bardwell presentation final

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)


Im7HdeA LinkerHis TagSS

N Terminal His Tag

1510: Wild Type L18A L19A L37AHis Tag



N Terminal His Tag


Load B9 B8 B7 B6 B5 B4 B3 B2 B1 C1 C2 C3 C4 C5 C6 C7 C8

25

20

37

Kda


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


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

* * * * * ** * * * * * **


* ** * *

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


**

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


N Terminal His 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


**

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!!!!!

bardwell presentation final

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