Affinity Protein Separation by Pulse Electrophoresis in Functionalized Anodic Aluminum Oxide (AAO)...
1
Affinity Protein Separation by Pulse Electrophoresis in Functionalized Anodic Aluminum Oxide (AAO) Membranes Zhiqiang Chen, Tao Chen , Xinghua Sun , Bruce. J. Hinds* Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY 40506 480 500 520 540 620 640 660 680 700 0.1u g /m l 0.1u g/m l P erm eate so lu tio n 2.56 u g /m l P erm eate so lu tio n F eed so lu tio n 7u g /m l ( Texas R ed conjugated B SA ) F eed so lu tio n 7ug/m l (F lu o resecen ce g reen p ro tein ) W avelen g th (n m ) N o rm a lize d E m issio n (A .U .) Introduction 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 -0.00 00 5 0.00000 0.00005 0.00010 0.00015 0.00020 0.00025 0.00030 0.00035 1 2 3 4 5 I/A E /V vs A g/A g + 0 20 40 60 80 100 120 Im id azo le releasin g G FP b lo ckin g B efo re G FP b lo ckin g F lu x (u g /h /cm ^2) The downstream protein separation and purification cost can be as high as 80% of the total cost of the recombinant therapeutics protein production [1] . Immobilized metal ion affinity chromatography (IMAC) for affinity protein separation is most widely used but suffers from limitations of expense, slow complex binding, purge, desorption cycles, long intra-particle diffusion time and column regeneration. [2] Affinity membrane chromatography with convection flow through the functionalized pores improve mass transport to binding site but has low binding capacity area (thin membranes) thus requiring numerous binding/purge/release cycles and suffers from indiscriminate flow induced fouling [3] . Electrophoresis through inorganic nanoporous membranes has been used for non-affinity protein separation but still suffers from poor selectivity/fouling of sized-based exclusion for proteins of the same charge. [4-5] Needed is a transformative idea in membranes that selectively bind proteins to pore entrance and pumps them through a membrane in a continuous manner. (1)Ghosh R, Cui Z.F. Journal of Membrane Science. 2000, 167, 47–53. (2)Ghosh R. Journal of Chromotography A. 2002, 952, 13-17. (3)Jain P et al. Biomacromolecules. 2010,11, 1019-1026. (4)Sun X et al. Langmuir. 2011,27, 3150-3156. (5)Yu S et al. Anal. Chem. 2003, 75, 1239-1244 (6)Lam P et al. J. Electrochem. Soc.,1999,124,2517. (7)Wirtz M et al. Analyst. 2002, 127,871. (8)Liu J et al. Langmuir. 2000, 16, 7471-7476. (9)Arnold F. Nature Biotechnology. 1991, 9, 151-156. Objectives 1. Produce multi-electrode membranes with protein receptor chemistry at entrance to 10nm diameter pores. 2. Demonstrate continuous affinity protein separation using repeated binding and release/pumping electrophoretic voltage pulses. Bare AAO membrane SEM image AAO Membrane Electrode Pore Entrance Size Control Electroless-plating AAO membrane SEM image A 5 nm gold seed layer was sputtered on top of AAO membrane for electroless-plating. Phosphate buffer containing 1.6 mM sodium gold (I) thiosulfate and 2.68 mM ascorbic acid, was used and is a modified from Lam’s method [6] and is similar to the process of plating Au on AAO membranes [7] . The pore entrance is controllably reduced from 20nm to the required 10 nm. Electrochemical functionalization of AAO Membranes Cyclic Voltammograms on the upper side of gold/AAO membrane in LiClO 4 ethanol solution with10 mM NTA for different cycles. Scan rate is 10mv/s. The oxidation potential is about 0.85V. Cyclic Voltammograms on bare and NTA oxidation