PHB-intein-mediated protein purification strategy.
A method has been developed that eliminates the need for complex chromatographic apparatus in the purification of recombinant proteins expressed in Escherichia coli. This method is similar to conventional affinity-tag separations, but the affinity resin is replaced by polyhydroxybutyrate (PHB) particles prodced in vivo in the E. coli expression host during protein expression. A PHB-binding protein known as a phasin is genetically fused to the product protein via an engineered pH and temperature dependent self-cleaving intein linker. Thus the phasin-sion acts as a self-cleaving purification tag, with affinity for the co-expressed PHB granules. The PHB particles and tagged target protein are purified by lysing the cells and washing the granules with sequential rounds of centrifugation and resuspension. The native target protein is then released from the bound tag through an intein-mediated self-cleavage reaction, induced by a mild pH shift. A final round of centrifugation removes the granules and associated tag, allowing the purified target to be recovered in the supernatant. This method has been shown to yield 35-40 microg of purified product per milliliter of liquid cell culture and is likely to be applicable to a wide range of expression hosts.
Gillies
Alison R
AR
Department of Chemical Engineering, Princeton University, Princeton, NJ, USA.
Mahmoud
Reza Banki
RB
Wood
David W
DW
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003594:Cytoplasmic Granules / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006885:Hydroxybutyrates / Q000378:metabolism*; D047668:Inteins; D037121:Plant Lectins / Q000235:genetics / Q000378:metabolism; D011091:Polyesters / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013696:Temperature; D014170:Transformation, Genetic; D000094122:Polyhydroxybutyrates
D002352:Carrier Proteins; D006885:Hydroxybutyrates; D037121:Plant Lectins; D011091:Polyesters; D011993:Recombinant Fusion Proteins; C008825:phasin; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates