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Expression and purification of membrane proteins: Initial screening of Thermotoga maritima α-helical membrane proteins for NMR structural studies. Functional Annotations of the Membrane Proteome. ABC transporters. 20%. 55%. Proteins with unknown function. 25%. Other functionally

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The membrane proteome of T. maritima

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The membrane proteome of t maritima

Expression and purification of membrane proteins: Initial screening of Thermotoga maritima α-helical membrane proteins for NMR structural studies


The membrane proteome of t maritima

Functional Annotations of the Membrane Proteome

ABC transporters

20%

55%

Proteins with

unknown function

25%

Other functionally

annotated proteins

Membrane proteins

24%

76%

Cytosolic proteins

The membrane proteome of T. maritima

Electron micrograph of T. maritima. The arrow points to the outer membrane, the “toga”, for which the organism is named.

Percentage of Membrane Proteins in T. maritima


The membrane proteome of t maritima

Overall Approach to Preparing Membrane Protein

Samples for NMR Studies


The membrane proteome of t maritima

# of

proteins

0 200 400 600 800 1000 1200 1400 1600

Protein length (amino acids)

Target Selection

NMR sample requirements

600 mL of 1 mM protein

uniform 15N and 13C-labeling

monodisperse

MW < 30 kD (for standard NMR experiments, development of TROSY has extended the MW limit for NMR studies)

Size distribution of the membrane proteome

Selected fifty targets less than 16 KD (~130 aa) that contained one to four predicted transmembrane segments.


Expression assay

Para

PT7

Sma I

Pme I

RBS

6-aa

6-his

CAC GTG TM gene TTA ATT AA

Nco I

Pml I

Pac I

kD

21.5

14.4

6.0

Expression Assay

96 x 65 mL Fermentor

Expression Vector

Eleven out of fifty targets overexpressed using arabinose induction.


The membrane proteome of t maritima

Localization of target membrane proteins

TM1554

TM1634

IB S M

IB S M

In order to verify that the target proteins are membrane proteins, the E. coli membranes were isolated using ultracentrifugation and extracted with n-decyl--D-maltoside .

Of the eleven expressing targets, two expressed exclusively in the insoluble fraction (IB) and nine expressed to both the insoluble fraction and the n-decyl--D-maltoside solubilized membrane (M) fraction. None of the proteins were found in the soluble (S) fraction.


The membrane proteome of t maritima

Soluble, monodisperse, and folded

Is that too much to ask?

Which detergent will do all this? For now, there is no paradigm. There isn’t one detergent that works for all membrane proteins; therefore, for each protein, many detergents need to be screened.


Detergent screen

Detergent Screen

NG

DG

OM

DM

OG

P S P S P S P S P S

TM0361

Aromatic side

chain protons

Backbone

amide protons

DPC

LDAO

DoDM

DHPC

CHAPS

W indole side

chain protons

P S P S P S P S P S

1H (ppm)


Soluble folded

9.5

9.0

8.5

8.0

7.5

7.0

6.5

Soluble ≠ folded!

1H (ppm)


The membrane proteome of t maritima

Optimization of solution conditions for structure determination: 2D spectroscopy of 15N-labeled protein

9.0

8.5

8.0

7.5

7.0

11.0

10.0

9.0

8.0

7.0

105

TM0361

TM1634

110

110

115

115

15N

(ppm)

120

15N

(ppm)

120

125

125

130

130

1H (ppm)

1H (ppm)

Using the 96 x 65 mL fermentor, these samples were prepared with less than 1L of commercial media.


Summary

Summary

  • Approximately 20% of the membrane protein targets express in HK100 E. coli cells with arabinose induction.

  • 2. Detergent screening has been successful in preparing solubilized membrane protein samples, but will be revised as we gain experience.

  • 3. Similar to soluble proteins, 1H 1D NMR spectroscopy is suitable for evaluating the overall fold of the protein.

  • 4. TM1634 has been optimized and NMR structure determination is in progress.


Acknowledgements

Acknowledgements

Kurt Wüthrich

Scott Lesley

Heath Klock

Bernhard Geierstanger

Joanna Hale

This work is funded by the NIH protein structure initiative grant P50 GM62411. LC is funded by NIH grant 1F32GM068286. KW is funded by the endowment of Cecil H. and Ida M. Green (TSRI).


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