Engineering Yeast to Develop a Detection Method for the Pancreatic Cancer Biomarker Glypican-1

Methods

Summary

In order to express the CR1 receptor protein in our desired yeast strain, S. cerevisiae, we custom synthesized our construct from IDT which includes the protein coding gene sequence and an N terminal hemagglutinin (HA) tag. 

Upon restriction enzyme digestion and ligation of our construct with a plasmid shuttle vector, the plasmid DNA will be isolated from colonies grown up by 5-alpha E coli. and transformed into our desired yeast strain using a lithium acetate/PEG/single stranded DNA carrier method adapted from a Nature protocol. 

After the yeast cells are transformed, they will be fixed and freeze fractured to visualize CR1 expression by probing the HA tag.

The recombinant yeast cells will be transformed with a c-src yeast expression plasmid to study downstream phosphorylation of the c-src mitogen activated protein kinase upon binding of CR1 with glypican-1. 

Challenges

The main challenge that we foresee with this project lies in successful insertion of CR1 into the yeast membrane without subsequent interference from the extracellular matrix or cell wall of S. cerevisiae. Due to the possibility of intracellular cleavage of the HA tag from the N terminus. we plan to supplement our freeze fracture test with Western blots using anti-CR1 as a secondary means of validating CR1 expression. We also plan to utilize site directed mutagenesis to introduce a mutation into the peptide signal sequence of CR1 to potentially circumvent N terminal cleavage of our tag by a peptidase. 

As the N terminal HA tag may interfere with the biological activity of the CR1 receptor, our construct was synthesized with unique restriction sites flanking the HA tag to facilitate removal via restriction enzyme digestion once we can confirm membrane expression. 

Pre Analysis Plan

In order to select for transformants containing our recombinant CR1 plasmid, a URA3 marker in our shuttle vector will be used as we will grow our strains on ura- media. The genotype of the plasmid containing our CR1 construct will be confirmed via sequencing and digestion using a unique cutter located only in our construct. We will validate membrane expression of CR1 by fixing our yeast cells and using freeze fracture to test for presence of the HA tag. While we expect the fluorescence to be localized to the membrane, there is potential for it to be localized within the cell, indicating cleavage of the tag- we plan to address this via means described in our "Challenges" section. When testing for phosphorylation of the c-src mitogen kinase upon interaction of glypican with CR1, we plan to test this against cells lacking the CR1 membrane protein to determine baseline phosphorylation and potentially develop mathematical modeling for the correlation of c-src phosphorylation to CR1 receptor activation.