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Expression vector and method for detecting membrane protein interaction in bacteria

A technology for expressing vectors and membrane proteins, applied in the field of genetic engineering, can solve the problems of affecting the interaction of membrane proteins, spending a lot of time, etc., to achieve the authenticity and reliability of test results, improve water solubility, and reduce degradation effects

Active Publication Date: 2020-12-11
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Membrane proteins have a single or multiple transmembrane structure and are prone to aggregation, making it difficult to dissolve in host bacteria, and it is usually difficult to obtain proteins with active functions, which affects subsequent research on membrane protein interactions
At present, the commonly used method of purifying membrane protein is to use detergent to dissolve membrane protein, but due to the different types of membrane protein, the type of detergent suitable for its dissolution is also different, and it takes a lot of time to explore the conditions; secondly, decontamination The detergent will affect the follow-up experiments, and the process of removing the detergent will also take a lot of time

Method used

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  • Expression vector and method for detecting membrane protein interaction in bacteria
  • Expression vector and method for detecting membrane protein interaction in bacteria
  • Expression vector and method for detecting membrane protein interaction in bacteria

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0042] Example 1, construction of expression vector pMAL-Th8H

[0043] This implementation is to construct the protein expression vector pMAL-Th8H, using the commercial expression vector pMAL-c2X as the backbone, such as figure 1 As shown, the design adds the sequence of encoding thrombin (Thrombin) recognition site and 8×His tag (the number of His can be 6-10, the preferred 8 His in this embodiment) at the downstream of its MBP coding gene, and at the same time at the Thrombin position Eight common restriction endonuclease sites were inserted between the dot and 8×His, and a translation stop codon was inserted downstream of the 8×His tag. The total length of the sequence is 1275bp (including the MBP coding gene). The specific nucleotide sequence is shown in the sequence table Shown in SEQ ID NO.1.

[0044] S1. Synthesis of Th-MCS-8×His fragment. Forward and reverse primers were designed for both ends of the Th-MCS-8×His fragment, and then sent to a third-party Sangon Bioeng...

Embodiment 2

[0055] Example 2. Expression and purification of membrane protein PP_0337 with pMAL-Th8H

[0056] PP_0337 is a phosphodiesterase in the model strain Pseudomonas putida KT2440, which localizes on the cell membrane and is responsible for degrading the second messenger c-di-GMP. It is difficult to obtain soluble PP_0337 protein by conventional expression methods. In this example, the vector pMAL-Th8H constructed in Example 1 was used to express and purify the PP_0337 protein. The specific operation steps are as follows:

[0057] S1. PCR amplification of the gene encoding PP_0337. Using the genome of Pseudomonas putida KT2440 as a template, the PP_0337 coding gene was amplified by PCR, and the primer sequences used were as follows:

[0058] PP_0337S: 5'-CGGGATCCATGAAAAGCCAACCCGATG-3' (SEQ ID NO.5)

[0059] PP_0337A: 5'-CCCTCGAGACGCGGATAGCGCTTCAG-3' (SEQ ID NO. 6)

[0060] The product was recovered with a PCR recovery kit, and the length of the amplified product was 2676 bp, in...

Embodiment 3

[0066] Example 3. Using His pull down assay to detect the interaction between membrane protein PP_0337 and CheA protein

[0067] CheA protein is a chemotactic kinase widely present in various Gram-negative bacteria and plays a key regulatory role in bacterial chemotaxis motility. Previous studies have used the bacterial two-hybrid method to confirm the interaction between CheA and PP_0337 protein in Pseudomonas, but this result was observed in the host bacteria Escherichia coli, and there is no more direct and intuitive in vitro result. In this example, the PP_0337 protein obtained in Example 2 is used to visually detect whether there is an interaction between PP_0337 and the CheA protein by using pull down technology. The operation process is as follows Figure 7 shown. The specific operation steps are as follows:

[0068] S1. Expression and purification of CheA protein. First, using the genome of Pseudomonas putida KT2440 as a template, the CheA coding gene was amplified ...

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Abstract

The invention discloses an expression vector and a method for detecting membrane protein interaction in bacteria. The method comprises the following steps of firstly, constructing a double-label protein expression vector with a maltose binding protein label (MBP) and a histidine label (8*His), and expressing and purifying insoluble membrane protein by using the vector to increase the solubility ofthe membrane protein; and then, detecting the interaction between the membrane protein and other proteins by utilizing a pull down technology. Meanwhile, the method can also be used for screening proteins interacting with the membrane protein in bacterial whole-cell proteins. The method for detecting the interaction of the membrane proteins in the bacteria breaks through the technical difficultyof insolubility of the membrane proteins in prokaryotes, provides an experimental means for researching the functions of the bacterial membrane proteins, and has important application value.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an expression vector and a method for detecting the interaction of membrane proteins in bacteria. Background technique [0002] The interaction between proteins refers to the process in which two or more protein molecules form protein complexes through non-covalent bonds, which is the basis of all known cell life activities. Studying the interaction between proteins is of great significance for in-depth exploration of protein functions and revealing the mechanism of life activities. The current research methods for protein interaction mainly include yeast / bacteria two-hybrid, phage display technology, surface plasmon resonance technology, fluorescence energy transfer technology, co-immunoprecipitation technology, pull down experiment, etc. Among them, due to the advantages of simple operation, low cost, and low requirements for equipment, the pull down experiment has...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/62C07K19/00C07K1/22G01N33/68
CPCC12N15/70C12N15/62G01N33/6845C07K2319/24C07K2319/50C07K2319/21Y02A50/30
Inventor 陈雯莉肖玉杰廖浩聂海玲
Owner HUAZHONG AGRI UNIV
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