AAO membrane in 0.1M K 3 Fe(CN) 6 PBS solution. Scan rate is 100mv/s. Change in CV demonstrate success of electrochemical oxidation 0.0 0.1 0.2 0.3 0.4 0.5 0.6 -0.0 010 -0.0 008 -0.0 006 -0.0 004 -0.0 002 0.0000 0.0002 0.0004 0.0006 0.0008 G old/A A O m em brane N TA o xidation g o ld/A A O m em brane I (A ) E (V ) vs A g/A g + Demonstration of Membrane Functionalization Nα,Nα-Bis(carboxymethyl)-L-lysine (NTA) electrochemical oxidation grafting process. NTA binds to Ni2+ that reversibly binds to hystidine tagged protein [8] Schematic shows the interaction between His-tagged proteins and Ni-NTA-gold. Ni-NTA-gold was achieved by incubate the NTA-gold membrane in 0.1M NiCI 2 for 30min. His-tagged Green fluorescent protein specifically bind with Ni-NTA- gold membrane. Gate Keeper Blocking of Functionalized Membrane Electrophoretic pumping flux of BSA before, after his-tagged GFP blocking the pore and after imidazole releasing the GFP. Imidazole can occupy the nickel coordination sites, displacing the his-tagged GFP [9] Schematic of pore entrance blocked by His- tagged GFP in a sequential manner. Direct Electrophoretic Pumping Process Table 1. Electrophoretic flux and Mobility of Texas-BSA and His-tagged GFP proteins through Ni-NTA-Gold/AAO membrane at different imidazole release agent concentration Pulse Electrophoretic Pumping Process Schematic of pulse electrophoresis process across AAO membrane (1)Release and pumping cycle: Imidazole from the permeate solution is pumped to the feed solution to release the His-tagged GFP, which acts steric hindrance at the pore entrance. The released His-tagged GFP is selectively electrophoretic pumped to the permeate solution (2)Binding cycle: By applying an opposite voltage across the AAO membrane, the imidazole at the feed pore is repelled and His-tagged GFP specifically bind and block the pores again Fluorescence spectra showing the emission intensity of the feed and permeate solutions after pulse electrophoresis. The release and pumping cycle time is 14s, the biding cycle time is 1s. The concentration of imidazole in the permeate solution is 10 mM Table 2. Electrophoretic Mobility and flux of BSA and His-tagged GFP proteins through different pumping process. The imidazole concentration in the permeate side is 10 mM a Feed solution contains 1000ug/ml BSA and 10ug/ml His-tagged GFP. b Feed solution contains 7ug/ml Texas red conjugated BSA and 7ug/ml His-tagged GFP. Conclusion References The asymmetric nature of the commercial AAO membranes allows thin 10 nm diameter porous electrode layer to act as gate keeper while bulk 200 nm diameter channels provides high flux during the separation process. Hys-tagged proteins bound to pore entrance block other proteins in a sequential/hopping manner, allowing selective transport. Electrophoretic pumping eliminates fouling associated with pressure flow, while pores closed by gatekeeper prevents fouling by other charged proteins. Pulsed electrophoresis binding/release/pumping cycle allows for a continuous protein separation process. This would revolutionize protein separations where expressed proteins can be directly removed from fermentation baths with this membrane separation system. Acknowledgements The authors would like to thank Jonathon Wagner and Dr. Ji Wu for helpful discussion. The authors would like to thank funding source of NIH and the department of CME and CeNSE. Bruce J. Hinds* [email protected] u Zhiqiang Chen [email