Academic Interests

Research Interests: Bioseparations in Two-Phase Aqueous Micellar Systems,

Affinity-Enhanced Extractions, Surfactant Science and Engineering
Thesis Advisors: aniel !" #" $ang and aniel Blan%schtein
Research Description
&iquid-liquid extraction in two-phase aqueous complex-fluid systems has 'een
proposed as a scala'le, (ersatile, and cost-effecti(e purification method for the
downstream processing of 'iotechnological products, such as proteins and (iruses"
Many nonionic surfactants )such as those 'elonging to the al%yl poly)ethylene oxide*,
#iEi family* and +witterionic surfactants )such as dioctanoyl phosphatidyl choline, #,-
lecithin* exhi'it phase separation in water at suita'le temperatures into a micelle-rich
phase coexisting with a micelle-poor phase, thus forming two-phase aqueous micellar
systems" The low surfactant concentrations and low phase separating temperatures,
com'ined with the relati(ely mild and nondenaturing en(ironment, suggests that
these systems are potentially useful as extractant phases for the purification and
concentration of proteins and other 'iomaterials" !n particular, this type of liquid-liquid
extraction, also %nown as cloud point extraction, should 'e particularly suita'le for
large-scale production due to its lower cost and easier scale-up, when compared to
chromatographic operations"
#areful choices of the phase-forming surfactants or surfactant mixtures allow these
two-phase aqueous micellar systems to separate 'iomolecules 'ased on
hydropho'icity, si+e, charge, or specific affinity" !n one of the first applications
en(isioned for this type of systems, mem'rane-'ound proteins were extracted
preferentially to the micelle-rich phase, 'y (irtue of the hydropho'ic interactions
'etween the oily micellar core and the hydropho'ic patches on the surface of the
protein" -#" Bordier, J. Biol. Chem., 256(4), ./01 ).2,.*3" Pre(ious experimental and
theoretical in(estigation of the partitioning 'eha(ior of se(eral water-solu'le proteins,
as well as of se(eral 'acteriophages, showed that 'iomolecules tend to partition
preferentially into the micelle-poor phase, 'ased on their si+e, where they experience
less excluded-(olume interactions" -#"&" &iu, 4" 5i%as, and " Blan%schtein, Biotechnol.
Bioeng., 52, .,6 ).22/*3" 7urther impro(ement of the separation was achie(ed 'y
introducing electrostatic interactions" By adding a small amount of the anionic
surfactant sodium dodecylsulfate )SS* to the nonionic surfactant system, the
negati(ely-charged mixed micelles that form attract the positi(ely-charged protein
lyso+yme, leading to an impro(ed yield in the micelle-rich phase -" 8amei, 9" 8ing, "
$ang, and " Blan%schtein, Biotechnol. Bioeng., 80, :;; ):00:*3" Similarly, the
negati(ely-charged en+yme glucose-/-phosphate dehydrogenase )</P* was shown
to partition more extremely in two-phase aqueous micellar systems with added
cationic cosurfactants, al%yltrimethylammonium 'romide )#iTAB* -#" 4agui, =" &am,
" 8amei, " $ang, A" Pessoa-9r, and " Blan%schtein, Biotechnol. Bioeng., 82, 116
):00;*3"
!n the present study, we attempted to impro(e the yield and specificity of the
'ioseparation 'y introducing specific affinity" !n particular, we ha(e in(estigated the
affinity-enhanced partitioning of a model affinity-tagged protein - green fluorescent
protein fused to a family 2 car'ohydrate-'inding module )#BM2-<7P* - in a two-phase
aqueous micellar system generated from the nonionic surfactant n-decyl >--
glucopyranoside )#.0<.*, which acts simultaneously as the phase-forming surfactant
and the affinity ligand" !n this simple system, #BM2-<7P was extracted preferentially
into the micelle-rich phase, in spite of the opposing tendency of the steric, excluded-
(olume interactions operating 'etween the protein and the micelles" $e o'tained
more than a six-fold increase )from 0"1? to ;".* in the protein partition coefficient )8p*
as compared to a control case where the affinity interactions were @turned off@ 'y the
addition of a competiti(e inhi'itor )glucose*" !t was demonstrated conclusi(ely that
the o'ser(ed increase in 8p can 'e attri'uted to the specific affinity 'etween the #BM2
domain and the affinity surfactant #.0<., suggesting that the method can 'e generally
applied to any #BM2-tagged protein"
To rationali+e the o'ser(ed phenomenon of affinity-enhanced partitioning in two-phase
aqueous micellar systems, we formulated a theoretical framewor% to model the
protein partition coefficient" The modeling approach accounts for 'oth the excluded-
(olume interactions and the affinity interactions 'etween the protein and the
surfactants, and considers the contri'utions from the monomeric and the micellar
surfactants separately" The model was shown to 'e consistent with the experimental
data, as well as with our current understanding of the #BM2 domain" -=" &am, M"
8a(oosi, #" =aynes, " $ang, and " Blan%schtein, Biotechnol. Bioeng., 89(4), ;,.
):006*3 More studies are underway to apply this promising system to a mixture
deri(ed from a real E. coli cell lysate, as well as to further impro(e and optimi+e the
'ioseparation"