Optimization of Protein Folding for Improved Secretion of Human Serum Albumin Fusion Proteins in Saccharomyces cerevisiae

Abstract

The successful expression and secretion of recombinant proteins in cell factories significantly depend on the correct folding of nascent peptides, primarily achieved through disulfide bond formation. Thus, optimizing cellular protein folding is crucial, especially for proteins with complex spatial structures. In this study, protein disulfide isomerases (PDIs) from various species were introduced into Saccharomyces cerevisiae to facilitate proper disulfide bond formation and enhance recombinant protein secretion. The impacts of these PDIs on recombinant protein production and yeast growth metabolism were evaluated by substituting the endogenous PDI1. Heterologous PDIs cannot fully compensate the endogenous PDI. Furthermore, protein folding mediators, PDI and ER oxidoreductase 1 (Ero1), from different species were used to increase the production of complex human serum albumin (HSA) fusion proteins. The validated folding mediators were then introduced into unfolded protein response (UPR)-optimized strains, resulting in a 7.8-fold increase in amylase-HSA and an 18.2-fold increase in albiglutide compared with the control strain. These findings provide valuable insights for optimizing protein folding and expressing HSA-based drugs.

Efficient Escorting Strategy for Aggregation-Prone Notch EGF Repeats with Sparcl1

“…The effect on EGF14-15-(G 3 S) 2 -His 6 secretion by co-transfection with mSparcl1-Flag, instead of generating mSparcl1 fusion proteins, was limited, making the chaperone-like activity of mSparcl1 unlikely. Thus, similar to fusion partners for the efficient secretion of recombinant proteins in yeast [ 23 , 24 ], the secretability of mSparcl1 may contribute to the increased secretion of aggregation-prone recombinant proteins. To assess the general utility of mSparcl1 fusion for efficient escort function, we tested delta-like homolog 1 as another fusion partner of mSparcl1, as its ectodomain was efficiently secreted from HEK293T cells into the culture medium [ 25 , 26 ].…”