A method for screening cleavage sites and its application

Abstract

The present invention provides a method for screening cleavage sites and its application. The method comprises the steps of: S1, database establishment: using computer language to write a program, predicting that after the intein is embedded in each of two adjacent amino acid residues in the amino acid sequence, a splicing reaction occurs to cut itself off, and the amino acid sequence formed after the adjacent peptide segments are connected constitutes a protein database; S2, experiment: inserting the intein sequence fragment into the gene fragment, molecular cloning is performed, and the peptide segment is obtained after translation, and the peptide segment is detected by mass spectrometry to see whether it contains a labeled amino acid sequence, and compared with the protein database to realize the confirmation of the cleavage site. The method of the present invention first constructs a protein database by computer programming, and then conducts experiments and realizes the detection and confirmation of the cleavage site by mass spectrometry; the final detection is realized by mass spectrometry, replacing high-throughput screening, and it is expanded to be able to search for cleavage sites for any active protein.

Description

Technical Field

[0001] The present invention relates to the field of protein cleavage site screening, and in particular to a method for screening cleavage sites and an application thereof. Background Art

[0002] Inteins are able to join flanking exoproteins (exonin) into a new protein fragment and cut themselves out, a process known as protein splicing. Inteins are found in many natural organisms, such as bacteria, fungi and lower plants, often embedded in important proteins. In nature, protein splicing produces two separate proteins (intein and exonin) and inteins precisely cut out the internal protein fragment (intein itself) and join the two flanking regions at the same time under the control of a gene. There are several forms of protein inteins in nature, including full-length inteins, mini-inteins and naturally occurring split inteins. Full-length inteins and mini-inteins are both cis-spliced ​​inteins, in which an endonuclease domain is present or absent. Split inteins are trans-spliced ​​inteins, containing two protein fragments and they are transcribed and translated from two independent genes. Trans-splicing requires the co-expression of two split intein fragments, namely N-protein (IN, fused to the C-terminus of N-exon) and C-protein (IC, fused to the N-terminus of C-exon). The split intein fragments then combine to restore their activity and catalyze the connection of N-exon and C-exon. In cis or trans splicing, the intein-mediated splicing chemistry does not require the help of any enzymes or cofactors, but only requires the expressed protein to have a correctly folded structure.

[0003] Existing scientific literature (“A systematic approach to inserting split inteins for Boolean logic gate engineering and basal activity reduction”, Baojun Wang et al., Nature Communications, 2021) records a method for protein split site screening, called Intein-assisted bisection mapping (IBM) split site screening, which is also based on the method of randomly inserting gene fragments by mini-Mu transposon mediated by phage infection mechanism, but it is limited to some fluorescent proteins or antibiotic resistance proteins that have successfully searched for split sites. These proteins themselves can excite fluorescence or have regulatory effects in inhibiting antibiotics and in transcription initiation elements, making them have the characteristics of being detected by high-throughput screening. However, most recombinant proteins, such as IL-2, IFN, EGF, bFGF and other high-value protein drugs, do not have characteristics that can be directly detected by high-throughput screening. Summary of the invention

[0004] In view of the above-mentioned problems existing in the prior art, one of the objects of the present invention is to provide a method for screening cleavage sites, firstly constructing a protein database through computer programming, then conducting experiments and realizing the detection and confirmation of cleavage sites through mass spectrometry; the final detection is realized by mass spectrometry, replacing high-throughput screening, and expanding it to search for cleavage sites of any active protein.

[0005] As a premise, the experimental principle of the present invention (i.e., the method of randomly inserting gene fragments by mini-Mutransposon mediated by phage infection mechanism) is the same as the above-mentioned scientific document "A systematic approach to inserting split inteins for Boolean logic gate engineering and basal activity reduction". If the description of the relevant action principle is not clear enough in the text of this application, this document can also be used as an explanation.

[0006] To achieve the above object, the first aspect of the present invention provides a method for screening cleavage sites, which comprises the steps of:

[0007] S1. Database establishment: Use computer language to write a program to predict that after the intein is embedded in each of the two adjacent amino acid residues in the amino acid sequence, a splicing reaction will occur to remove itself, and the amino acid sequence formed after the adjacent peptide segments are connected will constitute a protein database;

[0008] S2. Experimental process: insert an intein sequence into the gene fragment by molecular cloning experimental method, obtain a peptide segment after translation, detect the peptide segment by mass spectrometry to see if it contains a labeled amino acid sequence, and compare it with the protein database to confirm the break site.

[0009] In the present invention, the computer language may adopt any language that can implement programming functions, such as using Python to write scripts to complete the establishment of the database in step S1 of the present invention.

