Abstract
Protein secretion and subcellular localization in E. coli has been under investigation for more than 60 years. While many details about the molecular mechanisms of these processes have been revealed, several facets of protein translocation still remain unclear. Bacteria secrete numerous proteins such as pathogenicity factors, toxins or degradative enzymes (Fernandez and Berenguer 2000). Six different secretion mechanisms for extruding proteins into the extracellular environment have been identified to-date. In Gram-negative bacteria such as E. coli, secretion into the extracellular medium requires crossing of two biological membranes, the inner and outer membranes of the cell. However, systems for protein translocation into the extracellular medium are generally highly protein-specific and with very few exceptions have not yet been engineered for the efficient export of recombinant proteins. More relevant from a technical and engineering standpoint, is the translocation of polypeptides from the cytoplasm into the periplasmic space, the main secretory compartment which is equivalent to the endoplasmic reticulum of eukaryotic cells.
In the first part of this chapter, we discuss export via the general Sec pathway and the Twin-Arginine Translocase (Tat) pathway. Compartmentalized molecular chaperones facilitate folding, impose a quality control step on the maturation of certain secreted proteins, especially those exported via Tat, and further facilitate the decision which protein export route should be chosen. The second part of this chapter focuses on the design of genetic screens or selections that capitalize on protein secretion to aid the screening of libraries of protein variants for molecular recognition or catalysis. We will briefly summarize the major E. coli-based display technologies and introduce new methodologies particularly those utilizing the Twin-Arginine Translocase pathway.
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Strauch, EM., Georgiou, G. (2009). Mechanistic Challenges and Engineering Applications of Protein Export in E. coli . In: Lee, S.Y. (eds) Systems Biology and Biotechnology of Escherichia coli . Springer, Dordrecht. https://doi.org/10.1007/978-1-4020-9394-4_16
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