgtacaaagg tggttctggt 480

ggtgttgttt ctaaaggtga agaaaccatc acctctgtta tcaaaccgga catgcgtaac 540

aaactgcgta tggaaggtaa cgttaacggt cacgctttcg ttatcgaagg tgaaggttct 600

ggtaaaccgt tcgaaggtat ccagaccatc gacctggaag ttaaagaagg tgctccgctg 660

ccgttcgctt acgacatcct gaccaccgct ttccactacg gtaaccgtgt tttcaccaaa 720

tacccgcgt 729

<210> 14

<211> 729

<212> DNA

<213> Artificial sequence

<400> 14

atggttatcc cggactactt caaacagtct ttcccggaag gttactcttg gggtcgttct 60

atgacctacg aagacggtgg tatctgcatc gctaccaacg acatcaccat ggaaggtgac 120

tctttcatca acaaaatcca cttcgaaggt acccacttcc cgccgaacgg tccggttatg 180

cagaaacgta ccgttggttg ggaagcttct accgaagaaa tgtacggtcg tgacggtgaa 240

ctgaaaggtt acgttaaaat gaaactgcag ctgaaaggtg gtggtcacta ccgttgcgac 300

taccgtacca cctacaaagt taaacagaaa ccggttaaac tgccggacta ccactgcgtt 360

gaccaccgta tcgaaatcct gtctcacgac aaagactaca accgtgttaa actgtacgaa 420

cacgctgttg ctcgtaactc taccgacggt atggacgaac tgtacaaagg tggttctggt 480

ggtgttgttt ctaaaggtga agaaaccatc acctctgtta tcaaaccgga catgcgtaac 540

aaactgcgta tggaaggtaa cgttaacggt cacgctttcg ttatcgaagg tgaaggttct 600

ggtatgccgt tcgaaggtat ccagaccatc gacctggaag ttaaagaagg tgctccgctg 660

ccgttcgctt acgacatcct gaccaccgct ttccactacg gtaaccgtgt tttcaccaaa 720

tacccgcgt 729

Claims

1. A photo-cleavable protein mutant, wherein the mutant is a mutant of photo-cleavable protein phoCl, wherein the amino acid sequence of the photo-cleavable protein phoCl is shown as SEQ ID NO:1, and the mutant has mutation in at least one of the amino acid residues from 8 th to 232 th of the photo-cleavable protein phoCl.

2. The mutant photo-cleavable protein of claim 1, wherein said mutant photo-cleavable protein has a mutation at least one of amino acid residues 8, 9, 10, 18, 20, 28, 34, 43, 49, 52, 64, 73, 76, 80, 82, 84, 86, 90, 92, 98, 100, 108, 112, 113, 119, 125, 128, 135, 147, 148, 150, 151, 162, 165, 168, 179, 202, 207, 215, 232 of photo-cleavable protein phoCl.

3. The mutant photo-cleavable protein according to claim 1 or 2, wherein the mutant photo-cleavable protein has 10-15 mutation sites in amino acid residues 8-232 of the photo-cleavable protein phoCl.

4. The mutant photo-cleavable protein according to claim 3, wherein the mutant photo-cleavable protein comprises a protein with an amino acid sequence shown in SEQ ID NO 2 or SEQ ID NO 3.

5. The photo-cleavable protein mutant according to claim 1, wherein the photo-cleavable protein mutant further comprises an amino acid tag for purification.

6. Use of a photocleavable protein mutant according to any one of claims 1-5 for protein purification.

7. A fusion protein comprising a target protein and a photocleavable protein mutant according to any one of claims 1-5.

8. The fusion protein of claim 7, wherein the molecular weight of the target protein is 5-50 kDa.

9. A protein purification method comprising fusion-expressing a fusion protein comprising a target protein and a photocleavable protein mutant, purifying the fusion protein, and photocleaving the purified fusion protein to obtain a purified target protein, wherein the fusion protein is the fusion protein according to claim 7 or 8.

10. The method of claim 9, wherein the photocleavage conditions comprise a wavelength of 365 and 405nm for a period of 20-90 min.