[0010] The principle of using mass spectrometry for detection in the present application is that since the restriction sites left by molecular cloning will also be translated into amino acids when expressed, whether a labeled amino acid sequence translated from the marker sequence is generated can be used as a sign of whether the splicing reaction occurs. By detecting whether the peptide segment obtained after translation contains the labeled amino acid sequence by mass spectrometry, it is possible to find the cleavage site of any active protein, rather than being limited to some fluorescent proteins or antibiotic resistance proteins that have successfully searched for the cleavage site.

[0011] In some embodiments of the present application, the database establishment in step S1 includes:

[0012] S11, sequentially merging gene fragment 1, inserting the intein sequence fragment and gene fragment 2 to obtain a new DNA sequence;

[0013] S12, translating the new DNA sequence into a new amino acid sequence;

[0014] S13, searching for a target intein amino acid sequence in the new amino acid sequence (the program sets the new amino acid sequence to contain the target intein amino acid sequence), deleting the target intein amino acid sequence, and obtaining an output amino acid sequence;

[0015] S14, predicting that the inserted intein sequence fragment is inserted into each possible site of gene fragment 1 and gene fragment 2, and then repeating steps S11 to S13, and all the obtained output amino acid sequences constitute the protein data database.

[0016] According to the present invention, in step S13, the computer is programmed to automatically delete the target intein amino acid sequence in the new amino acid sequence, thereby obtaining an output amino acid sequence.

[0017] Figure 1This is a schematic diagram of the process of establishing a database by computer programming in some embodiments of the present invention, which can be referred to Figure 1 , M86 in the figure is the protein intein Ssp DnaBM86, which is modified from the Ssp DnaB intein found in Synechocystis spp. PCC6803.

[0018] In some embodiments of the present application, in step S11, several bases are inserted into the new DNA sequence to prevent frameshift mutations during translation. After the phage transposition, the first several (such as 5) bases inserted at the 5' end are replicated. Therefore, in order to prevent frameshift mutations, in the inserted intein sequence fragment, several (such as 1) bases are inserted at the 3' end of the replicated 5 bases (the 5' end of the inserted intein sequence fragment) so that the sum of the number of bases replicated after the phage transposition and the number of bases actively inserted satisfies a multiple of 3. Figure 1 is a schematic diagram of insert fragments in some embodiments of the present invention, which can be referred to Figure 2 , the 5 bases from i-5 to i are the first 5 bases copied during phage transposition, and after inserting the above-mentioned intein gene fragments with restriction sites and several bases added at the 5' and 3' ends, add these 5 bases downstream, and translate the entire gene into an amino acid sequence and then delete the intein sequence to obtain one of the amino acid sequences in the protein database. The above steps are written into a computer program language (such as a python program) to insert inteins between every two adjacent amino acids in the target protein and deduce them to obtain the final constructed protein database (see the description of step S1).

[0019] According to the present invention, the transposition mechanism of bacteriophage Mu will replicate the 5 bases upstream of the insertion position once at the downstream of the insertion end, thereby integrating the restriction site and the marker sequence ultimately left by the transposition mechanism.

[0020] The second aspect of the present invention provides an application of the method described in the first aspect in screening for cleavage sites, especially for screening for cleavage sites of Escherichia coli antigen protein Im7-6 and / or Cas9 protein.

[0021] The third aspect of the present invention provides an application of the method described in the first aspect to screening recombinant proteins such as IL-2 protein, IFN protein, EGF protein and bFGF protein.

[0022] Since high-throughput screening is used in the above-mentioned scientific literature, it is limited to some fluorescent proteins or antibiotic resistance proteins that have successfully searched for cleavage sites. These proteins can stimulate fluorescence or have regulatory effects in inhibiting antibiotics and in transcription initiation elements, making them have the characteristics of being detected by high-throughput screening. The present invention realizes the final detection through mass spectrometry, which is extended to the search for cleavage sites of any active protein.

[0023] The beneficial effects of the present invention include at least one of the following:

[0024] 1) The screening method provided by the present invention is reasonably combined with computer programming to construct a protein database;

[0025] 2) The screening method provided by the present invention realizes the final detection through mass spectrometry, which innovatively expands the existing cleavage site screening scheme and can be expanded to search for cleavage sites of any active protein;

[0026] 3) After the screening method provided by the present invention confirms the break site, experiments can be designed to perform protein assembly, providing a new idea for subsequent glycosylation experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A schematic diagram showing a process of establishing a database by computer programming in some embodiments of the present invention is shown;

[0028] Figure 2 A schematic diagram showing an insert fragment in some embodiments of the present invention;

[0029] Figure 3 A schematic diagram showing the operation steps of randomly inserting gene fragments in Example 1 of the present invention is shown;

[0030] Figure 4 A schematic diagram showing the nucleotide sequence of the intein Ssp DnaBM86 in Example 1 of the present invention is shown;

