The methylotrophic yeast Komagataella phaffii is often used as a host for the extracellular production of heterologous proteins in the biotechnology industry. The selection of a suitable promoter and signal sequence are of crucial importance for production efficiency, as they influence the final product titer. To evaluate the influence of the signal sequence and promoters on target protein production, we constructed 12 plasmids, which we successfully introduced into the PIPAg_0016 strain of K. phaffii. We evaluated the level of secretion of the target heterologous protein Ty1, and the plasmids used encoded different promoters (PAOX1 or PUPP) and signal sequences. By analysing the production of the target protein before and after cell sonification, we observed that most of the protein was secreted into the extracellular space and only a small portion remained intracellular, which is very desirable in industrial production processes. We confirmed the expression of the heterologous protein with the SDS-PAGE method and western blot immunodetection. The production of the heterologous protein Ty1 was higher when the PAOX1 promoter was used. The titer was the highest when this promoter was combined with the S3 signal sequence, which is a modified mating factor alpha (α-MF) from Saccharomyces cerevisiae. By applying PUPP promoter, we observed the highest protein titers when combined with the S1 signal sequence, which is the unmodified α-MF from S. cerevisiae. However, in both cases incomplete processing of the signal sequence occurred. Therefore, a signal sequence that leads to a lower titer, but complete processing of the target protein would be a better choice for heterologous protein production.
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