CN109652397B - Recombinant acetylated lysine arginine N-terminal protease and preparation method and application thereof - Google Patents

Recombinant acetylated lysine arginine N-terminal protease and preparation method and application thereof Download PDF

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CN109652397B
CN109652397B CN201710942714.8A CN201710942714A CN109652397B CN 109652397 B CN109652397 B CN 109652397B CN 201710942714 A CN201710942714 A CN 201710942714A CN 109652397 B CN109652397 B CN 109652397B
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lysine arginine
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徐平
王富强
张俊令
赵明治
常蕾
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Abstract

The invention provides a recombinant acetylated lysine arginine N-terminal protease, a preparation method thereof and application thereof in proteomics research. The recombinant acetylated lysine arginine N-terminal protease reduces self-digestion characteristics, improves self stability and has higher enzyme activity. The recombinant acetylated lysine arginine N-terminal protease can be used independently or in combination with other proteases, improves the coverage of protein identification sequences and the coverage of trypsin digestion on the sequence of the amino acid residues readable by the mirrored peptide fragments and the identified peptide fragments, and is a potential proteomic tool enzyme.

Description

Recombinant acetylated lysine arginine N-terminal protease and preparation method and application thereof
Technical Field
The invention belongs to the field of proteomics, and particularly relates to protease used in proteomics research.
The invention also relates to a preparation method of the protease and application of the protease in proteomics research.
Background
Proteomics is the sum of all proteins in a cell, tissue or organ and is the final result of gene transcription, translation and post-translational modifications. Studying the abundance, function, interaction, localization and regulation of proteins in cells is crucial to our understanding of the mysteries of life.
In the most common proteomics research based on mass spectrum at present, the identification of protein and post-translational modification requires that a protein sample is firstly enzymolyzed into a peptide fragment, a primary spectrogram is obtained through mass spectrum detection, then fragment ions are generated through collision of peptide fragment ions and inert gas in a mass spectrum trap, and a secondary spectrogram is obtained through mass spectrum detection. Then matching the experimental spectrogram with a theoretical spectrogram deduced based on an existing protein and genome database (called searching a library) to deduce the amino acid sequence of the peptide fragment to be detected. And utilizing the identified peptide fragment to reversely deduce the protein to be identified. This method is also known as shotgun (shotgun). The step of proteolysis into peptide fragments is the core step of proteomics research at present.
Trypsin is widely used in practice as the most commonly used protease in proteomics studies. On the one hand, the enzyme cutting efficiency and the specificity are high, and enzyme cutting hydrolysis can be carried out on the carboxyl terminals of lysine and arginine of a protein substrate specifically (but when the amino acid behind the lysine and the arginine is proline, the hydrolysis cannot happen). On the other hand, the short peptide ending in arginine or lysine formed by enzyme digestion is more suitable for ionization and fragmentation of peptide fragments to form stronger y and weaker b series ion pairs, and is suitable for separation and identification. However, since many peptide fragments formed by trypsin digestion cannot be identified by mass spectrometry, the overall coverage is low [ Swaney DL, Wenger CD & Coon JJ Value of using multiple proteins for large-scale mass spectrometry. J. protein Res 2010,9, 1323-; on the other hand, trypsin is easy to form missed cuts on enzyme cutting sites with posttranslational modifications (such as methylation, phosphorylation, ubiquitination and the like), so that the length of a peptide segment is increased, the ionization efficiency is reduced, the identification of the modification sites is influenced, and the functional analysis of important proteins is not facilitated.
The discovery, introduction and multi-enzyme combination of the novel protease can obviously improve the coverage of the proteome sequence and improve the identification efficiency of the post-translational modification site. For example, a more complete digestion of peptide fragments can be achieved by cascade digestion with lysyl endonuclease (Lys-C) capable of specifically hydrolyzing the carboxy-terminal lysine peptide bond of proteins and trypsin [ Sanders WS, Brudges SM, McCarthy FM, et al.preparation of peptides by mass spectrometry applied at the experimental set level. BMC biologicals, 2007,8Suppl 7: S23], greatly improving the efficiency of specific digestion. Glutamyl endonuclease Glu-C can be well applied to identification of phosphorylated peptides [ Seeley EH, Riggs LD, Regener FE. reduction of non-specific binding in Ga (III) immobilized metal affinity chromatography for peptides by using endogenous enzymes Glu-C as the differential enzyme J chromatography B,2005,817(1): 81-88 ], and in combination with trypsin, can effectively improve the sequence coverage of protein identification and increase the reliability of mass spectrometry detection [ precursor S, Tepp W, DatsGuta BR. Botuling of nucleic acid type B and E: purpose, limited nucleic acid enzyme and C and T-D of nucleic acid of reaction, and D-D.
The discovery of lysine arginine N-terminal protease (Lysagina) with complementary cleavage site and trypsin [ Huesgen PF, Lange PF, Rogers LD, et al, LysargNase mirrors trypsin for protein C-terminal and methylation-site identification. Nat Methods,2015,12(1): 55-58 ] provides a new powerful tool for proteomic research. LysagiNase specifically cleaves the N-terminus of lysine and arginine residues in proteins, mirroring the cleavage site of trypsin, and is called "mirror enzyme". The two enzymes are used together, so that a large number of peptide fragments with overlapped sequences can be identified, and as basic amino acid (K or R) in the peptide fragments cut by the LysagiNase is positioned at the N end, ions are easily distributed on the fragments of the series of molecular ions at the N end, the formed spectrogram has a b series ion peak which is stronger than that of the y series ions, and the b series ion peak is complementary with the stronger characteristic of the y series ion peak obtained by digesting the peptide fragments by trypsin. The method can not only complement the fragment ions which are possibly lost in the ion ions and improve the integrity of the fragment ions, but also ensure that the ion series has certain directionality, is convenient to identify and read, and can more effectively judge the accuracy of the identification sequence. The characteristics are fully utilized to develop a protein de novo sequencing technology (de novo sequencing) based on mass spectrum, and finally get rid of the dependence of shotgun proteomics on an annotated genome database, and the method has wide application prospects in the fields of unknown protein, posttranslational modification, identification of mutation-induced amino acid mutation sites and the like.
