Recombinant protein linker production as a basis for non-invasive determination of single-cell yeast age in heterogeneous yeast populations

Abstract

The physiological and metabolic diversity of a yeast culture is the sum of individual cell phenotypes. As well as environmental conditions, genetics, and numbers of cell divisions, a major factor influencing cell characteristics is cell age. A postcytokinesis bud scar on the mother cell, a benchmark in the replicative life span, is a quantifiable indicator of cell age, characterized by significant amounts of chitin. We developed a binding process for visualizing the bud scars of Saccharomyces pastorianus var. carlsbergensis using a protein linker containing a polyhistidine tag, a superfolder green fluorescent protein (sfGFP), and a chitin-binding domain (His6-SUMO-sfGFP-ChBD). The binding did not affect yeast viability; thus, our method provides the basis for non-invasive cell age determination using flow cytometry. The His6-SUMO-sfGFP-ChBD protein was synthesized in Escherichia coli, purified using two-stage chromatography, and checked for monodispersity and purity. Linker-cell binding and the characteristics of the bound complex were determined using flow cytometry and confocal laser scanning microscopy (CLSM). Flow cytometry showed that protein binding increased to 60 455 ± 2706 fluorescence units per cell. The specific coupling of the linker to yeast cells was additionally verified by CLSM and adsorption isotherms using yeast cells, E. coli cells, and chitin resin. We found a relationship between the median bud scar number, the median of the fluorescence units, and the chitin content of yeast cells. A fast measurement of yeast population dynamics by flow cytometry is possible, using this protein binding technique. Rapid qualitative determination of yeast cell age distribution can therefore be performed.