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False positives. False negatives. Characterization of novel membrane complexes in Escherichia coli Anna Albiniak 1 , Cristina F.R.O.Matos 1 , Colin Robinson 1 , Daniel O. Daley 2 Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK 1

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Presentation Transcript
slide1

False

positives

False

negatives

Characterization of novel membrane complexes

in Escherichia coli

Anna Albiniak1, Cristina F.R.O.Matos1, Colin Robinson1, Daniel O. Daley2

Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK1

Department of Biochemistry and Biophysics, Stockholm University, Stockholm, SE 106 91, Sweden 2

X

X

His

His

His

His

His

His

His

His

His

His

Co2+

Co2+

His

X

X

X

Co2+

Co2+

X

protein complex

Co2+

Co2+

X

X

part of the complex

non-specific

binding

non-specific

binding

part of the complex

Co2+

Co2+

Co2+

Co2+

co-purifying interactors

X

X

co-purifying interactors

Co2+

Co2+

+imidazole

2. Affinity purification to characterise novel membrane proteins

.

His6-tag is used as an “affinity handle” to purify the tagged protein along with co-purifying partners [2].

  • 1. Introduction
  • The functions of one third of predicted membrane
  • proteins remain unknown. In order to characterise
  • these 'orphans' we have to identify how and with what
  • they interact with.
  • Small membrane proteins
  • Small membranes proteins, below 100 amino acids are a challenge,
  • because of the lack of sufficient sequence for domain and homology determination.
  • Bioinformatic analysis indicates that 65% of all small ORFs (16-50 aa) contain
  • a transmembrane segment [1].
  • Aims:
  • to identify and characterise novel membrane protein complexes in Escherichia coli
  • to identify unknown small membrane proteins

+ imidazole

elutions

m/z

elutions

m/z

elutions

elutions

  • 3. Approach
  • expression of novel membrane proteins
  • affinity purification of proteins
  • identification of co - purifying proteins
  • characterisation of novel membrane complexes
  • Different types of contaminants are present in typical AP approach:
  • abundant proteins
  • proteins that bind to unfolded polypeptides
  • proteins that interact with affinity matrices [3]
  • Inclusion of proper controls allows to discriminate between contaminants and interactors:
  • 4. Testing the expression of ORFs
  • + 0.4mM IPTG induction for 2.5h
  • - cells without induction

BL21p – E.coli BL21 pET28a(+)

YiiP OppC DppC YfdH GsiC

YiiP OppC DppC YfdH GsiC

+ - + - + - + - + -

+ - + - + - + - + -

175

80

58

46

30

23

17

175

80

58

46

30

23

17

YiiP OppC DppC YfdH GsiC

- + - + - + - + - +

[kDa]

YiiP – 32.927 DppC – 32.308 OppC – 33.022

YfdH – 34.635 GsiC – 34.066 YhbN – 35.860

LepB – 35.960

30

5. Purification of ORFs

BL21p – E.coli BL21 pET28a(+)

Protocol No. 3

Protocol No. 1

Protocol No. 2

Protocol No. 3

LepB YfdH BL21p GsiC OppC

LepB YbhN BL21p YfdH

175

80

58

46

30

23

175

80

58

46

30

23

membranes

second elution

third elution

third elution

LepB YfdH BL21p GsiC OppC

LepB YbhN BL21p YfdH

*

*

175

80

58

46

30

23

*

80

58

46

30

23

17

7

C-

X

empty

vector

X

C+

*

*

*

*

*

*

*

*

*

*

*

*

LepB YfdH Ev GsiC OppC

175

80

58

46

30

23

17

7

second elution

His

  • 7. Conclusion
  • Characterisation of protein-protein interactions is crucial for understanding proteins function.
  • Novel membrane proteins have been expressed (YfdH, YhbN, GsiC, YfdC, YhbE, YbjE)
  • and purified (YfdH, YhbN, GsiC).
  • Additional bands are seen on SDS-PAGE gel after purification. Nevertheless, further
  • protocol‘s development is needed to eliminate contaminants.
  • Rigorous validation of true interactions will be supported by alternative techniques such as
  • BN-PAGE and 2D BN/SDS-PAGE.
  • Small membrane proteins soon to be studied and characterised.
  • 8. References and Acknowledgements
  • Hemm M.R., Paul B.J., Schneider T.D., Storz G., Rudd K.E. (2008) Small membrane proteins found by comparative genomics and ribosome binding site models. Molecular Microbiology 70(6), 1487-1501
  • Gingras A.-C., Gstaiger M., Raught B., Aebersold R. (2007) Analysis of protein complexes using mass spectrometry. Nature. Molecular Cell Biology 8: 645- 653
  • Howell J.M., Winstone T.L., Coorssen J.R., Turner R.J. (2006) An evaluation of in vitro protein-protein interaction techniques: Assessing contaminating background proteins. Proteomics 6, 2050-2069
  • Albiniak is funded by the European Commission under the 7th Framework Programme (FP7),
  • Marie-Curie ITN Project 215524.
  • The author would like to thank professor Colin Robinson for support and useful discussions and professor Daniel O. Daley
  • for help and providing the constructs.