The characteristics of the protease determine the self-digestion characteristics of the protease, so that the instability of the protease is increased, the activity of the protease is reduced, and meanwhile, the formed enzymolysis peptide fragment can also increase the noise in subsequent mass spectrometry. Methylation and acetylation of lysine residues have been found to significantly reduce trypsin self-digestion and increase trypsin stability (Sri Ram J, Terminallo L, Bier M, et al. on the mechanism of enzyme action. LVIII. Acetyl-trypsin, a stable trypsin derivative. Arch Biochem Biophys,1954,52: 464. 477; Rice RH, Means GE & Brown WD.stabilization of trypsin by reaction Acta,1977,492: 316. SP. 321; Heat M, Wu F.,. Xu P., expression of protease produced by reaction 120. mu. Protein, 2015,116. mu. Protein). However, no modification has been made to increase the stability of the lysine arginine N-terminal protease.
Disclosure of Invention
It is an object of the present invention to provide an acetylated recombinant lysine arginine N-terminal protease with higher stability and enzyme activity than the same enzyme that has not been acetylated.
Another objective of the invention is to provide a method for preparing an acetylated recombinant lysine arginine N-terminal protease.
Still another object of the present invention is to provide the use of an acetylated recombinant lysine arginine N-terminal protease in proteomic research.
According to one aspect of the invention, a recombinant acetylated lysine arginine N-terminal protease comprises a polypeptide having an amino acid sequence shown as SEQ ID No.1, wherein a lysine residue side chain of the polypeptide is modified by 1-5 acetyl groups, and the acetylated lysine arginine N-terminal protease has a molecular weight selected from the group consisting of: about 29048.121Da, about 29090.004Da, about 29132.211Da, about 29173.834Da, and about 29216.844 Da.
Lysine arginine N-terminal protease is the mirror image enzyme of trypsin, and can cleave the N-terminus of lysine and arginine residues of proteins; but also cleave its own lysine and arginine residues, which is responsible for its instability and low enzymatic activity.
Modification of the lysine as well as arginine of proteases is one direction of research to prevent self-digestion. Methylation modification (Rice et al, 1977) and acetylation modification (Sri Ram et al, 1954; Zhao et al, 2015) of trypsin effectively reduce its autodigestion, increasing enzyme stability without affecting its activity. However, lysine arginine N-terminal protease still cleaves efficiently at the methylation-modified lysine and arginine sites (Huesgen et al, 2015). Experiments show that acetylation can effectively prevent the self digestion of the N-terminal protease of lysine arginine and increase the stability of the N-terminal protease. Acetylation modification occurs on the free epsilon-amino group of protein lysine residues, and the enzyme activity and stability can be improved by acetylation modification of 1, 2, 3, 4,5, 6, 7 and/or 8 lysine residues of lysine arginine N-terminal protease, preferably acetylation modification of 1-5 amino acid residues.
The commercial recombinant N-terminal protease of lysine arginine can be used for acetylation modification, but experiments show that the recombinant N-terminal protease of lysine arginine obtained by the expression vector and the purification method constructed by the invention has better enzyme activity and stability.
The invention adopts molecular weight analysis of high resolution mass spectrum to represent acetylation of the enzyme, and mass spectrum analysis data shows that acetylation modification can occur on 1-5 lysine residues of the N-terminal protease of lysine arginine.
According to another aspect of the invention, the system of the invention establishes a method for preparing an acetylated recombinant lysine arginine N-terminal protease, comprising:
1) operably linking a gene expressing the lysine arginine N-terminal protease to an expression vector; transferring the expression vector into escherichia coli to obtain an escherichia coli strain for expressing recombinant lysine arginine N-terminal protease;
2) culturing the strain of step 1) and collecting the thalli;
3) crushing the thallus obtained in the step 2) to obtain recombinant lysine arginine N-terminal protease and purifying;
4) activating the recombinant lysine arginine N-terminal protease obtained in the step 3);
5) repurifying the recombinant lysine arginine N-terminal protease activated in the step 4);
6) performing acetylation modification on the recombinant lysine arginine N-terminal protease re-purified in the step 5) to obtain the recombinant acetylated lysine arginine N-terminal protease.
According to a further aspect of the present invention, it relates to the use of the recombinant acetylated lysine arginine N-terminal protease of the invention in proteomic studies. Comprises the steps of singly using acetylated recombinant lysine arginine N-terminal protease to cleave the lysine arginine N-terminal of a target protein, jointly using trypsin for cleaving the lysine arginine C-terminal of the target protein, and jointly using other proteases such as lysyl endonuclease.
Has the advantages that: the purified recombinant lysine arginine N-terminal protease prepared by the invention has high enzyme activity and stability; can provide suitable tool enzyme for proteomics research.
Drawings
FIG. 1 shows the N-terminal protease gene coding sequence and the corresponding amino acid sequence of lysine arginine of Methanosarcina acetophaga;
the grey shaded portion of the figure is the portion that is excised upon activation of the enzyme, and the introduced 6 × His tag and the enterokinase cleavage site are marked with solid and dashed lines, respectively.
FIG. 2 shows recombinant lysine arginine N-terminal protease zymogen His affinity chromatography and electrophoresis detection;
a, affinity chromatography chromatogram, wherein the left ordinate is ultraviolet absorbance at 280nm, the right ordinate is buffer solution B linear gradient (%), and the abscissa is volume (mL);
and b, SDS-PAGE detection, wherein a lane M is a protein molecular weight standard with the molecular weight of 10-220kD, and lanes super, flow through, wash and elution are respectively thalli lysis supernatant, sample flow-through, washing and elution samples.
