Protein Purification
The invention provided herein relates to methods for protein synthesis, characterisation and purification on a digital microfluidic device.
1 . A method for protein synthesis comprising
a. taking a digital microfluidic device having a planar array of electrodes;
b. synthesising a protein of interest having a binding tag in droplets on the device;
c. capturing the proteins via the binding tags, thereby immobilising the proteins;
d. moving the droplets using the electrodes, thereby removing the synthesised proteins from the droplet;
e. optionally washing the immobilised proteins; and
f. optionally releasing the proteins into further droplets.
2 . The method according to claim 1 comprising;
a. taking a digital microfluidic device having a planar array of electrodes;
b. using a variety of different conditions to synthesise a protein of interest having a tag in one or more droplets on the device, thereby identifying the optimal conditions for the expression of soluble protein having the tag;
c. removing the droplets from the device;
d. using the optimal conditions to synthesise a protein of interest in a further population of droplets on the device;
e. capturing the proteins via affinity to magnetic beads, thereby immobilising the proteins;
f. moving the droplets using the electrodes, thereby removing the immobilised synthesised proteins from the droplet;
g. optionally washing the immobilised proteins; and
h. removing the proteins from the device, either by releasing the proteins into further droplets and removing the protein droplets from the device or by removing the magnetic beads from the device.
3 . The method according to claim 1 , wherein the immobilised protein is released into further droplets for removal from the device.
4 . The method according to claim 1 , wherein protein synthesis is performed using cell free lysates.
5 . The method according to claim 1 , wherein protein synthesis performed using assembled components for transcription and translation in a system of purified recombinant elements (PURE).
6 . The method according to claim 1 , wherein multiple proteins of interest are synthesised in parallel on the device.
7 . The method according to claim 1 , wherein the yield of soluble protein is determined by fluorescence complementation.
8 . The method according to claim 7 , wherein the expressed proteins comprise GFP 11 .
9 . The method according to claim 1 , wherein the binding tags and detection tags are the same.
10 . The method according to claim 1 , wherein affinity purification uses binding tags selected from:
Alfa-tag
(SRLEEELRRRLTE)
Avi-tag
(GLNDIFEAQKIEWHE)
C-tag
(EPEA)
Calmodulin-tag
(KRRWKKNFIAVSAANRFKKISSSGAL)
Dogtag
(DIPATYEFTDGKHYITNEPIPPK)
E-tag
(GAPVPYPDPLEPR)
FLAG
(DYKDDDDK)
G4T
(EELLSKNYHLENEVARLKK)
HA
(YPYDVPDYA)
His
(HHHHHH)
Isopeptag
(TDKDMTITFTNKKDAE)
lanthanide binding tag
(LBT)
(FIDTNNDGWIEGDELLLEEG)
Myc
(EQKLISEEDL)
NE-Tag
(TKENPRSNQEESYDDNES)
Poly Glutamate-tag
(EEEEEEE)
Poly Arginine-tag
(RRRRRRR)
Rho1D4-tag
(TETSQVAPA)
SBP-tag
(MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP)
Sdytag
(DPIVMIDNDKPIT)
SH3
(STVPVAPPRRRRG)
Snooptag
(KLGDIEFIKVNK)
Softag 1
(SLAELLNAGLGGS)
Softag 3
(TQDPSRVG)
Spot-tag
(PDRVRAVSHWSS)
Spytag
(AHIVMVDAYKPTK)
S-tag
(KETAAAKFERQHMDS)
Strep-tag
(WSHPQFEK)
Strep-tag II
(AWAHPQPGG)
T7tag
(MASMTGGQQMG)
TC-tag
(EVHTNQDPLD)
Ty-tag
(CCPGCC)
VSV-tag
(YTDIEMNRLGK)
Xpress-tag
(DLYDDDDK).
11 . The method according to claim 1 , wherein additional droplets are passed over the immobilised proteins and removed, further purifying the proteins.
12 . The method according to claim 1 , wherein an assay to determine the total amount of purified protein is performed.
13 . The method according to claim 12 , wherein the assay uses Coomassie.
14 . The method according to claim 1 , wherein the digital microfluidic device comprises an oil-filled or humidified gaseous environment, wherein the humidified gaseous environment is achieved by enclosing or sealing the digital microfluidic device and providing on-board reagent reservoirs.
15 . The method according to claim 14 , wherein the oil is mineral oil, silicone oil, an alkyl-based solvent, or a fluorinated oil, wherein the oil optionally contains a surfactant.
16 . (canceled)
17 . The method according to claim 1 , wherein the screening step identifies the optimal conditions for purification on the device.
