Into the Membrane and Beyond : Improving Membrane Protein Over-Expression in Escherichia coli
Sammanfattning: Membrane proteins fulfil a wide variety of essential functions in the cell and many are (potential) drug targets. Since their natural abundance is usually very low, most membrane proteins have to be over-expressed for functional and structural studies. T7 RNA polymerase (T7RNAP) based Escherichia coli strains, like BL21(DE3), are very popular protein production hosts. Unfortunately, over-expression of membrane proteins in E. coli is usually toxic to the cells. During my Ph.D. I have tried to understand the reasons for this toxicity by studying the consequences of membrane protein over-expression using a combination of proteomics and more focused biochemical and genetic methods. First, we had to improve the existing 2D BN/SDS-PAGE protocol to perform reliable comparative analysis of membrane proteomes. With the new protocol I have studied the effects of the expression of membrane proteins, including the human KDEL receptor, on BL21(DE3) and its derivatives, C41(DE3) and C43(DE3) (a.k.a. the Walker strains). The latter two were isolated for their improved membrane protein over-expression characteristics. Saturation of the Sec translocon, a cytoplasmic membrane associated protein conducting channel that mediates the insertion/biogenesis of membrane proteins, appeared to be the prime reason for the toxicity of membrane protein over-expression. Therefore, it was not surprising that we have identified mutations in the promoter governing the expression of the T7RNAP in the C41(DE3) and C43(DE3) strains that weaken it compared to the one in BL21(DE3). Based on this observation, we have engineered a plasmid (pLemo) with the gene encoding the natural inhibitor of T7RNAP, T7 lysozyme, under the control of the titratable rhamnose promoter. With the help of this plasmid the activity of the T7RNAP can be precisely set thereby avoiding saturation of the Sec translocon upon membrane protein over-expression. However, we have identified more changes in the Walker strains. Notable examples are the up regulation of peptide transporters in C41(DE3) and the expression of the Lon protease in C43(DE3). To study peptide import in E. coli I have characterized the in C41(DE3) strongly up regulated periplasmic binding protein OppA using a combination of biochemical and structural methods. The obtained data have resulted in many leads and ideas to further improve membrane protein over-expression yields in E. coli.
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