大豆疫霉菌分泌蛋白的制备和纯化

Yeqiang Xia; Yan Wang; Yuanchao   Wang

doi:10.21769/BioProtoc.3045

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Peer-reviewed

Preparation and Purification of Proteins Secreted from Phytophthora sojae

大豆疫霉菌分泌蛋白的制备和纯化

YX Yeqiang Xia

Yan Wang

Yuanchao Wang email

发布: 2018年10月20日第8卷第20期 DOI: 10.21769/BioProtoc.3045 浏览次数: 6323

评审: Zhibing LaiJuan DuAnonymous reviewer(s)

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Abstract

Phytophthora sojae, the causal agent of soybean root and stem rot, is responsible for enormous economic losses in soybean production. P. sojae secrets various effectors to reprogram host immunity. The plant apoplastic space is a major battleground in plant-pathogen interactions. Here we describe a protocol for purification and isolation of secreted proteins from P. sojae, including precipitation of secreted proteins from P. sojae culture filtrate, chromatographic purification of the secreted proteins and analysis of the proteins by Mass spectrometry. With this protocol, it will be easier to identify potential apoplastic effectors in Phytophthora and will benefit our understanding of plant-microbe interactions.

Keywords: Phytophthora sojae (大豆疫霉菌)

Soybean root and stem rot (大豆根腐及茎腐病)

Secreted proteins (分泌蛋白)

Chromatographic puriﬁcation (色谱纯化)

ÄKTA (ÄKTA)

Apoplastic effectors (非原生质体效应)

Background

Purification of proteins secreted from Phytophthora species is essential for understanding Phytophthora pathogenesis. In the past, V8 juice and Plant (tomato, and Lima bean) juice medium have been used to culture Phytophthora and the culture filtrated was used for the subsequent analysis of Phytophthora secreted proteins. The drawback of these protocols is the culturing media contain huge amount of plant proteins, which represent a large proportion of the detected proteins. In this protocol, we made use of the Synthesis Liquid Medium, which does not contain any proteins. This medium significantly reduces the background of the Phytophthora culturing filtrate. Furthermore, making use of the Gel Filtration desalting and sieving columns instead of Ion Exchange columns, allows efficient and large-scale purification of Phytophthora secreted proteins.

Materials and Reagents

Pipette tips
Scalpel
Filter paper (Whatman ﬁlters) (No. 1)
0.22 μm Millex-GV ﬁlter (Merck, catalog number: SLGP033NS )
Amicon Ultra-15 Centrifugal Filter Unit 3KD (Merck, catalog number: UFC900308 )
P. sojae strain (P6497)
V8 juice (https://www.campbells.com/v8/vegetable-juice/)
Calcium carbonate (CaCO₃) (Sigma-Aldrich, catalog number: 398101 )
Potassium Dihydrogen Phosphate (KH₂PO₄) (Merck, catalog number: 1048730250 )
Yeast extract (Sigma-Aldrich, catalog number: Y1625-1KG )
Magnesium sulfate heptahydrate (MgSO₄•7H₂O) (Sigma-Aldrich, catalog number: RES0089M-A702X )
D-glucose (Sigma-Aldrich, catalog number: G8270-100G )
L-asparagine (Sigma-Aldrich, catalog number: A0884-100G )
β-sitosterol (Sigma-Aldrich, catalog number: S1270-10MG )
Thiamine(VB1) (Sigma-Aldrich, catalog number: T4625-5G )
Milli-Q-ﬁltered H₂O
Ammonium sulfate (NH₄)₂SO₄(Sigma-Aldrich, catalog number: A4418-100G )
Tris-aminomethane (Tris-HCl) (Sigma-Aldrich, catalog number: 154563-1KG )
Ethylene Diamine Tetraacetic Acid (EDTA) (Sigma-Aldrich, catalog number: 80849-10MG )
30% acrylamide
10% sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771-100G )
10% ammonium persulfate (Sigma-Aldrich, catalog number: A3678-25G )
N,N,N',N'-Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281-25ML )
Coomassie brilliant blue (CBB-R-250) (Beyotime Biotechnology, catalog number: P0017 )
Ethanol (ALADDIN-E, catalog number: A112719 )
Acetic acid (ALADDIN-E, catalog number: A116174 )
Methanol (ALADDIN-E, catalog number: M116127 )
V8 juice with 2% agar (see Recipes)
Synthesis liquid medium (see Recipes)
TE buffer (see Recipes)
SDS-PAGE Gel (see Recipes)
R-250 staining solution (see Recipes)
Destaining solution (see Recipes)

Equipment

Pipettes
Conical flask (1 L)
Beaker (5 L)
Measuring cylinder (1 L)
Constant temperature incubator (CTI) (25 °C)
Constant temperature shaker (CTS) (25 °C, 100 rpm)
Centrifuge
Liquid chromatograph (GE Healthcare, model: ÄKTA avant 25 )
Column Sephadex G-25S (GE Healthcare, catalog number: 17140801 )
Chromatography column (GE Healthcare, model: Superdex^TM 200 Increase, catalog number: 28990944 )

Note: The materials, reagents and equipment not provided company and catalog number can be ordered from any qualified company for using in this experiment.

Procedure

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如何引用

Xia, Y., Wang, Y. and Wang, Y. (2018). Preparation and Purification of Proteins Secreted from Phytophthora sojae. Bio-protocol 8(20): e3045. DOI: 10.21769/BioProtoc.3045.