E. coli PROTEIN PURIFICATION WITH THE HALOTAG - Promega
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Features & BeneFits Protocol ExPrEss & Purify difficult ProtEins ProtEin Activity BEttEr rEcovEry MorE inforMAtion E. coli Protein Purificaon with the HaloTag ® plaorm ✓ HaloTag is a highly-effecve solubility tag ✓ Yields more acve protein ✓ Improved protein recovery due to highly specific, covalent binding
Transcript of E. coli PROTEIN PURIFICATION WITH THE HALOTAG - Promega
F eat u r es & B e n eF i ts
Protocol
ExPrEss&PurifydifficultProtEins
ProtEinActivity
BEttErrEcovEry
MorEinforMAtion
E. coli Protein Purificationwith the HaloTag® platform
✓ HaloTag is a highly-effective solubility tag
✓ Yields more active protein
✓ Improved protein recovery due to highly specific, covalent binding
E. col i p r O t e i n p u r i F i c at i O n w i t h t h e h a l O ta g ® p l at F O r m
fEAturEs&BEnEfits
p rOtO cO l
ExPrEss&PurifydifficultProtEins
ProtEinActivity
BEttErrEcovEry
MorEinforMAtion
Rapid HaloTag® protocol
HaloLink
HaloLink
HaloTag
POI
POI
HaloLin
k
HaloTag
POI
HaloLink
HaloTag
POI
HaloLink
HaloTag
POI
HaloTag
POI
HaloTag
POI
HaloTag
POI
Capture HaloTag fusion proteins
with HaloLink™ resinWash away
unbound proteinsProceed with
tag-free protein
Cleave with TEV, leaving HaloTag
bound to the resin
Remove TEV with HisLink™
resin
9695
MB
HaloLinkHisLink
HaloTag
4–5 HOURS
POI
thisprotocolcanbefurthersimplifiedbyusinganoptimizedon-columntEv,calledHalotEvprotease.
E. col i p r O t e i n p u r i F i c at i O n w i t h t h e h a l O ta g ® p l at F O r m
fEAturEs&BEnEfits
Protocol
e x p r es s & p u r i F y d i F F i c u lt p rOt e i n s
ProtEinActivity
BEttErrEcovEry
MorEinforMAtion
Enhance protein solubility
# Soluble Proteins (of 23 tested)
9678
MA
HaloTag®
MBP
GST
His6Purif
icat
ion
(Fus
ion)
Tag
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Generate more soluble protein with HaloTag®
Expressionof23highlyinsolubleproteinsshowsthatHalotagisabettersolubilitytagoverconventionaltags.
ohana,r.f.et al. (2009)Halotag7:ageneticallyengineeredtagthatenhancesbacterialexpressionofsolubleproteinsandimprovesproteinpurification.Protein Expr. Purif.68(1):110-20.
E. col i p r O t e i n p u r i F i c at i O n w i t h t h e h a l O ta g ® p l at F O r m
fEAturEs&BEnEfits
Protocol
ExPrEss&PurifydifficultProtEins
p rOt e i n ac t i v i t y
BEttErrEcovEry
MorEinforMAtion
Recover more active proteinHaloTag® purified proteins are highly functional
cPKAactivitywasdeterminedusingKinase-Glo®assayafterpurificationwithmultiplefusiontags,includingHalotag.
HaloTag® GST MBP His6
cPKA
act
ivity
(uni
ts/µ
g)18
16
14
12
10
8
6
4
2
0 9717
MA
Gentlepurificationconditionsusingasinglenativebufferandfewerstepsintheprotocolresultsinhighlyactiveprotein
No dialysis necessary!
E. col i p r O t e i n p u r i F i c at i O n w i t h t h e h a l O ta g ® p l at F O r m
fEAturEs&BEnEfits
Protocol
ExPrEss&PurifydifficultProtEins
ProtEinActivity
B e t t e r r ecOv e ry
MorEinforMAtion
Recovery where other tags failHigher expression and greater recovery
HaloTag® GST MBP His6 tag
TCTP SMPX MAPK14 MAPK8 PRKCG
Yiel
d (m
g)
35
30
25
20
15
10
5
0 9677
MA
Improved yield with challenging proteins
purification of 6 difficult-to-express human proteins.covalent,nonreversiblebindingofHalotagtotheHalolink™resinmaximizespurificationyieldswhencomparedtootherfusiontags.
E. col i p r O t e i n p u r i F i c at i O n w i t h t h e h a l O ta g ® p l at F O r m
fEAturEs&BEnEfits
Protocol
ExPrEss&PurifydifficultProtEins
ProtEinActivity
BEttErrEcovEry
m O r e i n FO r m at i O n
ProMEGAcorPorAtion •2800WoodsHolloWroAd •MAdison,Wi53711-5399usA •tElEPHonE608-274-4330www.promega.com •©2011ProMEGAcorPorAtion•AllriGHtsrEsErvEd•PricEsAndsPEcificAtionssuBjEcttocHAnGEWitHoutPriornoticE•PrintEdinusA2/11•19769•PArt#is036
References
Hata,t.andnakayama,M.(2007)rapidsingle-tubemethodforsmall-scaleaffinitypurificationofpolyclonalantibodiesusingHalotagtechnology.J Biochem. Biophys. Methods;70(4);679-82.
Motejadded,H.et al.(2010)Expression,one-steppurification,andimmobilizationofHalotag™fusionproteinsonchloroalkane-functionalizedmagneticbeads.Appl. Biochem. Biotechnol.;162(7);2098-110.
ohana,r.f.et al.(2009)Halotag7:ageneticallyengineeredtagthatenhancesbacterialexpressionofsolubleproteinsandimprovesproteinpurification.Protein Expr. Purif.;68(1);110-20.
Ordering Information
p r O d u c t s i z e c ata l O g #
halotag® protein pur i f icat ion system 1each G6280
halotag® protein pur i f icat ion system sample pack
1each G6270
HalotagandKinase-GloareregisteredtrademarksofPromegacorporation.HalolinkisatrademarkofPromegacorporation.
