Simplified, enhanced protein purification using an inducible, autoprocessing enzyme tag.
We introduce a new method for purifying recombinant proteins expressed in bacteria using a highly specific, inducible, self-cleaving protease tag. This tag is comprised of the Vibrio cholerae MARTX toxin cysteine protease domain (CPD), an autoprocessing enzyme that cleaves exclusively after a leucine residue within the target protein-CPD junction. Importantly, V. cholerae CPD is specifically activated by inositol hexakisphosphate (InsP(6)), a eukaryotic-specific small molecule that is absent from the bacterial cytosol. As a result, when His(6)-tagged CPD is fused to the C-terminus of target proteins and expressed in Escherichia coli, the full-length fusion protein can be purified from bacterial lysates using metal ion affinity chromatography. Subsequent addition of InsP(6) to the immobilized fusion protein induces CPD-mediated cleavage at the target protein-CPD junction, releasing untagged target protein into the supernatant. This method condenses affinity chromatography and fusion tag cleavage into a single step, obviating the need for exogenous protease addition to remove the fusion tag(s) and increasing the efficiency of tag separation. Furthermore, in addition to being timesaving, versatile, and inexpensive, our results indicate that the CPD purification system can enhance the expression, integrity, and solubility of intractable proteins from diverse organisms.
Shen
Aimee
A
Department of Pathology, Stanford School of Medicine, Stanford, California, United States of America.
Lupardus
Patrick J
PJ
Morell
Montse
M
Ponder
Elizabeth L
EL
Sadaghiani
A Masoud
AM
Garcia
K Christopher
KC
Bogyo
Matthew
M
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D003066:Coenzyme A Ligases / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000378:metabolism; D053508:Matrix Metalloproteinase 12 / Q000378:metabolism; D051379:Mice; D009842:Oligopeptides / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine; D010447:Peptide Hydrolases; D053508:Matrix Metalloproteinase 12; D003066:Coenzyme A Ligases; C051127:biotin-CoA ligase