Polycistronic expression and purification of the ESCRT-II endosomal trafficking complex.
Eukaryotic cells use sophisticated mechanisms to direct protein traffic between subcellular compartments. In eukaryotic cells, transmembrane proteins are delivered for degradation in the lysosome or yeast vacuole via multivesicular bodies. The sorting of proteins into lumenal vesicles within multivesicular bodies is directed by the three ESCRT protein complexes. Here we describe the expression and purification of the ESCRT-II complex using the polycistronic expression vector pST39 developed by Tan. In a modification of Tan's procedure, Pfu polymerase amplification with overlapping oligonucleotides was used to generate the translation cassettes for subcloning into pST39 expression vector in a single step. This approach reduces the number of restriction sites and subcloning steps required to express a heterooligomeric protein complex, facilitating rapid screening of multiple complexes and complex variants for crystallization or biochemical characterization.
Aitor
A
Hierro
Jaewon
J
Kim
James H
JH
Hurley
D001483:Base Sequence; D002850:Chromatography, Gel; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes / Q000378:metabolism*; D005822:Genetic Vectors; D021381:Protein Transport; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins