An Efficient Genome-Wide Fusion Partner Screening System for Secretion of Recombinant Proteins in Yeast

Abstract

To produce rarely secreted recombinant proteins in the yeast Saccharomyces cerevisiae, we developed a novel genome-wide optimal translational fusion partner (TFP) screening system that involves recruitment of an optimal secretion signal and fusion partner. A TFP library was constructed from a genomic and truncated cDNA library by using the invertase-based signal sequence trap technique. The efficiency of the system was demonstrated using two rarely secreted proteins, human interleukin (hIL)-2 and hIL-32. Optimal TFPs for secretion of hIL-2 and hIL-32 were easily selected, yielding secretion of these proteins up to hundreds of mg/L. Moreover, numerous uncovered yeast secretion signals and fusion partners were identified, leading to efficient secretion of various recombinant proteins. Selected TFPs were found to be useful for the hypersecretion of other recombinant proteins at yields of up to several g/L. This screening technique could provide new methods for the production of various types of difficult-to-express proteins.

A novel fusion partner for enhanced secretion of recombinant proteins in Saccharomyces cerevisiae

“…In this regard, we developed a novel fusion partner by using the S. cerevisiae ER membrane protein, Voa1p. In a previous report (Bae et al 2015), we discovered that Voa1p is secreted at high levels (grams per liter) into the extracellular medium by S. cerevisiae when the TM domain was removed. Therefore, we expected that the truncated protein would have great potential as a fusion partner for soluble expression of target proteins in S. cerevisiae.…”

“…To analyze the secreted proteins from fed-batch fermentation, 5–10 μl of culture supernatant was directly used for SDS-PAGE after mixing with 2× SDS-PAGE sample buffer, and then stained with Coomassie blue. Total intracellular yeast protein was prepared from the cells by post-alkaline extraction (Bae et al 2015). A polyclonal antibody to hIL-2 (R&D Systems Inc., Minneapolis, MN, USA) and an anti-goat IgG alkaline phosphatase conjugate (Sigma Chemical Co., St. Louis, MO, USA) was used for western blot analysis.…”

“…We previously developed a translational fusion partner (TFP) technology that provides optimal fusion partners for secretory production of otherwise poorly secreted proteins in yeast (Bae et al 2015). In the course of TFP screening from yeast genome, we discovered that the VOA1 protein was abundantly secreted into extracellular medium when the C-terminal transmembrane domain was deleted and overexpressed under the control of the strong promoter.…”

Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms

“…Due to cell compartmentalization, yeast micro-organisms are capable of correct protein folding and further have the ability to perform typical eukaryotic PTMs including N-and, to some extent, O-linked glycosylation, phosphorylation, sulfation, and ubiquitination as described and reviewed by others (Irani, Kerkhoven, Shojaosadati, & Nielsen, 2015;Kim, Yoo, & Kang, 2015;Ptacek et al, 2005). Heterologous protein secretion can be achieved by the use of an appropriate signal peptide (Bae et al, 2015;Čiplys et al, 2015). Heterologous protein secretion can be achieved by the use of an appropriate signal peptide (Bae et al, 2015;Čiplys et al, 2015).…”

“…Based on the lack of human-like glycosylation, yeast strains are mainly used for the commercial manufacturing of smaller proteins, hormones, and vaccines, with several products currently in the market (Cao et al, 2018;Mattanovich et al, 2011;Nielsen, 2013). Heterologous protein secretion can be achieved by the use of an appropriate signal peptide (Bae et al, 2015;Čiplys et al, 2015). In contrast to N-glycosylation in mammalian cells, yeast performs hypermannosylation and lacks the ability to generate sialylated N-glycans (Tang et al, 2016).…”