Part IPurification ofHdeA D20A D51AJason Wang, Linda Foit, Scott Horowitz, Ke Wan8/8/2013
Aim
Problem
Purification of the constitutively active mutant
HdeA D20A D51Afor use in NMR
Aggregation of protein during purification
Standard purification procedure
• PEM (rich) medium• 1. Cation exchange HiTrap SP HP, pH 4• 2. Anion exchange HiTrap Q HP, pH 8
• Theoretical pI = 5.01
Problem that previously occurred during purification of HdeA D20A D51A from minimal medium
load
25201510
25201510
Increasing NaCl concentration during elution from HiTrap SP HP
HdeA
bla
Purification setup RT vs 4C
Cell Culture
Periplasmic protein
extraction
Dialysis
Purification at Room Temp/No
Glycerol
Purification at4o C/ 5% Glycerol
Elution Profile at RT/No glycerol
0 10 20 30 40 50 60 70 80 90 1000
500
1000
1500
2000
0
20
40
60
80
100
mAU RT
%B
Elution volume [ml]
Abso
rban
ce @
280
nm
[mAU
]
Conc
entr
ation
B [%
]
A10 B11
B6
B1
C4-C9
C9-C10
Increasing NaCl concentration
Load
Eluction Fractions: RT/No Glycerol
HdeA
37
25
20
kDa
B
10 9 8 7 6 5 4 3 2
C
1 1 2 3 4 5 6 7 8 910
CB11 2 3 4 5 6 7 8 910
0 10 20 30 40 50 60 70 80 90 1000
500
1000
1500
2000
0
20
40
60
80
100
mAU RT
%B
Elution volume [ml]
Abso
rban
ce @
280
nm
[mAU
]
Conc
entr
ation
B [%
]
A10 B11
B6
B1
C4-C9
Essentially no HdeA elutes from the column
Elution Profile at 4oC and 5% glycerol vs at RT/No glycerol
0 10 20 30 40 50 60 70 80 90 1000
500
1000
1500
2000
0
20
40
60
80
100
mAU 4C
mAU RT
%B
Elution volume [ml]
Abso
rban
ce @
280
nm
[mAU
]
Conc
entr
ation
B [%
]
A10 B11
B6
B1
Elution Fractions at 4oC and 5% glycerol
Increasing NaCl concentration
Load
3017 19 21 23 25 27 28 29 31 33 35 37 40 41 43 4530
37
25
20
kDa
Elution Profile at 4oC and 5% glycerol vs at RT/No glycerol
0 10 20 30 40 50 60 70 80 90 1000
500
1000
1500
2000
0
20
40
60
80
100
mAU 4C
mAU RT
%B
Elution volume [ml]
Abso
rban
ce @
280
nm
[mAU
]
Conc
entr
ation
B [%
]
A10 B11
B6
B1
28-35
?-46
Purification at 4oC and 5% glycerol
0.0010.00
20.0030.00
40.0050.00
60.0070.00
80.0090.00
100.000
500
1000
1500
2000
0
20
40
60
80
100
mAU 4C
mAU RT
%B
Elution volume [ml]
Abso
rban
ce @
280
nm
[mAU
]
Conc
entr
ation
B [%
]
A10 B11
B6
B1
28-35
37-46
Purification at 4oC and 5% glycerol
0.0010.00
20.0030.00
40.0050.00
60.0070.00
80.0090.00
100.000
500
1000
1500
2000
0
20
40
60
80
100
mAU 4C
mAU RT
%B
Elution volume [ml]
Abso
rban
ce @
280
nm
[mAU
]
Conc
entr
ation
B [%
]
A10 B11
B6
B1
28-35
?-46
2. Column:Anion exchange (HiTrap Q XL)
load FT
Increasing NaCl concentration during elutionKe Wan
Future Directions Part IPurification of the constitutively active mutant HdeA D20A D51A
• Purify further with a Phenyl Column to attempt to remove remaining contaminating proteins
• Redo the entire protocol using bacteria grown in 15N Labeled media.
