Abstract
BackgroundProteins can be secreted from a host organism with the aid of N-terminal secretion signals. The budding yeast Pichia pastoris (Komagataella sp.) is widely employed to secrete proteins of academic and industrial interest. For this yeast, the most commonly used secretion signal is the N-terminal portion of pre-pro-α-factor from Saccharomyces cerevisiae. However, this secretion signal promotes posttranslational translocation into the endoplasmic reticulum (ER), so proteins that can fold in the cytosol may be inefficiently translocated and thus poorly secreted. In addition, if a protein self-associates, the α-factor pro region can potentially cause aggregation, thereby hampering export from the ER. This study addresses both limitations of the pre-pro-α-factor secretion signal.ResultsWe engineered a hybrid secretion signal consisting of the S. cerevisiae Ost1 signal sequence, which promotes cotranslational translocation into the ER, followed by the α-factor pro region. Secretion and intracellular localization were assessed using as a model protein the tetrameric red fluorescent protein E2-Crimson. When paired with the α-factor pro region, the Ost1 signal sequence yielded much more efficient secretion than the α-factor signal sequence. Moreover, an allelic variant of the α-factor pro region reduced aggregation of the E2-Crimson construct in the ER. The resulting improved secretion signal enhanced secretion of E2-Crimson up to 20-fold compared to the levels obtained with the original α-factor secretion signal. Similar findings were obtained with the lipase BTL2, which exhibited 10-fold enhanced secretion with the improved secretion signal.ConclusionsThe improved secretion signal confers dramatic benefits for the secretion of certain proteins from P. pastoris. These benefits are likely to be most evident for proteins that can fold in the cytosol and for oligomeric proteins.
Highlights
Proteins can be secreted from a host organism with the aid of N-terminal secretion signals
We reported that replacement of the α-factor signal sequence with the Ost1 signal sequence strongly increased secretion of monomeric superfolder GFP (msGFP) in P. pastoris [23]
It will be interesting to test whether the improved secretion signal enhances the secretion from P. pastoris of other proteins, proteins that can fold prior to translocation and proteins that oligomerize in the endoplasmic reticulum (ER), and whether these enhancements are seen at high cell densities in bioreactor-scale fermentations
Summary
Proteins can be secreted from a host organism with the aid of N-terminal secretion signals. The budding yeast Pichia pastoris (Komagataella sp.) is widely employed to secrete proteins of academic and industrial interest For this yeast, the most commonly used secretion signal is the N-terminal portion of pre-pro-α-factor from Saccharomyces cerevisiae. The α-factor signal sequence is removed by a signal peptidase in the ER lumen, and the pro region is cleaved by the Kex processing protease in the Golgi [10, 11] This bipartite secretion signal has proven to be effective for secreting multiple heterologous proteins in P. pastoris, but the level of secretion varies widely, prompting efforts to improve secretion efficiency [4, 12,13,14,15,16]. Several of those attempts focused on modifying the α-factor secretion signal [17,18,19,20]
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