Methods for expression of recombinant proteins using a Pichia pastoris cell-free system
File(s)cpps.115.pdf (1.71 MB)
Published version
Author(s)
Aw, Rochelle
Spice, Alex J
Polizzi, Karen
Type
Journal Article
Abstract
Cell‐free protein synthesis is a powerful tool for engineering biology and has been utilized in many diverse applications, from biosensing and protein prototyping to biomanufacturing and the design of metabolic pathways. By exploiting host cellular machinery decoupled from cellular growth, proteins can be produced in vitro both on demand and rapidly. Eukaryotic cell‐free platforms are often neglected due to perceived complexity and low yields relative to their prokaryotic counterparts, despite providing a number of advantageous properties. The yeast Pichia pastoris (also known as Komagataella phaffii) is a particularly attractive eukaryotic host from which to generate cell‐free extracts, due to its ability to grow to high cell densities with high volumetric productivity, genetic tractability for strain engineering, and ability to perform post‐translational modifications. Here, we describe methods for conducting cell‐free protein synthesis using P. pastoris as the host, from preparing the cell lysates to protocols for both coupled and linked transcription‐translation reactions. By providing these methodologies, we hope to encourage the adoption of the platform by new and experienced users alike.
Date Issued
2020-12-01
Date Acceptance
2020-09-22
Citation
Current protocols in protein science, 2020, 102 (1)
ISSN
1934-3655
Publisher
Wiley-Blackwell
Journal / Book Title
Current protocols in protein science
Volume
102
Issue
1
Copyright Statement
© 2020 The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
License URL
Sponsor
Engineering & Physical Science Research Council (EPSRC)
Engineering & Physical Science Research Council (EPSRC)
Grant Number
EP/K038648/1
EP/R013764/1
Subjects
Komagataella phaffii
Pichia pastoris
cell-free protein synthesis
eukaryotic cell-free expression system
in vitro transcription-translation
rapid prototyping
synthetic biology
Publication Status
Published
Article Number
ARTN e115
Date Publish Online
2020-10-27