• Purify that protein and use it in NMR analysis
Fusion Proteins: Characterizing Interactions of HdeA and Im7 by NMR
Jason Wang, Linda Foit, Scott Horowitz, Ke Wan8/8/2013
Aim
Problem
Co-structure ofHdeA and its substrate Im7
Precipitation of Im7when in excess
Purpose
Intro: Co-structure of HdeA and Im7: increase local concentration
HdeA
HdeA
HdeA
HdeA
HdeA
HdeA
Im7
Im7
Im7
Im7
Im7
Im7
Free HdeA and Im7 Fusion Protein
Variable 1: Linker Length
HdeA Im7HdeA-GS-Im7
HdeA-SGSGS-Im7
HdeA-(GGGGS)2-Im7
HdeA-(GGGGS)3-Im7
pI ≈ 4.5
9.7 kDa
9.7 kDa
9.7 kDa
9.7 kDa
9.9 kDa
9.9 kDa
9.9 kDa
9.9 kDa
0.4 kDa
0.6 kDa
1.0 kDa
0.1 kDa
Variable 2: Variations of Im7
I22V
L18AL19A
L37A
I54A
L53A
Largely destabilized Partially unfoldedModel for intermediate state
Model for unfolded state
Variable 3:N- or C- Terminal 6xHis-tag
ss-HdeA – (GGGGS)2 – Im7 – GSG – 6xHis
Im7Linker
ss-6xHis-GSG-HdeA – (GGGGS)2 – Im7
HdeA Im7Linker
His Tag
His Tag
HdeASS
SS
His Tag Rationale
• Protein was not completely pure after native purification• Still contaminating bands, probably degradation
products
His Tag Rationale• Previous experiments demonstrated significant contamination
of the periplasmic extracts with potential degradation products in addition to the fusion protein of interest
HdeA F35W-(GGGGS)2-Im7 L53A I54A ∆W(LF 1434)
*
Full lengthprotein
Degradationproducts?
**
20 ulload
10 ulload
His Tag Rationale• Previous experiments demonstrated significant contamination
of the periplasmic extracts with potential degradation products in addition to the fusion protein of interest
• Adding a His-tag creates a system designed to quickly screen multiple types of proteins using one purification technique for their suitability in NMR analysis
Screening for expression of the His-tagged fusion proteins
1. Is the his-tagged fusion proteins expressed at all?2. Is an N- or a C-terminal tag preferred?• expression• purification
Screening for expression of the His-tagged fusion proteins
HdeA Im7
1507 WT 3A
1508 WT I22V
1509 WT L53A I54A
1516 ΔW 3A H40W
1510 WT 3A
1511 WT I22V
1512 WT L53A I54A
1513 ΔW 3A H40W
Purification of His-Tagged HdeA-Im7 Fusion Protein LF 1508
Im7Linker
His Tag
HdeASS
1508: Wild Type I22V His Tag
C Terminal His Tag
Increasing Concentration of Imidazole
35
25
15
kDa
35
25
15
kDa
Elution Fractions of LF1508 Purification
Im7LinkerHis Tag
HdeASS
1508: Wild Type I22V His Tag
C Terminal
FusionProtein
*Pure Products
1. NMR attempt with a 15N HdeA-Im7 fusion(HdeA WT-(GGGGS)2-Im7 I22V-GSG-6xHis)
HiTrap Chelating15N minimal mediumPurification Ke Wan
HSQC spectrum ofHdeA WT-(GGGGS)2-Im7 I22V-GSG-6xHis
1H
15N
Expected # of peaks: 185Buffer50 mM KH2PO490 mM NaCl1 mM DSS0.5 mM EDTA1 mM chloroacetic acid5% D2OpH 2.5
Scott
HSQC 15N HdeA WT pH 2.5,1400 uM
dimer
Linda + Loic
HSQC 15N HdeA WT pH 2.5, 15 uM
monomer
Linda + Loic
HSQC spectra 15N Im7 I22V + HdeA
1H [ppm]
15N
[ppm
]
500 uM 15N Im7 I22V 500 uM 15N Im7 I22V + 571 uM HdeA
Linda + Loic
Im7 L18A L37A L38A might be a better substrate for NMR
90% 1H2O10% 2H2O0.2 M Na2SO4
10C
Pashley, C. L. et al. (2011): Journal of Molecular Biology (2011)
Purification of His-Tagged HdeA-Im7 Fusion Protein LF 1510
Im7HdeA LinkerHis TagSS
N Terminal His Tag
1510: Wild Type L18A L19A L37AHis Tag
Elution Fractions of LF1510 Purification
Im7HdeA LinkerHis TagSS
N Terminal His Tag
1510: Wild Type L18A L19A L37AHis Tag
Load B9 B8 B7 B6 B5 B4 B3 B2 B1 C1 C2 C3 C4 C5 C6 C7 C8
25
20
37
Kda
Purification of His-Tagged HdeA-Im7 Fusion Protein LF 1507
Im7Linker
His Tag
HdeASS
1507: Wild Type L18A L19A L37A His Tag
C Terminal His Tag
Im7LinkerHis Tag
HdeASS
1507: Wild Type L18A L19A L37A His Tag
C Terminal His Tag
Elution Fractions of LF1507 Purification
Load FT Wash A8 A9 A10 A11 A12 B12 B11 B10 B9 B8 B7
25 20
37
Kda
Do fusion proteins undergo proteolysis?
