Multiprotein expression strategy for structural biology of eukaryotic complexes.
The concept of the cell as a collection of multisubunit protein machines is emerging as a cornerstone of modern biology, and molecular-level study of these machines in most cases will require recombinant production. Here, we present and validate a strategy to rapidly produce, permutate, and posttranslationally modify large, eukaryotic multiprotein complexes by using DNA recombination in a process that is fully automatable. Parallel production of 12 protein complex variants within a period of weeks resulted in specimens of sufficient quantity and homogeneity for structural biology applications.
Fitzgerald
Daniel J
DJ
ETH Zürich, Institut für Molekularbiologie und Biophysik, ETH-Hönggerberg, CH-8093 Zürich, Switzerland.
Schaffitzel
Christiane
C
Berger
Philipp
P
Wellinger
Ralf
R
Bieniossek
Christoph
C
Richmond
Timothy J
TJ
Berger
Imre
I
D000818:Animals; D016367:Baculoviridae / Q000737:chemistry / Q000235:genetics; D002460:Cell Line; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D046912:Multiprotein Complexes / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D018411:Spodoptera / Q000737:chemistry / Q000235:genetics
D046912:Multiprotein Complexes