Stronger Binders, Better Probiotics: Evaluating Mucin Affinity in Yeast Strains for Probiotic Potential

Abstract

The goal of our lab is to determine if the yeast, Pichia pastoris, can survive and secrete recombinant proteins in the murine gut. To possibly increase the retention of P. pastoris in the gut two strains were created. Strain yJGGA contains an agglutinin protein and strain yJGG33 contains a fusion protein of agglutinin and mucin binding domain. Both agglutinin and mucin binding domain have affinity to mucin. Mucin is the major protein found in the mucus linings of the gut. The ability of these strains to bind mucin was determined by incubating the strains with mucin-coated glass coverslips. Coverslips were washed to remove unbound cells and bound cells were collected through vigorous pipetting. Bound cells were then counted by plating onto a selective medium. This binding assay suggested that yJGGA had higher affinity to mucin than yJGG33. Immunofluorescence microscopy was then used to examine the presence of agglutinin and mucin binding domain on the cell wall of the two P. pastoris strains. The results showed that only yJGGA expressed agglutinin on the cell wall, further supporting the results of the binding assay. The affinity of yJGGA to mucin suggests that this P. pastoris strain would have higher binding to the mucus lining of the gut. This adhesiveness may extend the time yJGGA lives in the gut compared to yJGG33 and wildtype P. pastoris