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A polyhedrin and expression vector technology, applied in the field of mutant polyhedrin, can solve problems such as low protease digestion efficiency
Inactive Publication Date: 2012-06-27
TIANJIN YAOYU BIOLOGICAL TECH
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[0003] In order to overcome the disadvantages of low protease digestion efficiency in the existing purification method, the present invention provides a mutant polyhedrin protein obtained by overlapping extension PCR method
Method used
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Embodiment 1
[0022] Example 1: Primer Design
[0023] To amplify the wild-type polyhedron gene, the primers were designed as follows:
[0024] Upstream primer F: 5'-CATG CCATGG CCAATTATTCATACACCC-3' (indicated in italics Nco I restriction site)
[0025] Downstream primer R: 5'-CG GGATCC ATACGCCGGACCAGTGAA-3' (indicated in italics Bam H I restriction site).
Embodiment 2
[0026] Example 2: PCR amplification of wild-type polyhedron gene (as shown in SEQ ID NO: 1)
[0027] The Bombyx mori nuclear polyhedrosis virus genome (purchased from Invitrogen), the upstream primer F and the downstream primer R obtained in Example 1 were used as primers to amplify the target fragment. The PCR reaction parameters were designed as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, annealing at 72°C for 1 min, 30 cycles, and extension at 72°C for 10 min.
[0028] In a 100 μL centrifuge tube, add the following components:
[0029] KOD Dash Buffer 5μL
[0030] 2.5mMdNTPs 1.5μL
[0031] KOD Dash 1 μL
[0032] F 1μL
[0033] R 1 μL
[0034] Template 1 μL
[0035] Add sterile double distilled water to 50 μL.
[0036] After mixing the components, put them into a PCR machine, and design 30 cycles according to the above reaction parameters. After the reaction was completed, the amplified fragment was identif...
Embodiment 3
[0037] Embodiment 3: the design of mutation primer
[0038] According to the principle described above, the N-terminal four consecutive basic amino acids KRKK of the wild-type polyhedrin protein (as shown in SEQ ID NO: 2) were mutated into four consecutive acidic amino acids ESEE, and the primers designed by Primer5.0 were used as follows:
[0039] Upstream primer F': 5'-GTGCTCCTCGCTCTCGGCGTTTTTGATAAG-3'
[0040] Downstream primer R': 5'- G AG A GC G AG G AGCACCTAGTCGAACATGA -3' (the bold part is the mutation site).
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Abstract
The invention relates to a mutant polyhedron with which a target protein yield can be improved. The invention related to the field of biology. According to the invention, a recombinant expression vector pET-28a-polh is constructed; the recombinant expression vector is mutated by using a commercialized kit, such that a complete recombinant expression vector containing mutant sites is obtained; the complete recombinant expression vector is subject to induction expression, such that the mutant polyhedron which can reduce a dissolving pH is obtained. When the mutant polyhedron provided by the invention is connected to a target protein gene, an obtained fusion protein can be purified through pH regulation and differential centrifugation methods with the property of the polyhedron, such that the purification steps are simplified. The obtained fusion protein can also be digested under an optimal protease active pH condition, such that target protein with a maximal amount can be obtained. Therefore, the yield of target protein is greatly improved.
Description
technical field [0001] The invention relates to a mutant polyhedrin protein, in particular to a mutant polyhedrin protein capable of improving the yield of a target protein and a preparation method thereof. Background technique [0002] Because polyhedrin has the ability to express efficiently, it is often used as a tag to fuse the gene of the protein that is not expressed or difficult to express, so as to express it or increase its expression level. At the same time, the solubility properties of polyhedron can be used to obtain a relatively pure fusion protein through simple pH adjustment and differential centrifugation, and then perform proteolytic digestion to obtain a pure target protein. However, the optimal pH range of protease is 7.0-8.5, and polyhedrin protein can only be dissolved at pH 10.8, so when carrying out enzyme digestion under this condition, protease loses activity or cannot reach the maximum activity, and the obtained The amount of protein of interest is...
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