TrxA and SUMO double-solubilized expression tag sequence and application thereof
A technology for expressing tag sequences and solubilizing tags, applied in recombinant DNA technology, biochemical equipment and methods, using vectors to introduce foreign genetic material, etc. The effect is not very satisfactory and other problems, to save time and cost of research, promote correct folding, and accurate enzyme digestion
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Embodiment 1
[0027] The base sequence of the TrxA and SUMO dual pro-solution expression tag is as follows: SEQ ID NO: 1
[0028]
[0029] (1) Information of SEQ ID NO: 1 (see sequence listing)
[0030] (a) Sequence features:
[0031] *Length: 750bp
[0032] *Type: nucleic acid
[0033] * Chain type: double chain
[0034] *Topology: Linear
[0035] (b) Molecular type: cDNA
[0036] (c) Assumption: No
[0037] (d) Antisense: No
[0038] (e) Original source: Synthetic
[0039] (2) PCR amplification of SUMO gene
[0040] (a) PCR primer design and reaction conditions:
[0041] A set of primers were designed according to the SUMO gene sequence and the DNA sequence of pET32a:
[0042] Forward primer: 5'-CGGGGTACCATGTCGGACTCAGAAGTCAAT-3' (synthesized by Beijing Huada Gene Company)
[0043] Reverse primer: 5'-CAGTCCATGGCACCAATCTGTTCTCTGTGAGC-3' (synthesized by Beijing Huada Gene Company)
[0044] The PCR reaction system was: 10xPyrobest buffer (purchased from Dalian Bao Biological Co...
Embodiment 2
[0052] Fusion connection of SUMO sequence and TrxA sequence
[0053] (1) Double enzyme digestion of the pET32a vector: The pET32a vector plasmid DNA (purchased from Novagen) was double-digested with restriction endonucleases Kpn I and Nco I, and the digested product was analyzed by 1.5% agarose gel electrophoresis, and the gel was recovered Large fragments of DNA.
[0054](2) Ligation and transformation of digested products: Mix the double-digested SUMO PCR product fragment and pET32a vector at a molar ratio of 3:1, and use T4 DNA ligase (purchased from Dalian Bao Biological Company) to ligate overnight at 16°C , to construct a new expression vector pET-32a-SUMO. The ligation product was transformed into Escherichia coli competent cell DH5α (purchased from Dalian Bao Biology Co., Ltd.) (transformation operation was carried out according to Page 39-40 of the third edition of "Molecular Biology Experiment Guide" edited by F. Osper et al.), using ampicillin Transformants were s...
Embodiment 3
[0057] Application and effect evaluation of TrxA and SUMO dual pro-solution expression tag sequence
[0058] (1) Ligate the exogenous gene scFv-Sag into the gene containing TrxA and SUMO
[0059] The expression vector pET-32a-SUMO of the dual solubilizing expression tag sequence: the pET-32a-SUMO plasmid DNA and the plasmid pET-32a-scFv-Sag containing scFv-SAg (see the reference for the construction process: Xu Mingkai et al. 2006. Fusionimmunotoxin anti-HER-2-scFv-SEC2 expressed in E.coli with an improved expression vector pASK75-EX: its construction and function. Advances in Biochemistry and Biophysics (English Edition). 33(8):781-788.) DNA respectively Using Nco I and Xho I (purchased from Dalian Bao Biological Co., Ltd.) double digestion, after 1.0% agarose gel electrophoresis, gel recovery of 1881bp scFv-SAg fragment and 6100bp pET-32a-SUMO plasmid fragment, with T4DNA ligase After ligation at 16°C overnight, the fusion protein expression vector pET-32a-SUMO-scFv-SAg was...
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