protected] du (1)The pore is mostly open at high imidazole concentration, showing high flux but no selectivity since GFP is not bound to receptor. (2)The pore is mostly blocked by His-tagged GFP at low imidazole concentration, showing good selectivity while low flux. The ideal separation factor for 1:1 ratio with a monolayer ‘gate keeper’ release is 1.5. Observed separation factor of 7.7 proves multiple GFPs are in the channel and some remain to block pore as GFP is released/pumped. (3)Pulsed electrophoretic pumping with binding/pumping cycle can solve the tradeoff challenge between flux and selectivity. (1)GFP:BSA separations as high as 16 are seen. In more realistic 1:100 feed solution, separation factor of ~9 is seen. Bound GFP generally block pores but if open, the BSA has 100 fold higher chance of entering pore. (2)Further work to optimize blocking of non-his tagged proteins is being explored. Optimization of Binding/Release cycle may allow Hys-tag blocking of pore at all times. 10 m M imidazole 1 m M imidazole 0.1 m M im idazole B S A flux (ug h -1 cm -2 ),f b 0.30 0.12 0.01 G FP flux (ug h -1 cm -2 ),f g 0.28 0.19 0.082 B S A effective m obility ( 10 -6 cm 2 s -1 V -1 ),u b 1.97 0.79 0.07 G FP effective m obility ( 10 -6 cm 2 s -1 V -1 ),u g 1.88 1.27 0.54 Selectivity,S = u g /u b 0.95 1.6 7.7 N O OH H 2 N HO O OH O e - - H + N O HN HO O OH O N O OH HN HO O OH O N O OH H 2 N HO O OH O gold gold gold gold HN N O O O O O O Ni 2+ N NH N NH H is-G FP H is-G FP G old/AAO m em brane, directpumping a , N i-N TA-G old/A AO m embrane, direct pumping a N i-N TA-G old/AAO membrane, pulse pum ping (1:100) a N i-N TA-G old/A AO membrane, pulse pum ping (1:1) b B S A flux (ug h -1 cm -2 ),f b 112.56 48.32 14.07 0.02 G FP flux (ug h -1 cm -2 ),f g 0.7306 0.34 1.29 0.32 B S A effective m obility ( 10 -6 cm 2 s -1 V -1 ),u b 5.2 2.2 0.65 0.13 G FP effective m obility ( 10 -6 cm 2 s -1 V -1 ),u g 3.4 1.6 6.0 2.12 Selectivity,S = u g /u b 0.65 0.73 9.23 16
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Transcript of Affinity Protein Separation by Pulse Electrophoresis in Functionalized Anodic Aluminum Oxide (AAO)...
Affinity Protein Separation by Pulse Electrophoresis in Functionalized Anodic Aluminum Oxide (AAO) Membranes
Zhiqiang Chen, Tao Chen , Xinghua Sun , Bruce. J. Hinds*Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY 40506
10 mM imidazole 1 mM imidazole 0.1 mM imidazole
BSA flux (ug h-1 cm-2 ), fb 0.30 0.12 0.01
GFP flux (ug h-1 cm-2 ), fg 0.28 0.19 0.082
BSA effective mobility (10-6cm2 s-1 V-1), ub
1.97 0.79 0.07
GFP effective mobility (10-6cm2 s-1 V-1), ug
1.88 1.27 0.54
Selectivity, S= ug/ub 0.95 1.6 7.7
480 500 520 540 620 640 660 680 700
0.1ug/ml
0.1ug/ml
Permeate solution2.56 ug/ml
Permeate solution
Feed solution 7ug/ml(Texas Red conjugated BSA)
Feed solution 7ug/ml(Fluoresecence green protein)
Wavelength (nm)
Norm
alize
d E
mis
sio
n (A
.U.)
Introduction
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
-0.00005
0.00000
0.00005
0.00010
0.00015
0.00020
0.00025
0.00030
0.00035
1 2 3 4 5
I/A
E/V vs Ag/Ag+
0
20
40
60
80
100
120
Imidazole releasingGFP blockingBefore GFP blocking
Flu
x (u
g/h
/cm
2̂)
N
O OH
H2N
HO
O
OH
O e-
-H+
N
O
HNHO
O
OH
O
N
O OH
HNHO
O
OH
O
N
O OH
H2N
HO
O
OH
O
gold
gold
gold
gold
HN N
O O
O
O
O
O
Ni2+
N
NH
N
NH
His-GFP
His-GFP
The downstream protein separation and purification cost can be as high as
80% of the total cost of the recombinant therapeutics protein production [1].