[0031] Figure 5 A schematic diagram of the screening method of Example 1 of the present invention is shown;

[0032] Figure 6 The results of polypeptide cleavage sites obtained by mass spectrometry detection in Example 1 of the present invention are shown;

[0033] Figure 7 A schematic diagram showing the nucleotide sequence of the split intein Split Ssp DnaBM86 of Example 2 of the present invention;

[0034] Figure 8 The first result of the polypeptide cleavage site obtained by mass spectrometry detection in Example 2 of the present invention is shown;

[0035] Fig. 9 The second result of the polypeptide cleavage site obtained by mass spectrometry detection in Example 2 of the present invention is shown. DETAILED DESCRIPTION

[0036] To make the present invention easy to understand, the present invention will be described in detail below in conjunction with the accompanying drawings. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing specific embodiments and are not intended to be limiting.

[0037] Where a numerical range is provided, it is understood that each intervening value between the upper and lower limits of the range and any other specified or intervening values ​​in the specified range is encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed within the present invention, subject to any explicitly excluded limits in the specified ranges. Where a specified range includes one or two limits, ranges excluding either or both of those included limits are also encompassed within the present invention.

[0038] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, preferred methods and materials are now described.

[0039] The relevant experimental principles of the method of randomly inserting gene fragments by mini-Mu transposon mediated by phage infection mechanism adopted in the present invention can be found in the scientific literature ("A systematic approach to inserting split inteins for Boolean logic gate engineering and basal activity reduction", Baojun Wang et al., Nature Communications, 2021), and the full text of this document can be cited as a reference.

[0040] Example 1

[0041] Please refer to Figure 3 and Figure 4 In this example, the above screening method was used to verify the polypeptide cleavage site of the Escherichia coli antigen protein (Im7-6).

[0042] like Figure 3 As shown, Figure 3This is the same experimental principle as the above-mentioned scientific literature "A systematic approach to inserting split inteins for Boolean logic gate engineering and basal activity reduction", that is, the method of randomly inserting gene fragments by mini-Mu transposon mediated by the phage transposition mechanism. The specific operation is as follows:

[0043] 1. Use transposase MuA to perform transposition experiment on the target gene fragment, randomly insert the transposon into the target fragment, and its principle ensures that only one transposon fragment is inserted into each target gene fragment. The transposon fragment is a complete expression system with promoter, terminator and other elements and expressing chloramphenicol resistance protein;

[0044] 2. Connect the transposed gene fragment to the pET28a expression vector through seamless cloning method, transform the E. coli Top10 amplification vector, and screen the colonies using chloramphenicol resistance plates;

[0045] 3. Collect all the colonies in the above plates, mix and culture them, and extract their plasmids. Since the upstream and downstream ends of the transposon each have a NotI restriction site, an enzyme digestion experiment is performed to replace the transposon fragment with the expression intein SspDnaBM86 (corresponding to Figure 3 The cis-intein version in 3. Substitution was used, and then transformed into E. coli Top10 competent cells for vector amplification, and colony screening was performed using kanamycin antibiotic plates. All colonies were then collected and cultured, and plasmids were extracted and transformed into E. coli BL21DE3 expression strains. All colonies on the plate were collected again and inoculated into LB medium for protein expression. Finally, the mixed expressed protein was purified and concentrated to obtain a sample.

[0046] like Figure 4 Shown is a schematic representation of the nucleotide sequence of the intein Ssp DnaBM86 with restriction sites and extra bases added to prevent frameshift mutations. Figure 4 In the two boxes 1, GCGGCCGC is the nucleotide sequence of the restriction site, the C bases in the two boxes 2 are additional bases added to prevent frameshift mutations, and the XXXXX (X represents a base in ATCG) in box 3 are the 5 bases copied after the phage transposition. It should be noted that Figure 4 Only part of the nucleotide sequence is shown, especially the 5 duplicated bases in box 3 are non-fixed.

[0047] like Figure 5As shown, the present invention is significantly different from the above-mentioned scientific literature (the literature directly performs high-throughput screening on samples), and the specific operation is as follows:

[0048] 4. First, use Python to write a script to predict that after the intein is embedded in each of the two adjacent amino acid residues in the amino acid sequence, a splicing reaction will occur to remove itself, and the amino acid sequence formed by connecting the adjacent peptide segments constitutes a protein database;

[0049] 5. The sample obtained in step 3 is detected by mass spectrometry.

