(608c) Quantitative Analysis of Protein-Protein Interactions Using a Yeast-Yeast Two Hybrid Model
AIChE Annual Meeting
Wednesday, November 13, 2019 - 4:06pm to 4:24pm
Current methods for studying PPIs include yeast two hybrid (Y2H) and protein fragment complementation (PCA) assays where the protein of interest (âbaitâ) and its putative binding partner (âpreyâ) are fused to two different protein domains (Y2H) or protein fragments (PCA). Binding of the bait to the prey causes colocalization of the domains (Y2H) resulting in the stimulation of a readable output, like reporter gene activation, or reconstitution of a functional protein, like luciferase, by complementation of protein fragments (PCA). However, these methods are limited in scope as the affinities of binding interactions cannot be efficiently quantitatively assessed or rank-ordered. Previous Y2H systems have incorporated immunofluorescence to quantitatively assess prey-bait binding when the bait is expressed as a cell surface fusion and the prey is solubly expressed. Bait-prey binding can be detected using an antibody specific to a tag fused to the soluble prey protein. However, immunofluorescent detection may too insensitive to identify low affinity PPIs due to nonspecific background binding of the antibodies to the cell surface. The use of spilt fluorescent proteins can overcome detection limitations as the fluorescent signal will only occur if the bait and prey bind. However, the correlation between the interaction binding affinity and assay readout is complex due to the use of a spilt protein.
Here we describe a quantitative yeast-yeast two hybrid (qYY2H) that uses a luciferase-based assay for identification of putative PPIs and quantitative assessment of binding strength. In our system, the bait and prey proteins are expressed as yeast cell surface fusions using the yeast display platform. The bait cells also co-display an iron oxide binding protein as a cell surface fusion, allowing magnetization of the bait cells. Prey cells can be incubated with magnetized bait cells and bait-prey complexes can be isolated using a magnet. The prey cells also co-express an engineered luciferase. Standard curves comparing luminescence and known quantities of prey cells can be generated enabling the use of the luciferase assays to estimate the exact number of prey cells complexed with the bait cells.
Using qYY2H, we can identify even low affinity interactions (micromolar KD) due to the high avidity of interaction between the bait and prey cells along with the high sensitivity of luciferase assays. Specifically, we co-expressed an engineered luciferase on the surface of prey yeast cells along with two previously identified Sso7d mutants for the Fc portion of human IgG (hFc), M1 and M2. The prey cells were incubated with magnetized bait cells expressing hFc and bait-prey complexes were isolated using a magnet. If the surface expression levels of the prey proteins are reasonably similar, luciferase activity can be used to quantify the strength binding interactions. Using this system, the binding affinities of M1 (KD~400 nM) and M2 (KD~5.3 Î¼M) could be ranked-ordered with M1 cells being captured at rate ~4 times higher than the M2 cells. The signal from M2 in this assay was ~10 fold higher than the detection limit of the luciferase assay. These results suggest that binding affinities even lower than KD~5.3 Î¼M may be assessed using this approach. We discuss extension of this system to estimate numerical binding affinity values for bait-prey interactions using a non-linear regression model as well as evaluating this system to rank order affinities of putative binders for a target protein isolated from a cDNA library. Lastly, we discuss the use of this system to analyze the interactions of proteins mediated by post-translational modifications, which cannot be studied with any current approaches for studying PPIs.