FIG. 3 is a SDS-PAGE electrophoresis of recombinant lysine arginine N-terminal pro-protease activation;
lane M is a protein molecular weight standard with a molecular weight of 10-220kD, lane Nie is recombinant lysine arginine N-terminal pro-protease after His affinity chromatography, lane G25 is desalted recombinant lysine arginine N-terminal pro-protease, lanes 0h, 4h, 8h and O/N are recombinant lysine arginine N-terminal protease samples activated for 0h, 4h, 8h and 16 h;
FIG. 4 shows the anion exchange column chromatography and electrophoresis detection of the recombinant lysine arginine N-terminal protease;
a: anion exchange chromatography;
the left ordinate is the ultraviolet absorbance at 280nm, the right ordinate is the conductivity, and the abscissa is the volume (mL);
b: SDS-PAGE electrophoresis detection;
lane M is a protein molecular weight standard with a molecular weight of 10-220kD, and lanes 1-5 are elution peak samples collected at different times, respectively;
FIG. 5 gel filtration chromatography and electrophoresis detection of recombinant lysine arginine N-terminal protease;
a: chromatogram, with the ordinate being the ultraviolet absorbance at 280nm and the abscissa being the volume (mL);
b: SDS-PAGE detection, lane M is a protein molecular weight standard with a molecular weight of 10-220kD, lane R29 is purified recombinant lysine arginine N-terminal protease;
FIG. 6 SDS-PAGE electrophoresis profiles of recombinant lysine arginine N-terminal protease and recombinant acetylated lysine arginine N-terminal protease;
lane M is a protein molecular weight standard with a molecular weight of 10-220kD, Lane r-LysagiNase is a recombinant lysine arginine N-terminal protease, Lane r-Ac-LysagiNase is a recombinant acetylated lysine arginine N-terminal protease;
FIG. 7 Mass spectrometric detection of recombinant lysine arginine N-terminal protease and acetylated forms thereof;
absolute intensity: absolute signal strength;
a: recombinant lysine arginine N-terminal protease;
b: recombinant acetylated lysine arginine N-terminal protease;
FIG. 8 comparison of the activity of the product enzyme of the present invention and the commercial product enzyme;
r-lysargrnase: recombinant lysine arginine N-terminal protease, r-Ac-lysargina ase: recombinant acetylated lysine arginine N-terminal protease, Comm: a commercial recombinant lysine arginine N-terminal protease; TCL is total protein of yeast cells, 1:50, 1:200, 1:800 and 1:1600 represent the ratio (w: w) of enzyme amount to TCL substrate amount; lane M is a protein molecular weight standard with a molecular weight of 10-220 kD;
FIG. 9. enzyme stability assay for the products of the invention;
r-lysargrnase: recombinant lysine arginine N-terminal protease, r-Ac-lysargina ase: recombinant acetylated lysine arginine N-terminal protease;
a: SDS-PAGE electrophoresis detection;
b: separating and identifying self-digestion characteristic peptide fragment 'RSDEVDDTPNQADPN';
c: quantitative comparison of signal intensity of self-digested peptide fragment "RSDEVDDTPNQADPN";
d: separating and identifying self-digestion characteristic peptide fragment 'KIPVVVH';
e: quantitative comparison of signal intensity of self-digested peptide fragment "KIPVVVH".
FIG. 10. mirror image of the peptide fragment based de novo sequencing technique provides complete fragment ions, identifying the direction of the majority of the ions;
FIG. 11. ion coverage distribution of Trypsin spectrum (Trypsin), acetylated lysine arginine N-terminal protease (Ac-lysargiNase) spectrum and two-way enzyme digestion Mirror image spectrum (Mirror);
FIG. 12 comparison of the proportion of correctly resolved spectra of amino acid sequences obtained by the double restriction mapping strategy (pNovoM) with two trypsin-based single-cleavage methods (pNovo + and PEAKS);
the histogram on the left characterizes the number of mirror spectra each mirror peptide fragment contains.
Detailed Description
The present invention is described in detail below with reference to examples, but the present invention is not limited to the claims. The materials used in the present invention are publicly available.
Example 1 construction of recombinant lysine arginine N-terminal protease E.coli expression vector
The gene sequence of the recombinant lysine arginine N-terminal protease is derived from Methanosarcina acetovorans (GenBank No. AE010299), and is shown in SEQ ID No. 2. We introduced a 6 XHis tag and an enterokinase cleavage site DDDDDDK (15 amino acids added in total) at their N-terminus to obtain an Open Reading Frame (ORF) encoding 357 amino acids, i.e. the sequence shown in SEQ ID NO.3, and the corresponding protein sequence shown in SEQ ID NO.4, the gene coding sequence and the corresponding protein sequence also being shown in FIG. 1. The sequence shown in SEQ ID NO.3 was inserted between Nco I/Xho I cleavage sites of pET28a vector (Novagen, cat # 69864-3CN) to obtain recombinant expression vector of recombinant lysine arginine N-terminal protease, named pET-LysargiNase.
pET-LysargNase was introduced into competent cells of Escherichia coli BL21(DE3) by chemical transformation to obtain a recombinant strain containing a recombinant lysine arginine N-terminal protease gene, and this strain was named recombinant Escherichia coli BL21(DE3) -LysargNase (hereinafter abbreviated as BL 21-LysargNase strain).
Example 2 fermentation and Collection of cells of the [ BL21-LysargiNase Strain ]
The recombinant strain BL 21-LysargNase strain was activated on a solid LB/Amp plate to obtain a monoclonal of BL 21-LysargNase strain. A single clone of BL 21-LysargNase strain was inoculated into 50mL of LB liquid medium (ampicillin concentration 100. mu.g/mL) and shake-cultured at 37 ℃. OD of LB liquid culture System600Seed liquid of BL 21-LysargNase strain was obtained at 1.5-2.0 (LB liquid medium as a blank).
The seed solution of BL 21-LysargNase strain was inoculated alone into a 14L fermentor (BioFlo 310, New Brun) containing 5L of a high-density fermentation basal medium autoclaved at 121 ℃ by a wet-heat methodswick Scientific Co.), OD of the culture system after inoculation6000.01 (blank with high-density fermentation basal medium). Initial fermentation parameters: the culture temperature was 37 ℃, the stirring speed was 400rpm, the air flow was 6L/min, and the pH was 7.0. The pH of the fermentation medium was controlled by 30% ammonia (volume percent concentration). When Dissolved Oxygen (DO) (relative dissolved oxygen)<When the concentration is 15%, the rotation speed is linked, the maximum rotation speed is 1200rpm, and the dissolved oxygen content is kept to be 15% (relative dissolved oxygen content). When OD of the initial fermentation System600>20 (blank in high-density fermentation minimal medium), BL21-LysargiNase strain fermentation broth 1 was obtained.
