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Link application of protein tag

A protein and protease technology, applied in the field of protein labeling and its application, can solve the problems of enzyme protein denaturation, affecting the purity of reaction products, reducing and other problems

Inactive Publication Date: 2012-10-31
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

However, free enzymes are very sensitive to the environment, and are not stable enough in strong acids, strong alkalis, high temperatures, high ion concentrations, and some organic solvents, which can easily lead to denaturation of enzyme proteins, thereby reducing or even losing their catalytic activity.
At the same time, the free enzyme reaction is not easy to separate from the substrate and product, which affects the purity of the reaction product and is difficult to reuse, which greatly limits the wide application of enzymatic reactions.

Method used

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  • Link application of protein tag
  • Link application of protein tag
  • Link application of protein tag

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0290] Preparation of RbCl Competent Cells:

[0291] (1) Streak the DH5α strain on the LB plate, 37°C, 10-12h;

[0292] (2) Pick a single clone in 30ml S.O.B and culture overnight at 37°C;

[0293] (3) Inoculate the overnight bacteria into 200ml S.O.B at 1:100, culture at 37°C until OD550=0.35, about 2 hours;

[0294] (4) Quickly transfer the bacterial solution to an ice-salt bath (-3~-5°C), pre-cool for 15 minutes (shake gently once every ~3 minutes), and pre-cool the 500ml centrifuge cup at the same time;

[0295] (5) pre-cooling centrifuge;

[0296] (6) Quickly transfer the bacterial solution to a 500ml centrifuge cup;

[0297] (7) 4500rpm×5min, 4°C, discard the supernatant;

[0298] (8) Add 4×16ml Buffer1, lightly mix, suspend, and put in ice-salt bath for 15min;

[0299] (9) 4000rpm×5min, 4°C, discard the supernatant;

[0300] (10) Add 4×4ml Buffer2, suspend the cells, and place on ice;

[0301] (11) Immediately aliquot into 200 μl / tube (tube pre-cooled);

[0302]...

Embodiment 1

[0346] Embodiment 1, the acquisition of SBD fragment

[0347] Such as figure 1 Synthetic primers were designed for the coding sequence of the starch binding domain (SBD).

[0348] In the case that there is no template for gene amplification or the template is difficult to obtain, overlapping PCR technology is currently an effective method and approach for synthesizing genes. The present inventors artificially synthesized SBD gene fragments by stepwise overlapping PCR technique. A total of 10 primers were designed according to the sequence of the SBD, and there was a certain length of complementary overlapping sequence (20bp) between each two primers of 1-8. Use the middle two primers as templates (such as primer 2, primer 3), and the two sides as amplification primers (such as primer 1, primer 4), amplify 1-4, 5-8 by PCR, and compare the two sections. Long DNA fragments, and then these two fragments (SBD1-4, SBD5-8) after purification and recovery were used as templates, u...

Embodiment 2

[0350] Example 2. Purification application of fusion expression of SBD and solubilizing tag SUMO

[0351] The previously constructed pET28a His-SBD-SUMO-EGFP and pET28a His-SBD-SUMO-JNK2 were induced and expressed in E. coli BL21(DE3) by 0.5mM IPTG at 22°C for 20 hours, and the bacterial liquid was collected for sonication After centrifugation, the obtained supernatant protein crude extract was mixed with starch beads at 4° C. for 1 hour, and the solution was eluted twice to remove foreign proteins. Then Ulp1c enzyme (catalytic domain of Ulp1) was used for enzyme cleavage reaction.

[0352] Such as Figure 3-4 As shown, it can be seen that the SBD-SUMO-EGFP and SBD-SUMO-JNK2 fusion proteins are well bound to starch beads in one step, and can be effectively purified to the target protein after cleavage by Ulp1c enzyme. SBD itself, as a solid-phase label, can specifically bind to starch beads. This affinity binding is stronger than that of general anions and cations, and it is...

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Abstract

The invention relates to a link application of a tag. The invention develops a brand new protein tag, i.e., a starch binding domain (SBD). After a target protein is fused with the SBD which can be well combined with a starch substrate, the SBD can be applied to fixation or purification of the target protein as well as observation of mutual actions between the target protein and other proteins. The SBD is a cheap and reliable tag protein.

Description

technical field [0001] The invention belongs to the field of biotechnology; more specifically, the invention relates to a novel protein label and its application. Background technique [0002] Starch binding domain (SBD) [0003] Starch is the primary energy storage form of plants and the most important energy source for human food. From the perspective of chemical structure, starch can be divided into two categories: amylose and amylopectin. Amylose is a dextran linked by several hundred D-glucose groups with α-(1,4) glycosidic bonds, with a small molecular weight of about 50,000; amylopectin is composed of several thousand glucose groups with α-(1 , 4) The glucan formed by glucosidic linkages as the main chain and α-(1,6) glycosidic linkages as branch points has a molecular weight much larger than that of amylose, around 60,000 [1]. In natural starch, straight chain accounts for 20% to 26%, it is soluble, and the rest is amylopectin. There are many amylases that cataly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K17/10C07K1/14C07K19/00C12N9/26C12N15/63C12N11/10C12P21/06
CPCY02P20/50
Inventor 杨淑伟李建中
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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