Kit for research on ubiquitin-like system of bacteria and application of kit

A kit and ubiquitin-like technology, applied in the field of biology, can solve problems such as poor expression and solubility, and unclear reasons

Active Publication Date: 2018-08-17
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, Escherichia coli can express human insulin, antibacterial protein, etc., but its expression level and solubility are often poor when expressing foreign proteins. Current studies have found that the fusion of ubiquitin-like protein ThiS at t

Method used

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  • Kit for research on ubiquitin-like system of bacteria and application of kit
  • Kit for research on ubiquitin-like system of bacteria and application of kit
  • Kit for research on ubiquitin-like system of bacteria and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The kit for studying the ubiquitin-like system in bacteria in this example includes ThiS protein and ThiF protein, and the concentrations of ThiS protein and ThiF protein are 1 mg / mL respectively. The nucleotide sequence of ThiS protein is shown in SEQ ID NO.1, and the nucleotide sequence of ThiF protein is shown in SEQ ID NO.2. Align the amino acid sequence of ThiS protein with homologous protein sequences of other species, the results are as follows figure 1 As shown, ecol is the ThiS protein in this application, and the others are homologous sequences derived from Streptomyces and archaea. Through sequence comparison, it is found that ThiS has two very conserved glycines at the C-terminus, which are ubiquitin and ubiquitin-like Site of covalent attachment to substrate lysine.

[0035] The preparation steps of the ThiS protein and ThiF protein are as follows:

[0036] 1) Cloning the thiS gene and thiF gene into the commercial expression vector pET28-a respectively, ...

Embodiment 2

[0044] The kit for studying the ubiquitin-like system in bacteria in this example includes ThiS protein, ThiF protein, reaction buffer, DTT and ATP. ThiS protein and ThiF protein were purified by the method in Example 1, both at a concentration of 1 mg / ml. The reaction buffer includes MgCl 2 and Tris-HCl, Tris-HCl is 250mM pH 7.5, MgCl 2 It is 12.5mM in a bottle (named Reaction buffer). There is also a tube of 1M DTT and a tube of 100mM ATP. When in use, take 100 μl of Reaction buffer and add 0.5 μl of DTT to form a 5x buffer.

[0045] When the above kit is used: the mass concentration ratio of ThiS protein, ThiF protein and substrate protein is 4:2-4:1-4. The concentration of the ThiS protein is 0.15-0.25 μg / μL, preferably 0.02 μg / μL.

[0046] MgCl in the reaction buffer 2 The concentration used can be 2.3-2.7mmol / L, preferably 2.5mmol / L. The concentration of Tris-HCl can be 45-55mmol / L, preferably 50mmol / L, and the pH is 7.5. The concentration of ATP used can be 3.5-...

Embodiment 3

[0048] The ubiquitination reaction of ThiS protein and ThiF protein after overexpression in Escherichia coli.

[0049] With the pET28-a-thiS in embodiment 1 N-Flag Transform Escherichia coli Rosetta (DE3) with and / or pET28-a-thiF respectively, pick a single clone, culture in LB medium at 37°C overnight, dilute 1:100 into 1L of fresh LB medium, and culture until OD600 reaches 0.6-0.8 , adding (or not adding) the inducer IPTG with a final concentration of 0.5 mM, and induced overnight at 16°C. Collect the cells, add 2x SDS-PAGE loading buffer, heat at 95°C for 10 minutes to obtain a total protein sample, run SDS-PAGE gel (15%), without staining, use Anti-Flag mouse monoclonal antibody to incubate for 2 hours, after washing Then use the mouse secondary antibody to incubate for 1h, ECL exposure for 1min, the results are as follows: image 3 As shown in -A, it can be seen from the figure that after overexpressing ThiS protein and ThiF protein in Escherichia coli using IPTG induct...

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Abstract

The invention relates to a kit for research on ubiquitin-like system of bacteria and application of the kit and belongs to the technical field of biology. The kit comprises ThiS protein and ThiF protein, and can further comprise MgCl2 and Tris-HCl reaction buffer, DTT and ATP. As ThiF protein has a function of ligase, ThiS protein can be connected to substrate protein in the presence of ATP in vitro to form ubiquitin-like modification; the ubiquitin-like modification has a substrate selectivity, and accordingly, FtsA and PriC but MoaC can be modified by ThiS protein and ThiF protein. The kit can be applied to research on ubiquitin-like modification conditions of substrates effectively, and has the advantages of being convenient, rapid and effective.

Description

technical field [0001] The invention relates to a kit for studying the ubiquitin-like system in bacteria and its application, belonging to the technical field of biology. Background technique [0002] Escherichia coli ubiquitin-like protein ThiS is a small molecule protein with a molecular weight of 7.2kDa. Its structure is very similar to that of ubiquitin protein. They all have five β sheets and two α helices. Previous studies have found that it is involved in the synthesis of vitamin B1 ( Iyer, L.M., Burroughs, A.M. & Aravind, L. The prokaryotic antecedents of the ubiquitin-signaling system and the early evolution of ubiquitin-like β-grasp domains. Genome Biology 7, 91-96 (2006)). ThiS and ThiF proteins are mainly involved in the synthesis of vitamin B1 in vivo. ThiF can activate the two GGs at the end of ThiSC, and then participate in the sulfur transport process. The main function of ThiF protein is to adenylylate ThiS protein (activation function) when vitamin B1 is sy...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 徐西兵沈国民曹青沈滟
Owner HENAN UNIV OF SCI & TECH
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