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Method for screening splitting sites and application thereof

A site, amino acid technology, applied in the field of screening split sites, can solve problems such as lack of characteristics

Pending Publication Date: 2022-06-21
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most recombinant proteins, such as IL-2, IFN, EGF, bFGF and other high-value protein drugs, do not have the characteristics that can be directly detected by high-throughput screening

Method used

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  • Method for screening splitting sites and application thereof
  • Method for screening splitting sites and application thereof
  • Method for screening splitting sites and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0042] like image 3 shown, image 3 It is the same experimental principle as the above-mentioned scientific document "A systematic approach to insertingsplit inteins for Boolean logic gate engineering and basal activityreduction", that is, the method of randomly inserting gene fragments by mini-Mu transposon mediated by the phage transposition mechanism. The specific operations are as follows:

[0043] 1. Use the transposase MuA to carry out the transposition experiment on the target gene fragment, and insert the transposon into the target fragment randomly, and the principle ensures that only one transposon fragment is inserted into each target gene fragment. The transposon fragment is a complete expression line with promoter, terminator and other elements and expressing chloramph...

Embodiment 2

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

[0053] The steps 1-5 are roughly the same as those in Example 1, the difference is that in step 3, "replace the transposon fragment with the expression intein Ssp DnaBM86" by the restriction enzyme digestion Replace the transposon fragment with the N-terminal, terminator and promoter transcription elements of the split intein Ssp DnaBM86 and the gene fragment of the C-terminal of the split intein Ssp DnaBM86 (corresponding to image 3 3. The split intein version method in Substitution)"

[0054] like Figure 7 Shown is a schematic representation of the nucleotide sequence of the intein Ssp DnaBM86 with restriction sites and additional bases added to prevent frameshift mutations. Ditto Figure 4 said, Figure 7 GCGGCCGC in the two boxes 1 is the nucleotide sequence of the restriction enzyme cleavage site, the C bases in the two boxes 2 ar...

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Abstract

The invention provides a method for screening splitting sites and application of the method. The method comprises the following steps: S1, establishing a data writing a program by using a computer language, predicting that intein is subjected to splicing reaction after being embedded in every two adjacent amino acid residues in an amino acid sequence and is cut off, and forming a protein database by using the amino acid sequence formed by connecting adjacent peptide fragments; s2, performing an experiment: inserting intein sequence fragments into the gene fragments, then performing molecular cloning and translation to obtain peptide fragments, detecting whether the peptide fragments contain labeled amino acid sequences or not through mass spectrometry, and comparing the peptide fragments with the protein database to realize verification of the fracture sites. According to the method, a protein database is constructed through computer programming, then an experiment is carried out, and detection and verification of splitting sites are realized through mass spectrometry; final detection is achieved through mass spectrum, high-throughput screening is replaced, and the method is expanded to search for splitting sites of any active protein.

Description

technical field [0001] The invention relates to the field of protein cleavage site screening, in particular to a method for screening cleavage sites and its application. Background technique [0002] Inteins are able to join flanking exoproteins (external proteins) into a new protein fragment and excise itself out, a process known as protein splicing. Inteins are found in many natural organisms, such as bacteria, fungi and lower plants, and are often embedded in important proteins. In nature, protein splicing produces two separate proteins (intein and exonin) and under the control of one gene, the intein precisely excises the inner protein fragment (the intein itself) and simultaneously joins the two regions. Naturally occurring protein inteins exist in several forms, including full-length inteins, mini-inteins, and naturally occurring split inteins. Both full-length inteins and mini-inteins are cis-spliced ​​inteins with or without an endonuclease domain. Split inteins a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G16B35/20G16B50/30G16B20/30G16B30/10
CPCG16B35/20G16B50/30G16B20/30G16B30/10G16B35/10G16B40/10G16B15/00G16B40/00G16B20/00G01N33/6848
Inventor 王昊杨丽丽
Owner SHENZHEN INST OF ADVANCED TECH
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