Yeast Signal Peptide Testing

The yeast expression system is a highly efficient exogenous protein expression system that has been widely used to express and produce a variety of exogenous proteins. The signal peptide on the expression vector can guide the secretion of exogenous proteins well which brings convenience to the isolation and purification of the expressed proteins.

To meet your research needs, Lifeasible uses yeast signal peptide screening to verify the secretory function of signal peptides. The CDS region or signal peptide region of the gene which carrying the signal peptide is constructed into a suitable vector. A functional signal peptide ligated to the sucrose convertase gene of pSUC2 restores the secretory function of sucrose convertase, allowing the YTK12 strain to grow on cottonseed sugar-deficient medium as a carbon source. In addition, the TTC chromogenic reaction was used to verify the results obtained from the screening.

The experimental protocol we provide

• The signal peptide region of the candidate-secreted protein was predicted by SignalP, then constructed into the pSUC2 vector, which was resistant to ampicillin.
• The YTK12 strain was cultured on YPDA culture, then the constructed corresponding vector was introduced into YTK12 strain and coated on CMD-W medium plates.
• Positive clones were picked onto YPRAA medium with cottonseed sugar as a carbon source and cultured. If the transferred yeast cells can grow in the YPRAA medium, it proves that the signal peptide has a secretory function. Otherwise, it proves that the signal peptide has no secretory function.
• The positive transformants from the above step are cultured in a CMD-W medium, and the cell suspension is collected and centrifuged to remove the supernatant. The precipitate was resuspended in resuspension solution and incubated at 37°C for 10 min. after centrifugation, the supernatant was added to the tube containing TTC. The untransformed YTK12 yeast was used as a negative control, and the color change was observed and photographed. The signal peptide has a secretory function if the color turns red.

General service flow

• Fusion expression vector construction
• Yeast transformation and identification
• Spotting to show results

You will need to provide the gene CDS sequence and template, and Lifeasible will carry out the project according to your customized protocol. We will deliver detailed raw data, images of results, and standard lab reports to help you with your research. Please feel free to contact us for your exclusive proposal.