Gateway eukaryotic expression system for stably and efficiently expressing active fusion proteins

A technology for high-efficiency expression and fusion of proteins, which can be used in fusion with spectral/fluorescence detection, recombinant DNA technology, and peptides containing affinity tags. Accurate expression, powerful function, and the effect of promoting purification work

Pending Publication Date: 2021-09-03
谢文军
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing protein prokaryotic expression vector system often adopts an inefficient and outdated enzyme-cut ligation system, which is time-consuming and laborious, and it cannot guarantee that each expressed recombinant protein has a completely consistent side sequence, and the contrast is not strong.

Method used

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  • Gateway eukaryotic expression system for stably and efficiently expressing active fusion proteins
  • Gateway eukaryotic expression system for stably and efficiently expressing active fusion proteins
  • Gateway eukaryotic expression system for stably and efficiently expressing active fusion proteins

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Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1 (12xHis-GFP-GW) vector

[0034] Such as image 3 As shown, a group of A universal vectors in the Gateway eukaryotic expression system for stably and efficiently expressing active fusion proteins, the specific production methods include the following:

[0035] 1) using the pK7WGF2 vector as a substrate, digesting it with a restriction endonuclease SpeI to obtain a digested product, and purifying the digested fragment;

[0036] 2) Design and synthesize the forward primer 321-F1 (5'-TCGACCTGCAGGCGGCCGCACTAGTATGCACCATCATCATCATCATCACCACCACCACCA-3') and the reverse primer 321-R2 (5'-CTCCTCGCCCTTGCTCACCATACTAGTGTGGTGGTGGTGGTGGTGATGATGATGATGATGATG-3') with the recombination site and the partial coding sequence of the histone tag, Since the forward and reverse primers partially overlap with each other, they can serve as templates for each other to amplify and extend into a complete blunt-ended PCR product;

[0037] 3) detecting the PCR product, and purifying the P...

Embodiment 2

[0045] Embodiment 2 (12xHis-GW-GFP) vector

[0046] Such as Figure 4 As shown, a group of Gateway eukaryotic expression systems that stably and efficiently express active fusion proteins, the specific production methods include the following:

[0047] 1) using the pK7FWG2 vector as a substrate, digesting it with a restriction endonuclease SpeI to obtain a digested product, and purifying the digested fragment;

[0048] 2) Design and synthesize forward primer 321-F1 (5'-TCGACCTGCAGGCGGCCGCACTAGTATGCACCATCATCATCATCATCACCACCACCACCA-3') and reverse primer 321-R3 (5'-TTTGTACAAACTTGTGATATCACTAGTGTGGTGGTGGTGGTGGTGATGATGATGATGATGATG-3') with recombination site and partial coding sequence of histone tag, Since the forward and reverse primers partially overlap with each other, they can serve as templates for each other to amplify and extend into a complete blunt-ended PCR product;

[0049] 3) detecting the PCR product, and purifying the PCR product with the correct fragment length;

...

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Abstract

The invention relates to a group of Gateway eukaryotic expression system for stably and efficiently expressing active fusion proteins. The system comprises two universal destination vectors, namely, a 12xHis-GFP-GW (vector A) and a 12xHis-GW-GFP (vector B), and a user can select the two universal destination vectors to carry out LR reaction with an ENTR vector according to needs, so that a final expression destination vector can be obtained. According to the two universal vectors, a pK7WGF2 vector or a pK7FWG2 vector is used as an initial skeleton, and fusion and insertion are respectively carried out on a coding sequence of 12xHis by using restriction enzyme SpeI and recombinase MultiS. The invention provides a convenient and fast molecular cloning system as a necessary supplement for the previous patent (patent application number 202110472170. X), which is used for efficiently and accurately expressing a small number of eukaryotic proteins which cannot be expressed in a prokaryotic system, so that a protein expression and purification system is more comprehensive and perfect, has more powerful functions, and can powerfully promote the purification work of active proteins, and a basis is established for functional research of the proteins.

Description

technical field [0001] The invention relates to a group of Gateway eukaryotic expression systems for stably and efficiently expressing active fusion proteins. Background technique [0002] Gateway system is a set of efficient and accurate molecular cloning technology. The system only needs two simple LR or BP reactions, which can efficiently and accurately convert the target sequence between the expression vector (Destination vector) and the entry vector (ENTR vector), and can ensure the flanking of each target sequence The sequences are highly consistent. The active function of protein is the embodiment of vitality. Studying the structure, function and activity of proteins has always been one of the basic goals of biological research. Purifying a functional protein under non-denaturing conditions and testing its various biological properties is the main way to achieve this basic goal. However, the existing protein prokaryotic expression vector system often uses an ineff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79C12N15/67C12N15/62
CPCC12N15/79C12N15/62C07K2319/21C07K2319/60
Inventor 谢文军关素华
Owner 谢文军
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