BL 21-LysargNase strain fermentation broth 1 was fed at a rate of 16-18 mL/h. OD of fermentation system when supplemented600At 45 (blank with high-density fermentation basal medium with feed), the temperature was lowered to 32 ℃ and IPTG was added to a final concentration of 1mM for 10h of induction culture. The induction culture parameters are as follows: the culture temperature was 32 ℃, the stirring speed was 400rpm, the air flow was 6L/min, the pH was 7.0, and the pH of the fermentation medium was controlled by 30% ammonia water (volume percentage concentration). The dissolved oxygen amount and the rotation speed were controlled as above to obtain fermentation broth 2 of BL21-LysargiNase strain.
And centrifuging BL 21-LysargNase strain fermentation liquor 2 for 30min at 4000rpm by adopting a horizontal rotor to collect thalli, thereby obtaining BL 21-LysargNase thalli.
The medium and solution components were as follows:
high-density fermentation basal medium: 26.7g of glycerol, 40.0g of yeast powder, 1.8g of monopotassium phosphate, 1.8g of citric acid, 5mL of salt solution, 1mL of trace metal salt solution and 1mL of defoaming agent 204, distilled water is added to make up the volume of the solution to 1L, and the pH value is 7.2.
The salt solution consists of water and solutes, the solutes and their concentrations being: 250g/L MgCl2·6H2O、100g/L CaCl2·2H2O, 100g/L KCl and 2M Citric acid (Citric acid).
The trace metal salt solution: 6.8g ZnCl2、54.0g FeCl3·6H2O、16.2g MnCl2·4H2O、2.2g CuSO4·5H2O、4.8g CoCl2·6H2O、0.024g(NH4)6Mo7O24·4H2O, 0.2g KI, 119mL of 37% (vol.% concentration) concentrated hydrochloric acid, and distilled water to make up the volume of 1L.
The feed consists of water and solutes at concentrations: 275g/L of glycerol and 225g/L of yeast powder.
Example 3 [ purification of recombinant lysine arginine N-terminal protease ]
200g of BL21-LysargiNase cells obtained in example 2 were resuspended in 20mM Tris pH 7.5, 150mM NaCl buffer at a ratio of 1:10(w/v), homogenized with a high-speed tissue cutter, and disrupted by high-pressure homogenization (APV-1000 homogenizer, SPX FLOW, Inc.). The high pressure homogenization pressure was set at 800bar, 13000g after 3 successive disruptions, centrifugation at 4 ℃ for 30min, and the supernatant collected was subjected to metal chelate affinity chromatography on AKTA Purifier (GE healthcare) (HisTrapFF 5mL pre-column, GE healthcare, cat # 17-5255-01).
The column was pre-equilibrated with buffer A (20mM Tris,150mM NaCl, pH 7.5) and sample loaded at a flow rate of 5 mL/min. After the loading was completed, the hetero-protein was eluted with 10 column volumes of buffer A and 10 column volumes of 5% buffer B (20mM Tris,150mM NaCl,0.5M imidazole, pH 7.5), followed by linear gradient elution with 15 column volumes of 5-80% buffer B. All purification work was carried out at 20-25 ℃. The purified recombinant lysine arginine N-terminal protease solution was stored at 4 ℃. The results of the chromatographic and electrophoretic measurements are shown in FIG. 2.
Example 4 [ activation to recombinant lysine arginine N-terminal protease ]
The purified recombinant lysine arginine N-terminal protease solution obtained in example 3 was desalted by G-25 (HiPrep desalting column, GE healthcare, cat # 17508701, buffer 20mM Tris, pH 7.5), then concentrated by ultrafiltration through a 10kD ultrafiltration tube (Amicon Ultra-15mL,10kDa Centrifugal Filter Unit) to obtain a recombinant lysine arginine N-terminal protease solution with a protein concentration of 0.5-1mg CaCl/mL, and then added with CaCl2To 10mM, activated at 30 ℃ for 0h, 4h, 8h and 16h, and sampled respectively for SDS-PAGE electrophoresis detection. The results are shown in FIG. 3, activation resultsThe recombinant lysine arginine N-terminal protease of (3) has a molecular weight of 29.0 kD.
Example 5 [ purification of recombinant lysine arginine N-terminal protease ]
The activated recombinant lysine arginine N-terminal protease solution obtained in example 4 was purified by anion exchange chromatography (HiTrappQ HP 5mL pre-packed column, GE healthcare Cathe # 17-1154-01). The column was pre-equilibrated with equilibration buffer (20mM Tris, pH 7.5) and sample loaded. After the completion of the sample loading, the sample was eluted in a linear elution system using an elution buffer (20mM Tris,1M NaCl, pH 7.5) containing 1M sodium chloride, wherein the elution gradient was set to a linear gradient of 25mM to 500mM sodium chloride at a flow rate of 5mL/min for 20min (FIG. 4 a).
Elution peaks containing the target protein were pooled based on SDS-PAGE (FIG. 4b), and concentrated by ultrafiltration through a 10kD ultrafiltration tube (Amicon Ultra-15mL,10kD Centrifugal Filter Unit) to give a recombinant lysine arginine N-terminal protease solution at a concentration of 5-10 mg/mL. Further, purification was carried out by gel filtration chromatography (HiLoad Superdex 75PG, GE healthcare, cat. No. 28989334). The eluate was chromatographed by gel filtration (20mM Tris,150mM NaCl,10mM CaCl)2pH 7.5) was used. The separation range of the chromatography medium used for gel filtration chromatography covers 3-70 kD. And collecting the elution peak of the activated recombinant lysine arginine N-terminal protease to obtain a pure recombinant lysine arginine N-terminal protease (figure 5).