Immobilized metal ion affinity chromatography (IMAC) for affinity protein
separation is most widely used but suffers from limitations of expense, slow
complex binding, purge, desorption cycles, long intra-particle diffusion time
and column regeneration.[2] Affinity membrane chromatography with
convection flow through the functionalized pores improve mass transport to
binding site but has low binding capacity area (thin membranes) thus requiring
numerous binding/purge/release cycles and suffers from indiscriminate flow
induced fouling[3]. Electrophoresis through inorganic nanoporous membranes
has been used for non-affinity protein separation but still suffers from poor
selectivity/fouling of sized-based exclusion for proteins of the same charge. [4-5]
Needed is a transformative idea in membranes that selectively bind proteins to
pore entrance and pumps them through a membrane in a continuous manner.
(1) Ghosh R, Cui Z.F. Journal of Membrane Science. 2000, 167, 47–53.
(2) Ghosh R. Journal of Chromotography A. 2002, 952, 13-17.
(3) Jain P et al. Biomacromolecules. 2010,11, 1019-1026.
(4) Sun X et al. Langmuir. 2011,27, 3150-3156.
(5) Yu S et al. Anal. Chem. 2003, 75, 1239-1244
(6) Lam P et al. J. Electrochem. Soc.,1999,124,2517.
(7) Wirtz M et al. Analyst. 2002, 127,871.
(8) Liu J et al. Langmuir. 2000, 16, 7471-7476.
(9) Arnold F. Nature Biotechnology. 1991, 9, 151-156.
Objectives
1. Produce multi-electrode membranes with protein receptor chemistry at entrance to 10nm diameter pores.
2. Demonstrate continuous affinity protein separation using repeated binding and release/pumping electrophoretic voltage pulses.
Bare AAO membrane SEM image
AAO Membrane Electrode Pore Entrance Size Control
Electroless-plating AAO membrane SEM image
A 5 nm gold seed layer was sputtered on top of AAO membrane for
electroless-plating. Phosphate buffer containing 1.6 mM sodium gold (I)
thiosulfate and 2.68 mM ascorbic acid, was used and is a modified from Lam’s
method [6] and is similar to the process of plating Au on AAO membranes [7].
The pore entrance is controllably reduced from 20nm to the required 10 nm.
Electrochemical functionalization of AAO Membranes
Cyclic Voltammograms on the upper side of gold/AAO membrane in LiClO4 ethanol solution with10 mM NTA for different cycles. Scan rate is 10mv/s. The oxidation potential is about 0.85V.
Cyclic Voltammograms on bare and NTA oxidation AAO membrane in 0.1M K3Fe(CN)6 PBS solution. Scan rate is 100mv/s. Change in CV demonstrate success of electrochemical oxidation
0.0 0.1 0.2 0.3 0.4 0.5 0.6-0.0010
-0.0008
-0.0006
-0.0004
-0.0002
0.0000
0.0002
0.0004
0.0006
0.0008
Gold/AAO membrane NTA oxidation gold/AAO membrane
I (A
)
E (V) vs Ag/Ag+
Demonstration of Membrane Functionalization
Nα,Nα-Bis(carboxymethyl)-L-lysine (NTA) electrochemical oxidation grafting process. NTA binds to Ni2+ that reversibly binds to hystidine tagged protein [8]
Schematic shows the interaction between His-tagged proteins and Ni-NTA-gold. Ni-NTA-gold was achieved by incubate the NTA-gold membrane in 0.1M NiCI2 for 30min. His-tagged Green fluorescent protein specifically bind with Ni-NTA-gold membrane.
Gate Keeper Blocking of Functionalized Membrane
Electrophoretic pumping flux of BSA before, after his-tagged GFP blocking the pore and after imidazole releasing the GFP. Imidazole can occupy the nickel coordination sites, displacing the his-tagged GFP [9]
Schematic of pore entrance blocked by His-tagged GFP in a sequential manner.