18 - 19 . (canceled)
20 . The method according to claim 1 comprising
a. taking a digital microfluidic device having a planar array of electrodes;
b. using a variety of different conditions to synthesise and purify a protein of interest having a tag in one or more droplets on the device, thereby identifying the optimal conditions for the expression and purification of soluble protein having the tag by measuring the total concentration of purified protein and the yield of soluble protein to determine the soluble yield and purity of the synthesised protein;
c. removing the droplets from the device;
d. using the optimal conditions to synthesise a protein of interest in a further population of droplets on the device;
e. capturing the proteins via affinity to magnetic beads, thereby immobilising the proteins;
f. moving the droplets using the electrodes, thereby removing the immobilised synthesised proteins from the droplets;
g. washing the immobilised proteins; and
h. removing the proteins from the beads by releasing the proteins into further droplets.
21 . (canceled)
22 . The method according to claim 1 wherein the expressed protein in step b contains a GFP 11 peptide amino sequence tag selected from:
KRDHMVLLEFVTAAGITGT
KRDHMVLHEFVTAAGITGT
KRDHMVLHESVNAAGIT
RDHMVLHEYVNAAGIT
GDAVQIQEHAVAKYFTV
GDTVQLQEHAVAKYFTV
GETIQLQEHAVAKYFTE
and a binding tag selected from
Alfa-tag (SRLEEELRRRLTE)
Avi-tag (GLNDIFEAQKIEWHE)
C-tag (EPEA)
Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL)
Dogtag (DIPATYEFTDGKHYITNEPIPPK)
E-tag (GAPVPYPDPLEPR)
FLAG (DYKDDDDK)
G4T (EELLSKNYHLENEVARLKK)
HA (YPYDVPDYA)
His (HHHHHH)
Isopeptag (TDKDMTITFTNKKDAE)
lanthanide binding tag (LBT)
(FIDTNNDGWIEGDELLLEEG)
Myc (EQKLISEEDL)
NE-Tag (TKENPRSNQEESYDDNES)
Poly Glutamate-tag (EEEEEEE)
Poly Arginine-tag (RRRRRRR)
RholD4-tag (TETSQVAPA)
SBP-tag (MDEKTTGWRGGHVVEGLAGE
LEQLRARLEHHPQGQREP)
Sdytag (DPIVMIDNDKPIT)
SH3 (STVPVAPPRRRRG)
Snooptag (KLGDIEFIKVNK)
Softag 1 (SLAELLNAGLGGS)
Softag 3 (TQDPSRVG)
Spot-tag (PDRVRAVSHWSS)
Spytag (AHIVMVDAYKPTK)
S-tag (KETAAAKFERQHMDS)
Strep-tag (WSHPQFEK)
Strep-tag II (AWAHPQPGG)
T7tag (MASMTGGQQMG)
TC-tag (EVHTNQDPLD)
Ty-tag (CCPGCC)
VSV-tag (YTDIEMNRLGK)
Xpress-tag (DLYDDDDK).
23 . The method according to claim 1 for synthesizing a protein of interest (POI) comprising:
a. taking a digital microfluidic device having a planar array of electrodes;
b. synthesising a protein of interest having a GFP 11 binding tag and a protease cleavage site in droplets on the device;
c. capturing the POI via the binding tags to form GFP 1-11 , and capturing the GFP 1-11 , thereby immobilising the POI,
d. moving the droplets using the electrodes, thereby removing the synthesised fluorescent proteins from the droplet;
e. optionally washing the immobilised fluorescent proteins; and
f. releasing the proteins of interest into further droplets using a protease.
24 . The method according to claim 22 , wherein the GFP 1-11 is immobilised via a binding tag attached to GFP 1-10 wherein the GFP 1-10 contains a binding tag selected from:
Alfa-tag (SRLEEELRRRLTE)
Avi-tag (GLNDIFEAQKIEWHE)
C-tag (EPEA)
Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL)
Dogtag (DIPATYEFTDGKHYITNEPIPPK)
E-tag (GAPVPYPDPLEPR)
FLAG (DYKDDDDK)
G4T (EELLSKNYHLENEVARLKK)
HA (YPYDVPDYA)
His (HHHHHH)
Isopeptag (TDKDMTITFTNKKDAE)
lanthanide binding tag (LBT)
(FIDTNNDGWIEGDELLLEEG)
Myc (EQKLISEEDL)
NE-Tag (TKENPRSNQEESYDDNES)
Poly Glutamate-tag (EEEEEEE)
Poly Arginine-tag (RRRRRRR)
RholD4-tag (TETSQVAPA)
SBP-tag(MDEKTTGWRGGHVVEGLAG
ELEQLRARLEHHPQGQREP)
Sdytag (DPIVMIDNDKPIT)
SH3 (STVPVAPPRRRRG)
Snooptag (KLGDIEFIKVNK)
Softag 1 (SLAELLNAGLGGS)
Softag 3 (TQDPSRVG)
Spot-tag (PDRVRAVSHWSS)
Spytag (AHIVMVDAYKPTK)
S-tag (KETAAAKFERQHMDS)
Strep-tag (WSHPQFEK)
Strep-tag II (AWAHPQPGG)
T7tag (MASMTGGQQMG)
TC-tag (EVHTNQDPLD)
Ty-tag (CCPGCC)
VSV-tag (YTDIEMNRLGK)
Xpress-tag (DLYDDDDK).