Affinity-tagged green fluorescent protein (GFP) extraction from a clarified E. coli cell lysate using a two-phase aqueous micellar system.
Green fluorescent protein (GFP) has been proposed as an ideal choice for a protein-based biological indicator for use in the validation of decontamination or disinfection treatments. In this article, we present a potentially scalable and cost-effective way to purify recombinant GFP, produced by fermentation in Escherichia coli, by affinity-enhanced extraction in a two-phase aqueous micellar system. Affinity-enhanced partitioning, which improves the specificity and yield of the target protein by specific bioaffinity interactions, has been demonstrated. A novel affinity tag, family 9 carbohydrate-binding module (CBM9) is fused to GFP, and the resulting fusion protein is affinity-extracted in a decyl beta-D-glucopyranoside (C10G1) two-phase aqueous micellar system. In this system, C10G1 acts as phase forming and as affinity surfactant. We will further demonstrate the implementation of this concept to attain partial recovery of affinity-tagged GFP from a clarified E. coli cell lysate, including the simultaneous removal of other contaminating proteins. The cell lysate was partitioned at three levels of dilution (5x, 10x, and 40x). Irrespective of the dilution level, CBM9-GFP was found to partition preferentially to the micelle-rich phase, with the same partition coefficient value as that found in the absence of the cell lysate. The host cell proteins from the cell lysate were found to partition preferentially to the micelle-poor phase, where they experience less excluded-volume interactions. The demonstration of proof-of-principle of the direct affinity-enhanced extraction of CBM9-GFP from the cell lysate represents an important first step towards developing a cost-effective separation method for GFP, and more generally, for other proteins of interest.
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Priscila G
PG
Mazzola
Henry
H
Lam
Mojgan
M
Kavoosi
Charles A
CA
Haynes
Adalberto
A
Pessoa
Thereza Christina Vessoni
TC
Penna
Daniel I C
DI
Wang
Daniel
D
Blankschtein
D000345:Affinity Labels / Q000737:chemistry*; D000465:Algorithms; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D005960:Glucosides / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000302:isolation & purification*; D008823:Micelles; D044367:Phase Transition; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D013501:Surface-Active Agents / Q000737:chemistry; D020124:Thermotoga maritima / Q000201:enzymology / Q000235:genetics; D014867:Water / Q000737:chemistry; D014995:Xylosidases / Q000235:genetics
D000345:Affinity Labels; D005960:Glucosides; D008823:Micelles; D011993:Recombinant Fusion Proteins; D013501:Surface-Active Agents; D014867:Water; D049452:Green Fluorescent Proteins; D014995:Xylosidases; C504907:decyl glucoside