[0050] The mass spectrometry results are as follows Figure 6 As shown in the figure, it can be analyzed that for the Escherichia coli antigen protein (Im7-6), the amino acid sequence of AAALRPLY is a marker amino acid sequence translated from the marker sequence generated by the restriction site (the transposition mechanism of bacteriophage Mu will copy the 5 bases upstream of the insertion position once at the downstream of the insertion end, and the marker sequence finally left by the combined restriction site and the transposition mechanism is a sequence containing 8 amino acid residues: AAALRPXX, wherein XX is translated from the 5 copied bases and a base inserted to prevent frameshift mutation); then the protein database obtained in step 4 is searched, and when an ion fragment that fully covers or partially covers the marker sequence (partial coverage here means that at least two amino acids AL are searched to prove that the intein splicing reaction actually occurs) is found, it can be determined that a polypeptide breakage site of the Escherichia coli antigen protein (Im7-6) is Y61-Y62 (such as Figure 5 ).

[0051] Example 2

[0052] In this example, the above screening method was used to verify the two polypeptide breakage sites of the Cas9 protein.

[0053] Steps 1 to 5 are substantially the same as those in Example 1, except that in step 3, "subjecting an enzyme digestion experiment to replace the transposon fragment with the expression intein Ssp DnaBM86" is replaced by "subjecting an enzyme digestion experiment to replace the transposon fragment with the N-terminus, terminator and promoter transcription elements of the broken intein Ssp DnaBM86 and the gene fragment of the C-terminus of the broken intein Ssp DnaBM86 in sequence (corresponding to Figure 3 3. The split intein version method in Substitution)"

[0054] like Figure 7 Shown is a schematic diagram of the nucleotide sequence of the intein Ssp DnaBM86, with restriction sites and additional bases added to prevent frameshift mutations. Figure 4 As stated, Figure 7 In the two boxes 1, GCGGCCGC is the nucleotide sequence of the restriction site, the C bases in the two boxes 2 are additional bases added to prevent frameshift mutations, and the XXXXX (X represents a base in ATCG) in box 3 are the 5 bases copied after the phage transposition. Figure 7 Only part of the nucleotide sequence is shown, especially the 5 bases replicated in box 3 are non-fixed.

[0055] The mass spectrometry results are as follows Figure 8 and Fig. 9 As shown in the figure, it can be analyzed that for Cas9 protein, the amino acid sequences of AAALRPPD and AAALRPHV are respectively the marker amino acid sequences translated from the marker sequence generated by the restriction site (the transposition mechanism of bacteriophage Mu will copy the 5 bases upstream of the insertion position once at the downstream of the insertion end, and the marker sequence finally left by the combined restriction site and transposition mechanism is a sequence containing 8 amino acid residues: AAALRPXX, where XX is translated from the 5 copied bases and a base inserted to prevent frameshift mutation); then search the protein database obtained in step 4, and when the ion fragments that fully cover or partially cover the marker sequence (partial coverage here means that at least two amino acids AL are searched to prove that the intein splicing reaction actually occurs) are found, it can be determined that the two polypeptide breakage sites of Cas9 protein are D868-N869 ( Figure 6 ) and 181V-182D( Figure 7 ).

[0056] It can be seen from the above embodiments that the screening method provided by the present application is reasonably combined with computer programming to construct a protein database; and the final detection is achieved through mass spectrometry, which innovatively expands the existing cleavage site screening scheme and can be expanded to search for cleavage sites of any active protein; after confirming the break site, the next step is to design experiments for protein assembly, providing a new idea for subsequent glycosylation experiments.

[0057] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. A method for screening a cleavage site, comprising the steps of: S1. Database establishment: Use computer language to write a program to predict that after the intein is embedded in each of the two adjacent amino acid residues in the amino acid sequence, a splicing reaction will occur to remove itself, and the amino acid sequence formed after the adjacent peptide segments are connected will constitute a protein database; S2. Experimental process: inserting an intein sequence into a gene fragment by molecular cloning experimental method, obtaining a peptide segment after translation, detecting whether the peptide segment contains a labeled amino acid sequence by mass spectrometry, and comparing it with the protein database to confirm the break site; the labeled amino acid is an amino acid sequence translated from the base sequence of the restriction site left after molecular cloning, which can serve as a marker of whether a splicing reaction occurs; The database establishment in step S1 includes: S11, sequentially merging gene fragment 1, inserting the intein sequence fragment and gene fragment 2 to obtain a new DNA sequence; S12, translating the new DNA sequence into a new amino acid sequence; S13, searching for a target intein amino acid sequence in the new amino acid sequence, deleting the target intein amino acid sequence, and obtaining an output amino acid sequence; S14, predicting that the inserted intein sequence fragment is inserted into each possible site of gene fragment 1 and gene fragment 2, and then repeating steps S11 to S13, and all the obtained output amino acid sequences constitute the protein database.

2. The method according to claim 1, characterized in that In step S11, one base is inserted into the inserted intein sequence fragment.

3. An application of the method as described in any one of claims 1 to 2 in cleavage site screening to achieve the application of screening for Escherichia coli antigen protein Im7-6 and / or Cas9 protein cleavage sites.

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