Comparison of Different Signal Peptides for the Efficient Secretion of the Sweet-Tasting Plant Protein Brazzein in Pichia pastoris

Abstract

Brazzein is a small sweet-tasting protein found in the red berries of a West African evergreen shrub, Pentadiplandra brazzeana Baillon. Brazzein is highly soluble and stable over a large pH range and at high temperatures, which are characteristics that suggest its use as a natural sweetener. However, Pentadiplandra brazzeana culture is difficult at a large scale, limiting the natural source of brazzein. Heterologous expression of brazzein has been established in numerous systems, including bacteria, yeast, and transgenic plants. Brazzein requires four disulfide bonds to be active in eliciting an intense sweet taste, and the yeast Pichia pastoris appears to be one of the best options for obtaining functional brazzein in high quantities. Employing yeast secretion in the culture medium allows us to obtain fully active brazzein and facilitate purification later. To increase yeast secretion, we compared seven different signal peptides to successfully achieve brazzein secretion using the yeast P. pastoris. The brazzein proteins corresponding to these signal peptides elicited activation of the sweet taste receptor functionally expressed in a cellular assay. Among these tested signal peptides, three resulted in the secretion of brazzein at high levels.

Improved Production of Recombinant Myrosinase in Pichia pastoris

“…The deletion of both the N- and C-terminal native signal peptide sequences of alkaline phytase enhanced its (α-MF)-driven secretion in P. pastoris by 4-fold [ 19 ]. It has been previously reported that the selection of signal peptides is crucial for successful protein expression in P. pastoris and needs to be optimized for each protein individually [ 20 , 21 ].…”

“…The difference in size was probably the result of more extensive glycosylation either as a result of the extracellular expression or a different production strain. Previously, Härtel and Brandt [ 21 ] reported the extracellular production of MYR1 myrosinase from Brassica napus in P. pastoris GS115 (Mut + ). The size of the produced myrosinase corresponded well with the size of its glycosylated form, although a micro-heterogeneity of the pI was observed [ 22 ].…”

Expression of a triple mutational des‐pGlu brazzein in transgenic mouse milk

“…The highest expression level of the triple mutational des ‐pGlu brazzein in the milk of transgenic mice obtained in this study was 332.59 mg·L −1 , which is a bit low as a raw material to purify the recombinant brazzein protein. However, the expression level of randomly integrated transgenic animals is related to many factors, such as the integrations of the transgene [ 31 , 32 ], copy numbers, promoters [ 33 , 34 ] and the signal peptides [ 35 , 36 ]. The expression levels of brazzein in the milk of the mice produced in this study were very different, confirming this point.…”

Triterpenoid Saponins from the Cultivar “Green Elf” of Pittosporum tenuifolium

“…For functional experiments on the sweet taste receptor, HEK293T cells stably expressing the chimeric G-protein subunit Gα16gust44 were seeded into 96-well plates as previously described [16]. Then, 24 h after seeding, cells were transiently transfected with hTAS1R2 and hTAS1R3 cDNAs, cloned into pcDNA6 and pcDNA4 vectors (Life Technologies, Carlsbad, CA, USA), respectively, with plasmid pCMV-GCaMP5G (Addgene) used as a genetically encoded calcium indicator.…”