Characterization of novel membrane complexes in Escherichia coli

1. 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.