Method for screening sumoylation target protein of Aspergillus flavus based on in vitro reaction system

A reaction system, the technology of Aspergillus flavus, applied in the field of microbiology, can solve the problems of limited success rate and accuracy, low homologous comparison scores, protein instability, etc., achieve high success rate and accuracy, and reduce background noise , the effect of increased stability

Active Publication Date: 2019-02-22
FUJIAN AGRI & FORESTRY UNIV
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Problems solved by technology

However, Aspergillus flavus is a multicellular filamentous fungus, and it is difficult to screen and verify SUMOylated target proteins in vivo using existing genetic manipulation methods
[0004] According to the analysis of existing relevant data, the current screening methods for SUMOylated target proteins mainly use methods such as co-immunoprecipitation and tandem affinity purification for SUMO to directly analyze the complexes with SUMO proteins in eukaryotic cell / tissue lysates , using mass spectrometry to analyze the SUMOylated target protein in the complex, the difficulty and defects mainly lie in: 1. The SUMOylated modification itself is a two-way, dynamic, and spatiotemporal process, that is, the process of the target protein carrying the SUMOylated modification is unstable. (SUMOylated target proteins will be de-SUMOylated by SUMOylation-specific proteases), so the existing methods for direct analysis of SUMOylated targets in eukaryotic cell / tissue lysates can analyze SUMOylated protein information is a transient Many de-SUMOylated proteins cannot be screened and analyzed; 2. The abundance of various proteins in eukaryotic cells is uneven, and the existing analysis methods for proteins are very likely to lose some low-abundance proteins 3. Although the current mass spectrometry technology has higher and higher technical precision, the final protein identification is still based on the analysis of protein databases. For some non-model organisms with unclear genetic background, the success rate of final protein identification and Accuracy may be limited, and some proteins that cannot be identified due to low homologous alignment scores cannot be further analyzed

Method used

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  • Method for screening sumoylation target protein of Aspergillus flavus based on in vitro reaction system
  • Method for screening sumoylation target protein of Aspergillus flavus based on in vitro reaction system
  • Method for screening sumoylation target protein of Aspergillus flavus based on in vitro reaction system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: Cloning of Aspergillus flavus SUMOylation enzyme system and construction of prokaryotic expression vector

[0067]The inventor searched the The Fungal and Oomycete Genomics Resource database (http: / / fugidb.org / fungidb / ) and used bioinformatics comparative analysis to find the SUMOylation modification with Saccharomyces cerevisiae and Aspergillus nidulans in the genome sequence of Aspergillus flavus Enzyme genes with high homology genes are named as Aspergillus flavus Afsum O , Afaos A , Afuba B , ikB and AfsizA Gene (the nucleic acid sequence and amino acid sequence of its coding region are shown in SEQ ID NO.1-10). The inventor amplified from the cDNA of Aspergillus flavus by RT-PCR technology Afsum O , Afaos A , Afuba B , ikB and AfsizA The coding region fragment is cloned into the expression vector pColdI to prepare for the prokaryotic expression and purification of the protein components of the Aspergillus flavus SUMOylation modificatio...

Embodiment 2

[0081] Embodiment 2: prokaryotic expression of Aspergillus flavus SUMOylation modification enzyme system;

[0082] The prokaryotic expression vector of the Aspergillus flavus SUMOylation enzyme system constructed in Example 1 was transformed into an Escherichia coli expression strain to obtain the prokaryotic expression strain of the Aspergillus flavus SUMOylation enzyme system. The purified recombinant Aspergillus flavus SUMOylated enzymes AfSumO, AfAosA, AfUbaB, AfUbcI and AfSizA were obtained by cold shock-induced expression and Ni column purification.

[0083] The specific steps and conditions are as follows:

[0084] 1. Construction of the prokaryotic expression strain of Aspergillus flavus SUMOylation enzyme system: the above prokaryotic expression vector pCold- Afsum O , pCold- Afaos A , pCold- Afuba B , pCold- ikB and pCold- AfsizA Transform into Escherichia coli C41 (DE3) to obtain the prokaryotic expression strain of the corresponding gene;

[0085] 2. Ind...

Embodiment 3

[0088] Embodiment 3: the in vitro SUMOylation reaction of Aspergillus flavus

[0089] Using the recombinant Aspergillus flavus SUMOylation modified enzyme system components obtained in Example 2, carry out the SUMOylation in vitro reaction, the specific steps and conditions are as follows:

[0090] The solution system is a mixture of the following components: 50 mM HEPES (pH 7.2), 100 mM NaCl, 10 mMMgCl 2 , 0.1 mM dithiothreitol, 10 mM ATP, 50 μg / mL recombinant AfSumO (without His6-tag removal), 10 μg / mL recombinant AfAosA (without His6-tag), 10 μg / mL recombinant AfUbaB (without His6-tag), 10 μg / mL recombinant AfUbcI (without His6-tag) and 10 μg / mL recombinant AfSizA (without His6-tag). React at 30°C for 30 minutes.

[0091] The reaction product was immediately separated by SDS-PAGE, transferred to a membrane and analyzed by Western blot. The primary and secondary antibodies were respectively used Mouse anti-His6 Monoclonal antibody and HRP-conjugated Goat anti Mouse IgG (H+...

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Abstract

The invention relates to a method for screening aspergillus flavus SUMOylation target protein based on an in-vitro reaction system. The system is composed of a SUMOylation reaction solution system and comprises a modification molecule AfSumO in a prokaryotic expressed expression aspergillus flavus SUMOylation enzyme system, a SUMO E1 activating enzyme AfAosA-AfUbaB, a SUMO E2 conjugating conjugated enzyme AfUbcI, SUMO E3 ligase and protein to be detected or a medicine to be detected. By utilizing the system provided by the invention, a medicine molecule or a small molecule compound which influences an aspergillus flavus SUMOylation modification process can be rapidly identified / screened; meanwhile, whether certain protein can be modified by an aspergillus flavus SUMOylation system or not can be conveniently and rapidly detected in vitro by utilizing the system, so that the potential SUMOylation target protein is identified / screened.

Description

technical field [0001] The invention belongs to the field of microbiology and biotechnology, and in particular relates to a method for screening a flavus SUMOylated target protein based on an in vitro reaction system. Background technique [0002] Many reversible post-translational modifications, such as phosphorylation, glycosylation, ubiquitination, SUMO (small ubiquitin-related modifier) ​​and so on, are considered to be one of the important bridges connecting genetic background and environmental factors, mediating The complex interaction of the two ultimately affects the traits of organisms. As a very important protein post-translational modification, SUMOylation may affect the stability, cellular localization and protein activity of the target protein. SUMO proteins were first discovered in Saccharomyces cerevisiae, and the SUMOylation process is similar to the ubiquitination process. First, SUMO is activated by the E1 activating enzyme Aos1-Uba2, and then transferred...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68C40B50/06
CPCC40B50/06G01N33/68
Inventor 聂鑫怡李玲岳跃威汪世华
Owner FUJIAN AGRI & FORESTRY UNIV
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