Example 6 [ acetylation of recombinant lysine arginine N-terminal protease ]
Adding acetic anhydride with the concentration of 800mM (6mL/L reaction volume) into the recombinant lysine arginine N-terminal protease solution prepared in the example 5 to obtain a recombinant lysine arginine N-terminal protease acetylation reaction solution, and reacting at 20-25 ℃ for 30min to obtain the recombinant acetylated lysine arginine N-terminal protease. Wherein acetic anhydride (product of Beijing Hua Kaiyuan chemical Co., Ltd., catalog No. 10000318) is diluted with dioxane (1,4-dioxane, carbofuran scientific product, catalog No. 156496). In the reaction process, 0.5M sodium hydroxide is used for adjusting the pH value of the acetylation reaction liquid to be stable at 6.2-7.2.
The recombinant acetylated lysine arginine N-terminal protease solution was concentrated to 3mg/mL by ultrafiltration through a 10kD ultrafiltration tube (Amicon Ultra-15mL,10kDa Centrifugal Filter Unit).
Example 7 identification of recombinant lysine arginine N-terminal protease and recombinant acetylated lysine arginine N-terminal protease
The SDS-PAGE results of the recombinant lysine arginine N-terminal protease and the recombinant acetylated lysine arginine N-terminal protease are shown in FIG. 6. The molecular weight was found to be around 29kD, corresponding to the theoretical molecular weight of 29005.272.
Further, the prepared recombinant lysine arginine N-terminal protease and recombinant acetylated lysine arginine N-terminal protease are subjected to mass spectrum identification, and the mass spectrum conditions are as follows:
mass spectrometry: active PLus EMR;
liquid phase: RIGOL L-3000;
mobile phase: 20mM ammonium acetate;
flow rate: 0.3 mL/min;
resolution ratio: 35000.
as shown in FIG. 7a, the molecular weight of the recombinant lysine arginine N-terminal protease is 29006.022Da, which is consistent with its theoretical value of 29005.272 Da. As shown in FIG. 7b, the mass spectrum identification result of the N-terminal protease of the recombinant acetylated lysine arginine showed a total of 5 peaks, wherein the main peak was 29090.004Da and the peak with the largest molecular weight was 29216.844 Da. The molecular weight of the acetyl group is known to be 42Da, and the recombinant acetylated lysine arginine N-terminal protease obtained by us is supposed to be a mixture of 1-5 acetyl modified recombinant lysine arginine N-terminal proteases.
Example 8 [ assay of recombinant lysine arginine N-terminal protease and recombinant acetylated lysine arginine N-terminal protease Activities ]
The activities of the recombinant lysine arginine N-terminal protease and the recombinant acetylated lysine arginine N-terminal protease prepared in the foregoing examples were examined using yeast cell total protein (TCL).
Collecting 20 μ g of TCL, adding 1mM tricarboxymethylphosphonic acid (TCEP, Sigma, C4706), denaturing at 45 deg.C for 30min, and adding enzyme at a ratio of 1:50, 1:200, 1:800, and 1:1600Example, the reaction buffer (50mM HEPES,5mM CaCl) was added thereto and reacted at 45 ℃ for 4 hours21.5M Urea, pH 8.0). After the reaction is finished, the sample is subjected to SDS-PAGE electrophoresis detection. As shown in FIG. 8, the results showed that the digestion efficiency of TCL by the recombinant lysine arginine N-terminal protease and the recombinant acetylated lysine arginine N-terminal protease was equivalent when the substrate amount was 1:50 and 1:200, and that there was substantially no residual protein in the lanes, indicating that the substrate protein was completely degraded and the enzyme activity was high; in contrast, the commercial recombinant lysine arginine N-terminal protease (Structural Biology Unit of CSIC, Spain) has more significant substrate protein remained at the enzyme to substrate amount of 1:50, and more undegraded substrate protein remained at the enzyme to substrate amount of 1:200, indicating that the commercial recombinant lysine arginine N-terminal protease has lower activity. At enzyme to substrate amounts of 1:800 and 1:1600, the acetylated recombinant lysine arginine N-terminal protease lane fades out of residual protein, but the grayscale was significantly lower than the recombinant lysine arginine N-terminal protease digested sample, indicating that the activity of the acetylated recombinant lysine arginine N-terminal protease was significantly higher than the non-acetylated recombinant lysine arginine N-terminal protease.
Example 9 comparison of Mass Spectrometry identification of Yeast proteome samples Using recombinant lysine arginine N-terminal protease and recombinant acetylated lysine arginine N-terminal protease
Furthermore, we compared the activities of the recombinant lysine arginine N-terminal protease and the recombinant acetylated lysine arginine N-terminal protease prepared by the present application by using a liquid phase-mass spectrometry technology (LC-MS/MS) which is commonly used in the current proteomics research. A sample of total yeast cell protein (TCL) was first reductively alkylated with 10mM Dithiothreitol (DTT) and 30mM Iodoacetamide (IAA). The enzyme digestion buffer solution is 50mM HEPES, 0.05% RapiGest and 5mM CaCl2pH8.0, enzyme amount ratio 1:50 (enzyme: protein, w: w), and digestion at 37 ℃ overnight. The analysis was performed using an ultra-high pressure liquid chromatography system (Waters Corporation, Milford, MA, USA) with an LTQ Qrbitrap Velos mass spectrometer (Thermo Electron, San Jose, Calif., USA). Separating with home-made reversed phase chromatography column (75 μm × 15cm,3 μm,200A) for 60min, wherein the mobile phase A is 0.1% FA, 2% AcN, 98% H2O, flowPhase B is 0.1% FA, 98% AcN, 2% H2O; the flow rate was 300 nL/min. And (4) carrying out mass spectrum analysis on the eluted peptide fragments. The mass spectrum scanning range is 300-1600 m/z; the primary and secondary spectral accuracies were 30,000 and 7,500 (at 400m/z), respectively.
