Recombinant protein purification by self-cleaving elastin-like polypeptide fusion tag.
This unit presents a rapid and simple method for the nonchromatographic purification of recombinant proteins expressed in E. coli. This method relies on a thermally responsive elastin-like polypeptide (ELP) tag, where the tagged protein is precipitated using a mild temperature shift. The tag is then induced to self-cleave by a mild pH shift and is subsequently removed by a final thermal precipitation. The result is a purified native protein target, without the requirement for affinity apparatus or protease removal of the tag. This protocol describes the required cloning methods to insert a given target into the expression vector, as well as the general method for purifying the resulting expressed protein.
Wu
Wan-Yi
WY
Princeton University, Princeton, New Jersey.
Fong
Baley A
BA
Princeton University, Princeton, New Jersey.
Gilles
Allison G
AG
Princeton University, Princeton, New Jersey.
Wood
David W
DW
Princeton University, Princeton, New Jersey.
D003001:Cloning, Molecular / Q000379:methods; D004549:Elastin / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D011506:Proteins; D011993:Recombinant Fusion Proteins; D004549:Elastin