A toolbox of endogenous and heterologous nuclear localization sequences for the methylotrophic yeast Pichia pastoris

FEMS Yeast Research
Astrid WeningerThomas Vogl

Abstract

Nuclear localization sequences (NLSs) are required for the import of proteins in the nucleus of eukaryotes. However many proteins from bacteria or bacteriophages are used for basic studies in molecular biology, to generate synthetic genetic circuits or for genome editing applications. Prokaryotic recombinases, CRISPR-associated proteins such as Cas9 or bacterial and viral polymerases require efficient NLSs to function in eukaryotes. The yeast Pichia pastoris is a widely used expression platform for heterologous protein production, but molecular tools such as NLSs are limited. Here we have characterized a set of 10 NLSs for P. pastoris, including the first endogenous NLSs (derived from P. pastoris proteins) and commonly used heterologous NLSs. The NLSs were evaluated by fusing them in N- and C-terminal position to an enhanced green fluorescent protein showing pronounced differences in fluorescence levels and nuclear targeting. Thereby we provide a set of different NLSs that can be applied to optimize the nuclear import of heterologous proteins in P. pastoris, paving the way for the establishment of intricate synthetic biology applications.

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Citations

Dec 25, 2015·Fungal Genetics and Biology : FG & B·James M Wagner, Hal S Alper
Apr 5, 2017·Organic & Biomolecular Chemistry·M B QuinC Schmidt-Dannert
Nov 19, 2017·Scientific Reports·Kate E Royle, Karen Polizzi
Jan 20, 2018·G3 : Genes - Genomes - Genetics·Emily RoggenkampGregory C Finnigan
Jun 1, 2020·Applied Microbiology and Biotechnology·Luís C MartinsIsabel Sá-Correia
Dec 3, 2016·World Journal of Microbiology & Biotechnology·Zhen KangJian Chen
Feb 16, 2019·Fungal biology and biotechnology·Megan E GoeckelGregory C Finnigan
Apr 10, 2020·Applied Microbiology and Biotechnology·Endang Rahmat, Youngmin Kang
Aug 31, 2021·Applied Microbiology and Biotechnology·Nico D FessnerAnton Glieder

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Methods Mentioned

BETA
fluorescence microscopy
fluorescence spectroscopy

Software Mentioned

BLAST

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