Although the electrophoresis result shows that the digestion efficiency of the recombinant acetylated lysine arginine N-terminal protease and the recombinant lysine arginine N-terminal protease on yeast TCL is equivalent when the enzyme-substrate amount is 1:50 (figure 8, example 8), LC-MS/MS identification finds that the recombinant acetylated lysine arginine N-terminal protease is obviously higher than the recombinant lysine arginine N-terminal protease in relevant indexes such as Peptide-Spectrum matching number (PSM), total number of identified Peptide segments, number of K/R specific Peptide segments, protein identification number and the like, thereby having obvious advantages in proteomics research. In addition, a self-digesting peptide fragment was identified in the data for the recombinant lysine arginine N-terminal protease but not in the data for the recombinant acetylated lysine arginine N-terminal protease (table 1). These results indicate that acetylation can significantly improve lysine arginine N-terminal protease stability and activity.
Table 1: comparison of protein Mass Spectrometry identification results
Figure BDA0001430985360000121
Example 10 [ identification of stability of recombinant acetylated lysine arginine N-terminal protease ]
To investigate the stabilizing effect of acetylation on the recombinant lysine arginine N-terminal protease, the recombinant lysine arginine N-terminal protease and the recombinant acetylated lysine arginine N-terminal protease obtained in the above examples were diluted to 0.5mg/mL, and the same amount of the enzyme (electrophoresis results are shown in FIG. 9 a) was added to a reaction buffer (50mM HEPES,5mM CaCl) respectively21.5M Urea, pH8.0) and reacted at 30 ℃ for 18 hours.
The digestion products were quantitatively assayed for self-digestion-characterized peptides of recombinant lysine arginine N-terminal protease and recombinant acetylated lysine arginine N-terminal protease using an ultra-high pressure liquid chromatography system (Waters Corporation, Milford, MA, USA) in combination with an LTQ Qrbitrap Velos mass spectrometer (Thermo Electron, San Jose, Calif., USA). The sample separation and mass spectrometry conditions were the same as in example 9.
The results showed that after 18 hours of autodigestion, the amount of autodigestion-characteristic peptide stretch "RSDEVDDTPNQADPN" (SEQ No.5) in the recombinant acetylated lysine arginine N-terminal protease was only about 5% of the recombinant lysine arginine N-terminal protease (fig. 9b, 9 c); the quantitative analysis of another self-digestion characteristic peptide fragment "KIPVVVH" (SEQ No.6) also showed that the peptide fragment of the recombinant acetylated lysine arginine N-terminal protease was only about 14% of the recombinant lysine arginine N-terminal protease (FIGS. 9d, 9 e). This result further demonstrates that the recombinant acetylated lysine arginine N-terminal protease is structurally stable, resistant to self-digestion under conventional optimal digestion temperature conditions, and may further enhance its own activity in degrading other protein substrates by its self-digestion resistance.
Example 11 [ establishment of a mirror image peptide fragment-based protein De novo sequencing technology ]
The term "mirror image peptide" as used in this study refers to two peptides (one from trypsin and one from acetylated lysine arginine N-terminal protease) whose sequences are respectively designated A1A2…Al[K/R/-]And [ K/R/-]A1A2…AlWherein A isiAnd can be any of 20 amino acid residues, "-" indicates no amino acid. For example, inGLEWVAR and KGLEWVAAndGLEWVAr andGLEWVAare considered to be mirror image peptide fragments. The mirror image spectrogram refers to a mass spectrum spectrogram corresponding to the mirror image peptide fragment.
Based on the enzyme digestion characteristics of trypsin and acetylated lysine arginine N-terminal protease, the y-series ion peak of a trypsin digestion mass spectrum is stronger, and the b-series ion peak of acetylated lysine arginine N-terminal protease digestion is stronger, so that most ion types can be confirmed; further, conventional trypsin-based cleavage spectra are often limited by the absence of ions, particularly N-terminal ions, as shown in fig. 10, due to the absence of y when trypsin is used alone6Ion, the sequence of two amino acids at the N-terminus isGL or LG are not determinable. But when the corresponding mirror image of the N-terminal protease cleavage of acetylated lysine arginine is introduced, b2The presence of the ion can demonstrate that the N-terminus is GL rather than LG.
By using the strategy of combining trypsin with acetylated lysine arginine N-terminal protease, a new algorithm software pNovoM (in the software copyright application) for mirror image spectrum search and amino acid sequence analysis is developed, and de novo sequencing is carried out on a CHO recombinant expression monoclonal antibody PXL 1.
The result of comparing the amino acid sequence obtained by pnofom analysis with the actual sequence of PXL1 shows that the proportion of the full coverage spectrum of ions can reach 96% by using the matching of the peptide fragment of the double digestion mirror image of trypsin and acetylated lysine arginine N-terminal protease, while the full coverage of ions can be realized by only 48% and 44% of the spectra of trypsin or acetylated lysine arginine N-terminal protease (fig. 11).
Further analysis shows that the proportion of the correct amino acid sequence analysis spectrogram reaches 87 percent (FIG. 12) based on the double-enzyme digestion mirror image spectrogram, and only 4 amino acid errors occur in 416 amino acid sequences obtained by analysis, and the error rate is about 1 percent. If trypsin is used for single digestion, other de novo sequencing software available today, pNovo + (Chi H., Chen H., He K.et al. pNovo +: de novel peptide sequencing HCD and ETD standard Mass spectrum Res.201312: 615) and PEAKS (Ma B., Zhang K., Hendrie C.et al. PEAKS: power functional software for peptide de novel sequencing by Mass spectrometry.Rapid communication Mass Spectrometry.2003,17: 2332347-625) are used, and the proportion of correct amino acid sequence spectra is lower than 60% (FIG. 12). These results indicate that a bi-directional cleavage of trypsin and acetylated lysine arginine N-terminal protease in combination with pNovoM software algorithm can achieve accurate de novo sequencing of a single purified protein.