Direct Electrophoretic Pumping ProcessTable 1. Electrophoretic flux and Mobility of Texas-BSA and His-tagged GFP proteins through Ni-NTA-Gold/AAO membrane at different imidazole release agent concentration
Pulse Electrophoretic Pumping Process
Schematic of pulse electrophoresis process across AAO membrane
(1) Release and pumping cycle: Imidazole from the permeate solution is
pumped to the feed solution to release the His-tagged GFP, which acts
steric hindrance at the pore entrance. The released His-tagged GFP is
selectively electrophoretic pumped to the permeate solution
(2) Binding cycle: By applying an opposite voltage across the AAO
membrane, the imidazole at the feed pore is repelled and His-tagged GFP
specifically bind and block the pores again
Fluorescence spectra showing the emission intensity of the feed and permeate solutions after pulse electrophoresis. The release and pumping cycle time is 14s, the biding cycle time is 1s. The concentration of imidazole in the permeate solution is 10 mM
Table 2. Electrophoretic Mobility and flux of BSA and His-tagged GFP proteins through different pumping process. The imidazole concentration in the permeate side is 10 mM
Gold/AAO membrane, direct pumpinga,
Ni-NTA-Gold/AAO membrane, direct pumpinga
Ni-NTA-Gold/AAO membrane, pulse pumping (1:100)a
Ni-NTA-Gold/AAO membrane, pulse pumping (1:1)b
BSA flux (ug h-1 cm-2 ), fb 112.56 48.32 14.07 0.02
GFP flux (ug h-1 cm-2 ), fg 0.7306 0.34 1.29 0.32
BSA effective mobility (10-6cm2 s-1 V-1), ub
5.2 2.2 0.65 0.13
GFP effective mobility (10-6cm2 s-1 V-1), ug
3.4 1.6 6.0 2.12
Selectivity, S= ug/ub 0.65 0.73 9.23 16
a Feed solution contains 1000ug/ml BSA and 10ug/ml His-tagged GFP. b Feed solution contains
7ug/ml Texas red conjugated BSA and 7ug/ml His-tagged GFP.
Conclusion
References
The asymmetric nature of the commercial AAO membranes allows thin 10
nm diameter porous electrode layer to act as gate keeper while bulk 200
nm diameter channels provides high flux during the separation process. Hys-tagged proteins bound to pore entrance block other proteins in a
sequential/hopping manner, allowing selective transport. Electrophoretic
pumping eliminates fouling associated with pressure flow, while pores
closed by gatekeeper prevents fouling by other charged proteins. Pulsed electrophoresis binding/release/pumping cycle allows for a
continuous protein separation process. This would revolutionize protein
separations where expressed proteins can be directly removed from
fermentation baths with this membrane separation system.
AcknowledgementsThe authors would like to thank Jonathon Wagner and Dr. Ji Wu for helpful
discussion. The authors would like to thank funding source of NIH and the
department of CME and CeNSE.
Bruce J. Hinds*
Zhiqiang Chen
(1) The pore is mostly open at high imidazole concentration, showing high flux but no selectivity since GFP is not bound to receptor.
(2) The pore is mostly blocked by His-tagged GFP at low imidazole concentration, showing good selectivity while low flux. The ideal separation factor for 1:1 ratio with a monolayer ‘gate keeper’ release is 1.5. Observed separation factor of 7.7 proves multiple GFPs are in the channel and some remain to block pore as GFP is released/pumped.
(3) Pulsed electrophoretic pumping with binding/pumping cycle can solve the tradeoff challenge between flux and selectivity.
(1) GFP:BSA separations as high as 16 are seen. In more realistic 1:100 feed solution, separation factor of ~9 is seen. Bound GFP generally block pores but if open, the BSA has 100 fold higher chance of entering pore.
(2) Further work to optimize blocking of non-his tagged proteins is being explored. Optimization of Binding/Release cycle may allow Hys-tag blocking of pore at all times.
