(789c) Detection of Peptide Biomarkers By Engineered Yeast Receptors

Access to low-cost, point-of-care (POC) diagnostics enables screening, diagnosis and treatment monitoring for a number of critical diseases. POC diagnostics, such as lateral flow sandwich immunoassays, nucleic acid amplification tests, and colorimetric chemical reactions, are available for proteins, DNA/RNA, and small molecule disease biomarkers. However, there is a technology gap for assaying peptides and small proteins biomarkers smaller than 15 kDa. These biomarkers are often too small to be simultaneously bound by two antibodies as required for sandwich immunoassays and instead require more sophisticated assays like immunoturbidimetric measurements and mass spectrometry, which can prove impractical and prohibitively expensive at the POC level. Thus, a cost-effective POC peptide diagnostic can improve early detection and treatment management for a range of diseases.

Bakerâ??s yeast (Saccharomyces cerevisiae) is a particularly promising vehicle for a POC peptide diagnostic because it is cheap, robust, and easily engineered with standard biological techniques. Active dry yeast is simple to manufacture and can be freeze-dried and reconstituted to provide easy distribution without requiring a cold-chain. Yeast contains a native G-protein coupled receptor (GPCR) which detects the 13 amino acid a-factor peptide. We hypothesized that Ste2p may be a useful starting point for directed evolution to produce receptors for peptide biomarkers that cannot currently be assayed at the POC.

Directed evolution has been used to evolve GPCRs to detect small molecules, but not peptides. Directed evolution experiments ideally start with proteins that have a low basal activity that can be improved upon. However, most peptide biomarkers have sequences that are very dissimilar from α-factor, so Ste2p is not likely to have basal activity for the peptide biomarker. The sequence dissimilarity, representing a large evolutionary distance, is challenging as many simultaneous mutations would likely be required for the receptor to detect the biomarker. To overcome this difficulty, we chose substrate walking as an alternative technique for covering large evolutionary distances. In substrate walking, an enzyme is evolved along a pathway of chimeric substrates which contains features of both the native and desired substrates before being evolved for the desired substrate. Substrate walking with peptides-receptor pairs has not been explored, so we investigated the feasibility and constraints of this strategy.

Here, we chose a peptide biomarker of chronic kidney disease (CKD). CKD, the 12^thleading cause of death worldwide, affects 10% of the adult American population and is a significant, emerging burden in low and middle income countries. As CKD worsens, cystatin C accumulates in the serum and urine. Clinically, cystatin C satisfies the criteria for a universal biomarker as it is independent of age, weight, and muscle mass and is more sensitive to early-onset renal failure compared to creatinine, a commonly used biomarker for CKD. Cystatin C is typically measured with a nephelometer, which is costly and requires substantial infrastructure to use and maintain. We used tryptic digest to ensure heterogeneous degradation products are cleaved into standard peptides. Trypsin digestion of cystatin C yields an 11 amino acid peptide that can serve as a CKD biomarker.

In this study, we demonstrate YBBs as a promising, versatile platform for POC diagnostics. We present a generalizable platform for producing peptide biomarker receptors using substrate walking and directed evolution, and validate the approach by developing a YBB for cystatin C, a biomarker for renal failure. Finally, we demonstrate the clinical feasibility of the YBB by showing functionality in pooled human urine, a relevant sample matrix. To our knowledge, this is the first demonstration of engineering a GPCR to detect a fully orthogonal peptide sequence.