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A self-cleaving protein label, modification method and application thereof
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A self-shearing, protein technology, applied in the field of protein purification, can solve the problems of secondary use load reduction, influence on medium reuse, incomplete elution, etc., to optimize regeneration effect, improve regeneration effect, and improve elution effect Effect
Active Publication Date: 2022-04-15
JIANGNAN UNIV
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[0006] During use, it was found that under weakly alkaline elution conditions, I C The -GFP complex cannot be completely eluted after being applied to the column; under strong basic elution conditions, part I N The protein will be denatured, both of which will lead to a decrease in the capacity of the secondary use, affecting the reuse of the medium
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Embodiment 1
[0073] Primer design:
[0074] The C-terminal splicing domain of the natural break-type intein Cfa DnaE was molecularly modified by PCR technology, and the modified I C Fragment Named I C * . According to the gene sequence provided by GeneBank, the molecular biologysoftware PrimerPremier 5 was used to design I based on overlapping PCR. C * Forward primers A_F, B_F, C_F, D_F and reverse primers A_R, B_R, C_R, D_R:
[0083] Among them, primers A_F, A_R can construct bacterial strain pET28a-I ...
Embodiment 2
[0086] recombinant vector pET28a-I C * The build:
[0087] Extract plasmid pET28a-I C , using the forward primers A_F, B_F, C_F, D_F and reverse primers A_R, B_R, C_R, D_R designed in Example 1 as primers, plasmid pET28a-I C As a template, use Prime STAR DNApolymerase to pET28a-I C * 12-3G-13, pET28a-I C * 12-6G-13, pET28a-I C * 12-12G-13, pET28a-I C * 12-18G-13 sequence for PCR. According to the instructions of Prime STAR DNApolymerase, select 50 μL system to carry out.
[0088] PCR reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing temperature at 65°C for 30 s, extension temperature at 72°C for 5 min and 30 s, and 30 cycles; finally, 72°C for 10 min.
[0089] Plasmid pET28a-I was recovered using an agarose gel recovery kit C * 12-3G-13, pET28a-I C * 12-6G-13, pET28a-I C * 12-12G-13, pET28a-I C * 12-18G-13. Transform the competent cell E.coliJM109 with the recombinant plasmid to obtain the recombinant strai...
Embodiment 3
[0091] recombinant vector pET-28a-I C * The build:
[0092] Overnight culture of strain E.coli JM109 / pET28a-I C * , extract the plasmid, transform the competent E.coli BL21(DE3), and obtain the transformed recombinant I C * Expression strain E.coli BL21(DE3) / pET28a-I C * spare.
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Abstract
The invention provides a modification method and product of a self-cleaving protein tag and its application, wherein, the construction of the affinity ligand is to Cfa DnaE-C, i.e. I C carried out on the basis of the transformation of the structure; in I C An appropriate amount of glycine is added to the appropriate position between No. 12 and No. 18 to reduce the I C The structural stability of the protein is used to optimize the elution conditions and improve the elution effect. The present invention changes the I C The protein did not have a significant impact on the intein recognition connection, and it can be seen that the fragmentation rate did not decrease significantly during the fragmentation reaction. In addition, the modification of the present invention has no obvious impact on the capacity of the affinity medium, and it can be seen that the capacity of the affinity medium decreases slightly during the static adsorption experiment. The affinity purification medium constructed by the invention has better purification effect and higher reuse rate.
Description
technical field [0001] The invention belongs to the field of protein purification, and relates to a modification method and product of a self-cleaving protein tag and its application. Background technique [0002] In recent years, with the continuous development of biotechnology, more and more people pay attention to purification technology. Affinity chromatography technology is one of the effective means for the separation and purification of recombinant proteins, which has the characteristics of easy operation and high purification efficiency, but affinity chromatography technology usually requires the introduction of special affinity tags into the target protein. The commonly used de-labeling methods in industry include endonuclease method, chemical method, etc., but the processing methods are often time-consuming, labor-intensive and very expensive. [0003] Intein is a gene sequence inserted into the precursor protein, which can undergo self-splicing during the process...
Claims
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