Topic
Pichia pastoris
About: Pichia pastoris is a research topic. Over the lifetime, 7937 publications have been published within this topic receiving 162645 citations. The topic is also known as: Komagataella pastoris.
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TL;DR: The methylotrophic yeast, Pichia pastoris, has been used to express both human and murine macrophage inhibitory cytokine-1 (MIC-1), a transforming growth factor beta (TGF-β) superfamily cytokine, and the protein is secreted in its correctly folded dimeric form at milligram per litre quantities, which is significantly higher than using mammalian expression systems.
51 citations
TL;DR: A method to purify the full-length human and rat 11beta-HSD1 with retention of their natural oxidoreductase activities and exhibited dehydrogenase and reductase activities with K(M) values in agreement with those reported in the literature.
51 citations
TL;DR: The details of the fusion of up to 66 N‐terminal targeting sequences of fungal type II membrane proteins to 33 catalytic domains of heterologous glycosylation enzymes are reported.
Abstract: To humanize the glycosylation pathway in the yeast Pichia pastoris, we developed several combinatorial genetic libraries and used them to properly localize active eukaryotic mannosidases and sugar transferases. Here we report the details of the fusion of up to 66 N-terminal targeting sequences of fungal type II membrane proteins to 33 catalytic domains of heterologous glycosylation enzymes. We show that while it is difficult to predict which leader/catalytic domain will result in the desired activity, analysis of the fusion protein libraries allows for the selection of the leader/catalytic domain combinations that function properly. This combinatorial approach, together with a high-throughput screening protocol, has allowed us to humanize the yeast glycosylation pathway to secrete human glycoprotein with complex N-glycosylation.
51 citations
TL;DR: This introduction focuses directly on key ways that the P. pastoris expression system is different from other eukaryotic systems, particularly Escherichia coli.
Abstract: The construction of Pichia pastoris expression strains and the general growth and manipulation of this yeast expression system are in many ways similar to those of bacterial expression systems, particularly Escherichia coli. Because of this, it is typically easy for researches experienced with bacterial systems to make the jump to this eukaryotic system. However, because the system is similar, users can be falsely fooled into assuming that the system is completely bacterial-like and may waste time and effort performing experiments that are unlikely to yield the desired results with this yeast. To aid in preventing P. pastoris users from falling into one or more or these traps, this introduction focuses directly on key ways that the P. pastoris expression system is different.
51 citations
TL;DR: A Sec12–Sec16 interaction has a conserved role in ER export and tER organization and it is demonstrated that human Sec12 is concentrated at tER sites, likely due to association with a C-terminal fragment of Sec16A.
Abstract: COPII vesicles bud from an ER domain known as the transitional ER (tER). Assembly of the COPII coat is initiated by the transmembrane guanine nucleotide exchange factor Sec12. In the budding yeast Pichia pastoris, Sec12 is concentrated at tER sites. Previously, we found that the tER localization of P. pastoris Sec12 requires a saturable binding partner. We now show that this binding partner is Sec16, a peripheral membrane protein that functions in ER export and tER organization. One line of evidence is that overexpression of Sec12 delocalizes Sec12 to the general ER, but simultaneous overexpression of Sec16 retains overexpressed Sec12 at tER sites. Additionally, when P. pastoris Sec12 is expressed in S. cerevisiae, the exogenous Sec12 localizes to the general ER, but when P. pastoris Sec16 is expressed in the same cells, the exogenous Sec12 is recruited to tER sites. In both of these experimental systems, the ability of Sec16 to recruit Sec12 to tER sites is abolished by deleting a C-terminal fragment of Sec16. Biochemical experiments confirm that this C-terminal fragment of Sec16 binds to the cytosolic domain of Sec12. Similarly, we demonstrate that human Sec12 is concentrated at tER sites, likely due to association with a C-terminal fragment of Sec16A. These results suggest that a Sec12–Sec16 interaction has a conserved role in ER export.
51 citations