Sequence listing
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Gly Glu Asp Ala Trp Pro Ala Asp Arg Tyr Leu Asn Ile Trp Val Cys
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His Val Leu Lys Ser Glu Ile Gly Gln Asp Ile Leu Gly Tyr Ala Gln
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atggcagaaa aatttgaaag tagagggata gaggaagcgt catctgaagt gccaactcaa 60
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gaacgcgatc aaattgaaaa tctggctctc aaatataaac aagggtttcg agcaatatct 180
cggatggaaa ttgtcaagat ccctgtggtt gttcatgtcg tatggaatga agaagaggag 240
aatatttctg acgctcagat ccagagccag atagatattc tcaataaaga cttccgcaag 300
ttgaattccg acgtgtcaca ggtgccttct gtctggagta atctcatagc agacctgggg 360
attgagtttt tcctcgcaac aaaagacccg aatggaaatc agactacagg aataactcgc 420
actcaaactt cagtgacctt tttcactacc agcgatgaag tgaaattcgc atccagtggg 480
ggtgaggatg catggccggc cgatcgttat ctgaatattt gggtatgtca tgtgcttaaa 540
agtgagatag gtcaagacat attgggttac gcgcaatttc caggtgggcc cgctgaaacc 600
gatggtgttg ttattgttga tgcagctttt ggtaccactg gaactgcctt accgccattt 660
gacaagggac gaacggcaac ccatgaaatc ggacactggt taaacctcta ccatatctgg 720
ggtgacgaat tgcgttttga ggatccatgt tcacgttcag atgaggttga tgatactcca 780
aatcaggcag atcctaattt tggctgtcca agttatccac atgtcagctg cagcaatgga 840
ccaaatggcg atatgttcat gaattacatg gattacgtag acgataaatg tatggttatg 900
ttcacacagg gccaggcaac tcgtgtaaat gcatgtcttg acggaccaag atcatcgttc 960
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atggaagttc accacaggct gctaaggtct gcgtcgtacg tcagagaacg cgatcaaatt 180
gaaaatctgg ctctcaaata taaacaaggg tttcgagcaa tatctcggat ggaaattgtc 240
aagatccctg tggttgttca tgtcgtatgg aatgaagaag aggagaatat ttctgacgct 300
cagatccaga gccagataga tattctcaat aaagacttcc gcaagttgaa ttccgacgtg 360
tcacaggtgc cttctgtctg gagtaatctc atagcagacc tggggattga gtttttcctc 420
gcaacaaaag acccgaatgg aaatcagact acaggaataa ctcgcactca aacttcagtg 480
acctttttca ctaccagcga tgaagtgaaa ttcgcatcca gtgggggtga ggatgcatgg 540
ccggccgatc gttatctgaa tatttgggta tgtcatgtgc ttaaaagtga gataggtcaa 600
gacatattgg gttacgcgca atttccaggt gggcccgctg aaaccgatgg tgttgttatt 660
gttgatgcag cttttggtac cactggaact gccttaccgc catttgacaa gggacgaacg 720
gcaacccatg aaatcggaca ctggttaaac ctctaccata tctggggtga cgaattgcgt 780
tttgaggatc catgttcacg ttcagatgag gttgatgata ctccaaatca ggcagatcct 840
aattttggct gtccaagtta tccacatgtc agctgcagca atggaccaaa tggcgatatg 900
ttcatgaatt acatggatta cgtagacgat aaatgtatgg ttatgttcac acagggccag 960
gcaactcgtg taaatgcatg tcttgacgga ccaagatcat cgttcctggc gagagtggaa 1020
gaaacagaaa agaaagaagc accatccaag cgtgaaatgc ctatgccaag gtaa 1074
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Leu Lys Tyr Lys Gln Gly Phe Arg Ala Ile Ser Arg Met Glu Ile Val
65 70 75 80
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Claims (9)

1. A recombinant acetylated lysine arginine N-terminal protease, wherein the lysine arginine N-terminal protease is a polypeptide with an amino acid sequence shown as SEQ ID No.1, the acetylated lysine arginine N-terminal protease is modified with 1-5 acetyl groups on a lysine residue side chain of the polypeptide, and the acetylated lysine arginine N-terminal protease has a molecular weight selected from the following under the condition of mass spectrum resolution 35000: about 29048.121Da, about 29090.004Da, about 29132.211Da, about 29173.834Da, and about 29216.844 Da;
modifying an acetyl group on a lysine residue side chain of the polypeptide by:
adding acetic anhydride into the activated recombinant lysine arginine N-terminal protease solution for acetylation, and reacting at 20-25 ℃ and pH6.2-7.2 to obtain the recombinant acetylated lysine arginine N-terminal protease.
2. The recombinant acetylated lysine arginine N-terminal protease of claim 1, wherein the recombinant acetylated lysine arginine N-terminal protease is prepared by the method of:
1) operably linking a gene expressing the lysine arginine N-terminal protease to an expression vector; transferring the expression vector into escherichia coli to obtain an escherichia coli strain for expressing recombinant lysine arginine N-terminal protease;
2) culturing the strain of step 1) and collecting the thalli;
3) crushing the thallus obtained in the step 2) to obtain recombinant lysine arginine N-terminal protease and purifying;
4) activating the recombinant lysine arginine N-terminal protease obtained in the step 3);
5) repurifying the recombinant lysine arginine N-terminal protease activated in the step 4);
6) performing acetylation modification on the recombinant lysine arginine N-terminal protease re-purified in the step 5) to obtain the recombinant acetylated lysine arginine N-terminal protease.
3. The recombinant acetylated lysine arginine N-terminal protease of claim 2, wherein the protease is prepared by a process comprising:
1a) introducing a 6 XHis tag and an enterokinase cleavage site DDDDK into the N end of a protease gene sequence expressing the N end of lysine arginine to obtain a recombinant gene sequence, inserting the recombinant gene sequence into a pET28a vector to obtain a recombinant expression vector of the protease gene expressing the N end of the lysine arginine, and transferring the expression vector into a competent cell of escherichia coli BL21(DE3) to obtain a recombinant strain containing a recombinant protease gene expressing the N end of the lysine arginine;
2a) fermenting and culturing the recombinant strain in the step 1a), and collecting;
3a) resuspending the thallus obtained in step 2a), crushing the thallus, centrifuging and collecting supernatant, performing metal chelate affinity chromatography, and purifying to obtain recombinant lysine arginine N-terminal protease;
4a) desalting the purified recombinant lysine arginine N-terminal protease of step 3a), concentrating by ultrafiltration, adding CaCl2Incubation and activation are carried out to obtain activated recombinant lysine arginine N-terminal protease;
5a) subjecting the activated recombinant lysine arginine N-terminal protease of step 4a) to anion exchange chromatography to obtain a purified recombinant lysine arginine N-terminal protease;
6a) adding acetic anhydride solution into the purified recombinant lysine arginine N-terminal protease obtained in the step 5a) for acetylation to obtain the recombinant acetylated lysine arginine N-terminal protease.