Degradation products (?) already occur in periplasmic extract I
HdeA ∆W ∆W ∆W WT ∆W ∆W WT∆Wlinker 15 10 5 2 15 10 5 2
Im7 I22V L53A I54A
∆W15 10 5
I22V
* * *
252015
10
Full lengthprotein
* * * * * ** * * * * * **
Degradationproducts?
* ** * *
HdeA – linker – Im7 (no His-tag)LINDA
2015
Degradation products (?) already occur in periplasmic extract II
*
Full lengthprotein
* * * * * *
* * * * **Degradation
products?
HdeA – linker – Im7(no His-tag)
HdeA ∆W ∆W∆W WT ∆W W16F∆Wlinker 15 10 5 2 15 10 2
Im7 AAA AAA H40W
*25
10
* * * *
LINDA
Degradation products (?) also occur after purification I
HdeA F35W-(GGGGS)2-Im7 L53A I54A ∆W(LF 1434)
*
Full lengthprotein
Degradationproducts?
**
20 ulload
10 ulload
LINDA
Degradation products (?) also occur after purification II
Selected elution fractions purificationHdeA WT-(GGGGS)2-Im7 L18A L19A L37A-GSG-6xHis(LF1507)
2015
25
10
***
Full lengthprotein
DegradationProduct(s)?
20
15
25
10
Selected elution fractions purificationHdeA WT-(GGGGS)2-Im7 I22V-GSG-6xHis(LF1508)
*
*
C-terminal His-tag cannot be detected for degradation products I
Selected elution fractions purificationHdeA WT-(GGGGS)2-Im7 L18A L19A L37A-GSG-6xHis(LF1507)
2015
25
10
***
Anti 6xHis antibody
2015
25
10
Full lengthprotein
DegradationProduct(s)?
*
C-terminal His-tag cannot be detected for degradation products II
N-terminal His-tag cannot be detected at all (even in full length constructs)
N-terminal His-tag is present however
Im7HdeA LinkerHis TagSS
N Terminal His Tag
1510: Wild Type L18A L19A L37AHis Tag
Elution fractions HiTrap Chelating column
Identify degradation products by mass spectrometry (UofM Bioconsortium)
HdeA F35W-(GGGGS)2-Im7 L53A I54A ∆W
(LF 1434)
*
Full lengthprotein
Degradationproducts?
**
20 ulload
10 ulload
Cut out bands Proteolytic digest Identification of peptides
His-tag-GSG-HdeA WT-(GGGGS)2-Im7 L18A L19A L37A
(LF1510)
Identify degradation products by mass spectrometry (Indiana University)
LF 1434 HdeA F35W-link-Im7 L53A I54A W75FLF 1508 HdeA WT -link-Im7 I22V-6xHisLF 1510 6xHis-HdeA WT -link-Im7 L18A L19A L37A
Cut out bands Proteolytic digest identification of peptides
Intact mass determination
Mass spec data turnaround
University of MichiganProteomics Core
Indiana University
Mass spec results…
Future experiments• 15N labeling of HdeA D20A D51A
structural studies with NMR• Identification of degradation products of the HdeA-Im7
fusions use stable degradation products for structural studies
instead of full length protein• Create fusion proteins between HdeA and peptides previously
identified as potential HdeA substrates in peptide array
Acknowledgements
Dr. Bardwell, Scott Horowitz, Ke Wan,
The Bardwell Lab, the Jakob Lab
Work studies!!!!!