New-type label protein and application thereof

A protein and ubiquitin-like technology, applied in the direction of using vectors to introduce foreign genetic material, peptides, chemical instruments and methods, etc., can solve problems such as low cutting efficiency

Inactive Publication Date: 2009-11-04
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In the Half SUMO complementary expression system, although the SUMO protein tag can be used in the eukaryotic expression system, the cleavage efficiency is low during the subsequent processing to remove the protein tag

Method used

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  • New-type label protein and application thereof
  • New-type label protein and application thereof
  • New-type label protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0132] Preparation of RbCl Competent Cells

[0133] (1) Streak Escherichia coli strains onto LB plates and incubate at 37°C for 10-12 hours.

[0134] (2) Pick a single clone in 30ml S.O.B (1:100 before adding 1M MgCl 2 , 1:100 by adding 1M MgSO 4 ), cultivate overnight at 37°C.

[0135] (3) 1:100 overnight bacteria into 200ml S.O.B (add 1M MgCl 1:100 before use) 2 , 1:100 added to 1M MgSO4), cultivated at 37°C until OD600=0.35.

[0136] (4) Quickly transfer the bacterial solution to an ice-salt bath (-3 to -5°C) and pre-cool for 15 minutes.

[0137] (5) Centrifuge at 4°C, 4500rpm×5min, and discard the supernatant. Add 64ml Competent Cell Buffer 1, suspend, and ice-salt bath for 15 minutes.

[0138] (6) Centrifuge at 4000rpm×5min at 4°C and discard the supernatant. Add 16ml Competent Cell Buffer 2 and suspend the cells on ice.

[0139] (7) Immediately aliquot into 200 μl / tube (tube pre-cooled).

[0140] (8) Quick-frozen in liquid nitrogen and stored at -70°C.

[0141]...

Embodiment 1

[0199] Example 1. Prokaryotic expression vector construction

[0200] The DNA sequences of SUMO, SUMO-CTHS, and Zipper-CTHS were inserted into pET28a through NcoI and XhoI sites respectively, and then the green fluorescent protein EGFP was inserted through EcoRI and XhoI as the target protein of the expression test, and the prokaryotic expression plasmid pET28a was constructed. - 6His-SUMO-EGFP, pET28a-6His-CTHS-EGFP, pET28a-6His-Zipper-CTHS-EGFP. And the 6His tag is fused at the N-terminus, which is convenient for purification and detection.

[0201] Construction of pET28a-6His-EGFP: Using pEGFP-N (Clontech Company) as a template, using primer1 (SEQ ID NO: 6) and primer2 (SEQ ID NO: 7) as primers, amplifying the EGFP gene, using EcoRI and XhoI After digestion, it was inserted into the pET28a vector (with 6His tag) that had undergone the same digestion.

[0202] Construction of pET28a-6His-SUMO-EGFP (SE): Using the Saccharomyces cerevisiae genome as a template, using primer3...

Embodiment 2

[0208] Example 2. Prokaryotic expression and solubility test

[0209] In this example, it is proved that in the E. coli expression system, Zipper (Zip)-CTHS has a good effect of promoting fusion protein solubility and enhancing fusion protein expression.

[0210] Expression of the fusion protein in E.coli: Transform the recombinant expression plasmids pET28a-6His-SUMO-EGFP, pET28a-6His-CTHS-EGFP, and pET28a-6His-Zipper-CTHS-EGFP into Escherichia coli (BL21 DE3 strain) respectively, and inoculate Monoclonal colonies were cultured in LB culture medium at 37°C for 12-14 hours; the above-mentioned culture liquid was inoculated into fresh LB medium at a ratio of 1:100, and cultured at 37°C until OD600 If it is 0.6-0.8, add isopropylthiogalactoside (IPTG) and cultivate at 22°C for 16-20h. The bacteria were collected by centrifugation at 6000rpm. Dissolve the bacteria in the bacteriostasis buffer containing 0.5mg / ml lysozyme, freeze and thaw with liquid nitrogen, repeat 3 times, add...

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Abstract

The invention provides a method for expressing a target protein, which comprises the following steps that the fusion protein of a leucine zipper 1, a carboxyl terminal region of a homothetic ubiquitin molecule and the target protein is expressed in a host cell; the fusion protein is mixed with the rebuilding protein of the homothetic ubiquitin molecule containing the amino terminal region of the homothetic ubiquitin molecule and a leucine zipper 2 in order to produce a rebuilding product which is processed by the specific prolease of the homothetic ubiquitin molecule, and thus, the target protein is obtained. The invention also provides a reagent kit for the expression of the target protein, the reagent kit comprises a structuring object for expressing the target protein, an encoding gene containing the encoding gene of the leucine zipper 1 and the carboxyl terminal region of the homothetic ubiquitin molecule and the rebuilding protein or the rebuilding structuring object of the homothetic ubiquitin molecule.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a novel tag protein. The tag protein can be used in prokaryotic and eukaryotic expression systems to obtain homologous recombination proteins with high activity in large quantities, economically and rapidly. Background technique [0002] In past studies, a large ubiquitin-related small protein family Ubls (ubiquitin like proteins) has been discovered, including NEDD8, ISG15, etc., as well as small ubiquitin-like modifier proteins (small ubiquitin-like modifier, SUMO). [0003] The homologue of SUMO, Smt3, was first discovered in the genetic mutation screen of Saccharomyces cerevisiae. In 1996, SUMO (Smt3) was first discovered to be able to covalently bind a small G protein activator protein RanGAP1, which affected the transport of this protein in the nucleus and nucleoplasm, and then this small protein was named SUMO, and then SUMOylated Modifications are found and elucidated in vario...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/63C07K19/00
Inventor 杨淑伟魏星
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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