4. The recombinant acetylated lysine arginine N-terminal protease of claim 3, wherein the chromatography of step 3a) is performed by equilibrating the column with buffer A containing 20mM Tris,150mM NaCl, pH 7.5, then loading at a flow rate of 5mL/min, eluting the hetero-protein with buffer A and 5% buffer B after loading, and then eluting with a linear gradient of 5-80% buffer B containing 20mM Tris,150mM NaCl,0.5M imidazole, pH 7.5.
5. The recombinant acetylated lysine arginine N-terminal protease of claim 3, wherein the anion exchange chromatography of step 5a) is performed using a HiTrap Q HP 5mL pre-packed column, pre-equilibrated the column with an equilibration buffer, loaded, and eluted linearly with an elution buffer at an elution flow rate of 5mL/min for 20min, wherein the equilibration buffer comprises 20mM Tris, pH 7.5, the elution buffer comprises 20mM Tris,1M NaCl, pH 7.5, and the gradient of the linear elution is 25 mM-500 mM NaCl.
6. A method of making the recombinant acetylated lysine arginine N-terminal protease of claim 1 comprising:
1) operably linking a gene expressing the lysine arginine N-terminal protease to an expression vector; transferring the expression vector into escherichia coli to obtain an escherichia coli strain for expressing recombinant lysine arginine N-terminal protease;
2) culturing the strain of step 1) and collecting the thalli;
3) crushing the thallus obtained in the step 2) to obtain recombinant lysine arginine N-terminal protease and purifying;
4) activating the recombinant lysine arginine N-terminal protease obtained in the step 3);
5) repurifying the recombinant lysine arginine N-terminal protease activated in the step 4);
6) performing acetylation modification on the recombinant lysine arginine N-terminal protease re-purified in the step 5) to obtain the recombinant acetylated lysine arginine N-terminal protease.
7. The method of claim 6, comprising the steps of:
(1) introducing a 6 XHis tag and an enterokinase cleavage site DDDDK into the N end of an expressed lysine arginine N-terminal protease gene to obtain a recombinant gene sequence, and inserting the recombinant gene sequence into a pET28a vector to obtain a recombinant expression vector of the recombinant lysine arginine N-terminal protease; introducing the recombinant expression vector of the recombinant lysine arginine N-terminal protease into competent cells of escherichia coli BL21(DE3) to obtain a recombinant strain containing a recombinant lysine arginine N-terminal protease gene, namely BL21(DE3) -LysargNase;
(2) inoculating BL21(DE3) -LysargNase strain, and performing shake flask culture by using LB culture medium to obtain a seed solution;
inoculating the seed solution in a fermentation tank, performing fed-batch fermentation, performing IPTG induced expression to obtain fermentation liquor, centrifuging the fermentation liquor, and collecting BL21(DE3) -LysargNase thallus;
(3) resuspending the thallus obtained in the step (2), crushing the thallus, centrifuging and collecting supernatant, and performing metal chelating and affinity chromatography;
wherein, in the chromatography process, the chromatography column is balanced by buffer solution A in advance, then the sample is loaded, the flow rate of the sample loading is 5mL/min, the buffer solution A contains 20mM Tris,150mM NaCl and pH 7.5,
after the loading is finished, eluting the hybrid protein by using a buffer solution A and a 5% buffer solution B, and then eluting by using a linear gradient of the 5-80% buffer solution B, wherein the buffer solution B contains 20mM Tris,150mM NaCl,0.5M imidazole and pH 7.5;
(4) desalting, ultrafiltering and concentrating the purified recombinant lysine arginine N-terminal protease solution obtained in the step (3), and adding CaCl2Incubating and activating to 10mM to obtain a recombinant lysine arginine N-terminal protease solution;
(5) carrying out anion exchange chromatography on the activated recombinant lysine arginine N-terminal protease solution obtained in the step (4) to obtain a repurified recombinant lysine arginine N-terminal protease solution;
wherein, anion exchange chromatography adopts a HiTrap Q HP 5mL prepacked column, the chromatographic column is balanced by an equilibrium buffer solution in advance, the sample is loaded, the elution is carried out linearly by an elution buffer solution, the elution flow rate is 5mL/min, the elution time is 20min, the equilibrium buffer solution contains 20mM Tris and pH 7.5, the elution buffer solution contains 20mM Tris,1M NaCl and pH 7.5, and the gradient of linear elution is 25 mM-500 mM sodium chloride;
(6) adding acetic anhydride solution into the repurified recombinant lysine arginine N-terminal protease solution obtained in the step (5) for acetylation to obtain recombinant acetylated lysine arginine N-terminal protease solution, and finally performing ultrafiltration concentration;
the ratio of the purified recombinant lysine arginine N-terminal protease solution to the acetic anhydride solution is 6mL/L reaction volume, the concentration of the acetic anhydride solution is 800mM, the acetic anhydride solution is obtained by diluting with dioxane, the reaction temperature is 20-25 ℃, the reaction lasts for 30min, the pH of the reaction solution is controlled to be 6.2-7.2, and the obtained recombinant acetylated lysine arginine N-terminal protease solution is concentrated by ultrafiltration through an ultrafiltration tube.
8. Use of the recombinant acetylated lysine arginine N-terminal protease of claim 1 in proteomics research.
9. The use of claim 8, wherein the recombinant acetylated lysine arginine N-terminal protease is used in combination with trypsin.
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