TEV protease mutant, gene, biomaterial, preparation method, reagent, or reagent kit and application
A biomaterial, mutant technology, applied in microorganism-based methods, biochemical equipment and methods, botanical equipment and methods, etc., can solve the problems of low yield and water solubility, reduced protease activity and stability, etc. Activity and stability, improved water solubility, good stability and specificity
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[0058] The present invention also provides a preparation method of the rTEV, comprising expressing the gene encoding the rTEV in a host.
[0059] The preparation method can be performed by expressing the above-mentioned gene encoding the rTEV in a host, for example, but not limited to, an Escherichia coli expression system, a yeast expression system, an insect expression system, a plant expression system or a mammalian expression system.
[0060] In some preferred embodiments, the gene expressing the rTEV is first inserted into the recombinant plasmid obtained by the pET-28a vector; then the recombinant plasmid is introduced into E. rTEV.
[0061] The present invention also provides a reagent or a kit containing the rTEV, and the reagent or the kit may also include reagents and / or consumables matched with the rTEV, for example, but not limited to, other enzyme-cutting DNA Protease, Buffer for dissolving protease, SDS-PAGE related reagents or experimental consumables for more ...
Embodiment 1
[0066] This embodiment provides a TEV protease mutant named rTEV, and TEVw is the wild-type TEV protease, which has the mutation sites as shown in the following table:
[0067] Table 1 Mutation sites of rTEV
[0068] amino acid position
TEVw amino acid name
rTEV amino acid name
17
T
S
56
L
V
68
N
D
77
I
V
135
S
G
219
S
V
233
Q
H
[0069] The preparation method of rTEV is as follows:
[0070] The whole gene was synthesized according to the nucleotide sequence of rTEV in Table 1, and subcloned into the final expression vector pET-28a with Nde I as the N-terminal restriction site and Xho I as the C-terminal restriction site. After sequencing by Shanghai Sangon Bioengineering Technology Service Co., Ltd., it was finally verified that the obtained recombinant plasmid sequence was consistent with the sequence of the theoretical seven-mutant TEV protease.
[0071] Expression an...
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