• Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study.

      Busso, Didier; Peleg, Yoav; Heidebrecht, Tatjana; Romier, Christophe; Jacobovitch, Yossi; Dantes, Ada; Salim, Loubna; Troesch, Edouard; Schuetz, Anja; Heinemann, Udo; et al. (2011-08)
      Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.
    • Structural basis for complex formation between human IRSp53 and the translocated intimin receptor Tir of enterohemorrhagic E. coli.

      de Groot, Jens C; Schlüter, Kai; Carius, Yvonne; Quedenau, Claudia; Vingadassalom, Didier; Faix, Jan; Weiss, Stefanie M; Reichelt, Joachim; Standfuss-Gabisch, Christine; Lesser, Cammie F; et al. (2011-09-07)
      Actin assembly beneath enterohemorrhagic E. coli (EHEC) attached to its host cell is triggered by the intracellular interaction of its translocated effector proteins Tir and EspF(U) with human IRSp53 family proteins and N-WASP. Here, we report the structure of the N-terminal I-BAR domain of IRSp53 in complex with a Tir-derived peptide, in which the homodimeric I-BAR domain binds two Tir molecules aligned in parallel. This arrangement provides a protein scaffold linking the bacterium to the host cell's actin polymerization machinery. The structure uncovers a specific peptide-binding site on the I-BAR surface, conserved between IRSp53 and IRTKS. The Tir Asn-Pro-Tyr (NPY) motif, essential for pedestal formation, is specifically recognized by this binding site. The site was confirmed by mutagenesis and in vivo-binding assays. It is possible that IRSp53 utilizes the NPY-binding site for additional interactions with as yet unknown partners within the host cell.