From Biogenesis to Over- expression of Membrane Proteins in Escherichia coli Samuel Wagner Stockholm University © Samuel Wagner, Stockholm 2008 ISBN 978-91-7155-594-6, Pages i-81 Printed in Sweden by Universitetsservice AB, Stockholm 2008 Distributor: Department of Biochemistry and Biophysics, Stockholm University To Claudia. Abstract In both pro- and eukaryotes 20-30% of all genes encode α-helical transmem- brane domain proteins, which act in various and often essential capacities. Notably, membrane proteins play key roles in disease and they constitute more than half of all known drug targets. The natural abundance of membrane proteins is in general too low to con- veniently isolate sufficient material for functional and structural studies. Therefore, most membrane proteins have to be obtained through overexpres- sion. Escherichia coli is one of the most successful hosts for overexpression of recombinant proteins, and T7 RNA polymerase-based expression is the major approach to produce recombinant proteins in E. coli. While the pro- duction of soluble proteins is comparably straightforward, overexpression of membrane proteins remains a challenging task. The yield of membrane lo- calized recombinant membrane protein is usually low and inclusion body formation is a serious problem. Furthermore, membrane protein overexpres- sion is often toxic to the host cell. Although several reasons can be postu- lated, the basis of these difficulties is not completely understood. It is gener- ally believed, that the complex requirements of membrane protein biogenesis significantly contribute to the difficulty of membrane protein overexpres- sion. Therefore, an understanding of membrane protein biogenesis is a pre- requisite for understanding membrane protein overexpression and for de- signing rational strategies to improve overexpression yields. The objective of my Ph.D. studies has been to improve membrane protein overexpression in E. coli by a) understanding membrane protein overexpres- sion from the perspective of membrane protein biogenesis, b) systematically investigating the physiological response to overexpression of membrane proteins and c) engineering strains that are optimized for membrane protein overexpression based on insights resulting from these studies. By working toward these objectives, I was able to identify and alleviate one of the major bottlenecks of membrane protein overexpression in E. coli: saturation of the Sec-translocon could be overcome by harmonizing transla- tion and membrane insertion of the recombinant membrane protein. This minimized the toxic effects of overexpression and thus resulted in increased membrane protein-producing biomass. List of Publications Primary publications This thesis is based on the following publications, which will be referred to by their roman numerals. I. Baars L., Wagner S., Wickström D., Klepsch M., Ytterberg A.J., van Wijk K.J., de Gier J.W. (2008). Effects of SecE depletion on the inner and outer membrane proteomes of Escherichia coli. J. Bacteriol. in press. II. Wagner S.*, Pop O.*, Haan G.J.*, Baars L., Klepsch M.M., Genevaux P., Luirink J., de Gier J.W. Biogenesis of MalF and the MalFGK2 maltose transport complex in Escherichia coli requires YidC. J. Biol. Chem. in revi- sion. III. Wagner S., Baars L., Ytterberg A.J., Klussmeier, A., Wagner C.S., Nord O., Nygren P.Å., van Wijk K.J., de Gier J.W. (2007). Consequences of membrane protein overexpression in Escherichia coli. Mol. Cell. Proteomics 6:1527-50. IV. Wagner S., Klepsch M.M., Schlegel S., Appel A., Draheim R., Tarry M., Högbom M., van Wijk K.J., Slotboom D.J., Persson J.O., de Gier J.W. Tuning Escherichia coli for membrane protein overexpression. Submitted manuscript. Additional publications Klepsch M.M., Schlegel S., Wickström D., Friso G., van Wijk K.J., Persson J.O., de Gier J.W., Wagner S. (2008). Immobilization of the first dimension in 2D blue native/SDS-PAGE allows the relative quantification of membrane proteomes. Methods 46 commissioned manuscript. Wagner S., de Gier J.W. Expression system for proteins. Patent application filed on 02/29/2008, Reference number: #P08207SE00. Wagner S.*, Lerch-Bader M.*, Drew D.*, de Gier J.W. (2006). Rationaliz- ing membrane protein overexpression. Trends Biotechnol. 24:364-71. Baars L., Ytterberg A.J., Drew D., Wagner S., Thilo C., van Wijk K.J., de Gier J.W. (2006). Defining the role of the Escherichia coli chaperone SecB using comparative proteomics. J. Biol. Chem. 281:10024-34. Marani P.*, Wagner S.*, Baars L., Genevaux P., de Gier J.W., Nilsson I., Casadio R., von Heijne G. (2006). New Escherichia coli outer membrane proteins identified through prediction and experimental verification. Protein Sci. 15:884-9. * authors contributed equally Contents Abstract ...........................................................................................................v List of Publications ......................................................................................... vi Primary publications ....................................................................................................... vi Additional publications.................................................................................................... vi Abbreviations ..................................................................................................x 1 Introduction ..................................................................................................1 Biological membranes .....................................................................................................2 Definition and classification of membrane proteins.........................................................3 Biogenesis of membrane proteins...................................................................................3 Targeting to the cytoplasmic membrane....................................................................4 Integration into the cytoplasmic membrane...............................................................4 Assembly and folding in the membrane.....................................................................6 How membranes shape protein structure..................................................................7 Quality control of membrane proteins .............................................................................9 Biogenesis of secretory proteins ...................................................................................11 Folding of cytoplasmic proteins & heat shock response...............................................11 Folding of cytoplasmic proteins ...............................................................................12 Heat shock response ...............................................................................................13 Recovery and degradation of aggregated proteins .................................................15 Host systems for the overexpression of proteins ..........................................................17 Protein overexpression in E. coli...................................................................................17 Promoter systems ....................................................................................................19 T7 RNA polymerase based expression ...................................................................21 Metabolic burden of protein overexpression............................................................21 Inclusion body formation and how to prevent it .......................................................23 Targeted/localized overexpression ..........................................................................25 Membrane protein overexpression in E. coli.................................................................26 Which properties make a well expressing membrane protein?...............................27 Monitoring membrane protein overexpression ........................................................27 Bottlenecks of membrane protein overexpression ..................................................28 Heterologous expression .........................................................................................30 Target protein optimization ......................................................................................32 Host strain optimization............................................................................................34 Optimization of expression conditions .....................................................................35 2 Methodology...............................................................................................37 Flow cytometry of bacterial cells ...................................................................................37 Aggregate purification ...................................................................................................39 Preparation of inner and outer membranes ..................................................................39 2-dimensional polyacrylamide gel electrophoresis .......................................................40 2-dimensional IEF/SDS polyacrylamide gel electrophoresis...................................41 2-dimensional blue native/SDS polyacrylamide gel electrophoresis.......................42 Image analysis of 2-dimensional polyacrylamide gels.............................................43 Statistical analysis of 2-dimensional polyacrylamide gels .......................................45 Identification of proteins by MALDI-TOF and peptide mass fingerprinting..............46