Characterization and exploitation of inner membrane proteins in Escherichia coli

This thesis illustrates the characterization and exploitation of inner membrane proteins in Escherichia coli and involves two parallel aspects. Firstly, in order to build the picture of the small inner membrane proteome in Escherichia coli K12, global search for E. coli K12 small inner membrane proteins was performed by means of bioinformatic analyses. The profile that was generated in this study consists of 108 short open reading frames encoding membrane proteins in E. coli str. K12. Additionally, a second list of 138 sequences was produced and is formed by recently identified genomic sequences with predicted transmembrane domains. The generated profile of small (characterized and predicted) membrane proteins facilitated further biochemical analysis of some predicted and hypothetical short open reading frames encoding inner membrane proteins. Two experimental approaches to study small membrane proteins were used. In the first, confirming genomic expression, the expression of short open reading frames encoding membrane proteins was analysed by means of RT-PCR. The second approach, involved cloning the predicted, hypothetical small membrane proteins under arabinose-inducible promoter, pBAD and detecting their membrane localization. These experiments confirmed bioinformatic results and furthermore demonstrated the uselfullness of the approach used. The Bacillus subtilis small inner membrane protein, TatAd was then used as the case study in the exploitation of inner membrane proteins. Recently, it has been shown that E. coli cells can export high levels of heterologous protein in Tat-dependent manner. In this thesis, B. subtilis TatAdCd system in E. coli lacking the native TatABC system was analysed by means of batch fermentation. It was showed, that a heterologus model protein, TorA-GFP, is efficiently exported by the TatAdCd system to the periplasm. Further analysis revealed that while the GFP is initially exported to the periplasm, the mature protein is progressively found in the extracellular medium. It was demonstrated that by the end of a 16 h batch fermentation, ~ 90 % of exported GFP was present in the medium as active mature protein. It was confirmed that total protein profiles of the medium and periplasm were essentially identical, confirming that the outer membrane became leaky during the fermentation process. The downstream processing and sample analysis revealed that GFP export levels were relatively high, with ~ 0.35 g GFP L-1 culture present in the medium. The analysis of bacterial cells under transmission electron microscopy revealed, that the cells remain intact and there is no large-scale release of cytoplasmic contents. Additionally, novel phenotype for E. coli !tat TatAdCd-expressing cells was observed. Cells overexpressing TatAdCd system displayed filamentous phenotype which became more prominent towards the end of the incubation time. The thesis concludes with a discussion of the pharmaceutical and biotechnological relevance of producing recombinant protein in E. coli by the TatAdCd system and harvesting directly from the medium, with potential advantages in terms of ease of purification and downstream processing. Additionally, the advantages and general applicability of global analysis of E. coli small inner membrane proteome are presented