Functional Evaluation of the Signal Peptides of Secreted Proteins
分泌蛋白信号肽功能评估
发布: 2018年05月05日第8卷第9期 DOI: 10.21769/BioProtoc.2839 浏览次数: 12264
评审: Xiaolin ChenAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Aug 2013
Abstract
Here, we describe a method that can be used to evaluate the function of predicted signal peptides. This method utilizes the yeast Saccharomyces cerevisiae YTK12 strain and pSUC2 vector in which the pSUC2 vector with fused predicted signal peptide is transformed into yeast. The function of the signal peptides can be evaluated by using different selective media and color reaction. In this protocol, we provide the detailed description of manipulation in order to implement easily.
Keywords: Secreted protein (分泌蛋白)
Signal peptide (信号肽)
Function (功能)
Yeast (酵母菌)
pSUC2 vector (pSUC2载体)
Background
Microbial eukaryotes, such as fungi and oomycetes, secret abundant of proteins to play a variety of functions. Currently, the most widely used method for prediction of signal peptide from amino acid sequences is to use software SignalP (Petersen et al., 2011). The yeast YTK12 strain is invertase negative and pSUC2 vector contains invertase gene but lack Methionine (Met) and signal peptide sequence, thus YTK12 strain and pSUC2 vector were widely used for biological evaluation of peptide secretion (Jacobs et al., 1997; Oh et al., 2009). In addition, the color reaction could be used to verify the result, because the invertase enzymatic activity can be detected by the reduction of 2,3,5-Triphenyltetrazolium Chloride (TTC) to insoluble red colored 1,3,5-Triphenylformazan (TPF). Although the method has been generally used to evaluate the function of signal peptides, there is no specific and detailed information about this method (Oh et al., 2009; Song et al., 2015). Here, we describe a modified high-efficiency yeast transformation method (Gietz and Schiestl, 2007) and detailed protocol to evaluate the function of signal peptides, which will be the primary part for the secretory proteins research.
Materials and Reagents
- Pipette tips
- 1.5 ml tubes
- 2 ml tubes
- 10 ml test tube
- Millipore filter units, 0.22 µm (Millex-GP, Merck, catalog number: SLGP033RB )
- Yeast YTK12 strain
- pSUC2 vector
- EcoRI restriction enzymes (Takara Bio, catalog number: 1040S )
- XhoI restriction enzymes (Takara Bio, catalog number: 1094S )
- Yeast extract (OXOID, catalog number: LP0021 )
- Peptone (BD, DifcoTM, catalog number: 211677 )
- Glucose (Sinopharm Chemical Reagent, catalog number: 10010518 )
- Agar A (Sangon Biotech, catalog number: A600010 )
- Adenine hemisulfate salt (Sigma-Aldrich, Vetec, catalog number: V900375 )
- Tris-HCl (Sigma-Aldrich, catalog number: V900312 )
- EDTA (Sinopharm Chemical Reagent, catalog number: 10009617 )
- NaOH (Sinopharm Chemical Reagent, catalog number: 10019718 )
- Single-stranded carrier DNA (salmon sperm DNA, Solarbio, catalog number: D8030 )
- LiAc (Sinopharm Chemical Reagent, catalog number: 30109760 )
- PEG (Polyethylene glycol, Sigma-Aldrich, catalog number: P3640 )
- DMSO (Dimethyl Sulfoxide, Sigma-Aldrich, catalog number: D5879 )
- YNB (yeast nitrogen base without amino acids, BD, DifcoTM, catalog number: 291920 )
- -Trp DO supplement (Clontech, catalog number: 630413 )
- Sucrose (Sinopharm Chemical Reagent, catalog number: 10021418 )
- Antimycin A (Sigma-Aldrich, catalog number: A8674 )
- Raffinose (D-(+)-Raffinose pentahydrate, Bomei, CAS Number: 17629-30-0)
- Sodium acetate (Sinopharm Chemical Reagent, catalog number: 10018718 )
- Acetic acid (Sinopharm Chemical Reagent, catalog number: 10000218 )
- KH2PO4 (Sinopharm Chemical Reagent, catalog number: 10017618 )
- Na2HPO4 (Sinopharm Chemical Reagent, catalog number: 10020318 )
- TTC (Tokyo Chemical Industry (TCI), CAS Number: 298-96-4)
- YPD medium (1 L) (see Recipes)
- 1x TE Buffer (100 ml) (see Recipes)
- Single-stranded carrier DNA (2 mg/ml) (see Recipes)
- 1.0 M LiAc (100 ml) (see Recipes)
- 50% (w/v) PEG (100 ml) (see Recipes)
- CMD-W medium (1 L) (see Recipes)
- Antimycin A stock solution (50 mg/ml) (see Recipes)
- YPRAA medium (1 L) (see Recipes)
- 10 mM acetic acid-sodium acetate buffer (100 ml, pH = 4.7) (see Recipes)
- Phosphate Buffer (150 ml) (see Recipes)
- TTC Stock solution (2%) (see Recipes)
Equipment
- Pipettes (Mettler-Toledo International, RAININ, model: XLS )
- Incubator (ZHICHENG, model: ZXDP-B2050 )
- Clean bench (AIRTECH, model: SW-CJ-2FD , catalog number: A11062689)
- Water bath (Shanghai Jinghong Laboratory Instrument, model: DK-8D )
- Vortex mixer (Kylin-Bell Lab Instruments, model: VORTEX-5 )
- Incubator shaker (Changzhou Zhiborui Instrument Manufacturing, model: THZ-D )
- Centrifuge (Eppendorf, model: 5424 )
Procedure
文章信息
版权信息
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Yin, W., Wang, Y., Chen, T., Lin, Y. and Luo, C. (2018). Functional Evaluation of the Signal Peptides of Secreted Proteins. Bio-protocol 8(9): e2839. DOI: 10.21769/BioProtoc.2839.
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分类
植物科学 > 植物免疫 > 宿主-细菌相互作用
分子生物学 > 蛋白质 > 检测
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