Signal peptide assay

Service Introduction

Detecting whether a protein's signal peptide has a secretory function and confirming whether a protein is a secreted protein is of central significance in both basic research and biotechnological applications. It directly affects whether a protein can be properly transported and perform its intended function. It not only addresses the basic biological question of 'where a protein works,' but also plays a key role in modern biotechnology in analyzing the pathogenic mechanisms of effector proteins from pathogenic microorganisms (such as bacteria and fungi).

Signal peptide secretion yeast assay:

Signal peptide secretion in yeast is detected using the pSUC2 yeast secretion system, which employs the YTK12 yeast strain that lacks the invertase gene. This strain cannot grow on media with sucrose as the sole carbon source. The selected vector plasmid pSUC2 contains the tryptophan synthesis gene and an invertase gene SUC2 that lacks a signal peptide and the start codon ATG. When the signal peptide to be tested is linked to the invertase gene in pSUC2, if the signal peptide has secretion function, it can restore the secretion of invertase, enabling the YTK12 strain to grow on defective media using raffinose as the carbon source. This can be further verified using the TTC colorimetric reaction.

Tobacco leaf signal peptide detection:

Detection of tobacco leaf signal peptides uses the non-toxic effector protein Avr3a from the oomycete pathogen Phytophthora and the corresponding potato resistance gene R3a in the host. R3a can recognize Avr3a within plant cells, triggering cell death. When Avr3a is secreted outside the plant cell by fusion with a signal peptide, it cannot be recognized by R3a and therefore does not induce cell death.

Service Advantages

▶Experimental results are reliable, and the images are visually appealing;

▶In addition to effector protein signal peptide detection experiments, services such as effector protein-induced/inhibited cell programmed death detection can also be provided, which can further enrich the experimental content.

Service Process

Signal peptide secretion yeast assay

Tobacco leaf signal peptide assay

Article Case

The Puccinia triticina effector Pt3372 suppresses wheat innate immunity by targeting wheat TaERP3 in TcLr2a and TcLr18

academic journal：Phytopathology Research

Pt3372 signal peptide has a secretory function. Functional validation of the putative N-terminal signal peptide of Pt3372 was carried out using the yeast invertase secretion assay and with 2,3,5-triphenyltetrazoliu chloride (TTC). a The recombinant vector pSUC2-SP3372 carrying Pt3372 signal peptide was transferred into YPRAA. pSUC2-SP3372 showed that only the signal peptide had secretory function could grow normally in YPRAA. b The Pt3372 ORF sequence and the ORF sequence without the signal peptide were fused in-frame to the invertase sequence in the pSUC2 vector. The pSUC2-Pt3372 and pSUC2-Pt3372ΔSP were used to indicate that only the yeast strain capable of secreting invertase could react with 2,3,5-triphenyl tetrazolium chloride (TTC) and reduce it to insoluble red 1,3,5-triphenyl formazan (TPF).

Applicable Fields

▶Identify the secretion function of target protein signal peptides and clarify the subcellular localization and transport mechanisms of proteins.

▶Perform high-throughput screening of efficient signal peptide sequences to optimize the secretion and expression efficiency of recombinant proteins (enzymes, antibodies, etc.).

▶Analyze the secretion mechanisms of pathogenic microbial effector proteins to support research on pathogenic mechanisms and the development of drug targets.