Recombinant Expression and Overproduction of Transmembrane ß-Barrel Proteins.

Transmembrane ß-barrel proteins reside in the outer membrane of Gram-negative bacteria and are thus in direct contact with the environment. Because of that, they are involved in many key processes stretching from cellular survival to virulence. Hence, they are an attractive target for the development of novel antimicrobials, in addition to being of fundamental biological interest. To study this class of proteins, they are often required to be expressed in Escherichia coli. Recombinant expression of ß-barrel proteins can be achieved using two fundamentally different strategies. The first alternative uses a complete coding sequence that includes a signal peptide for targeting the protein to its native cellular location, the bacterial outer membrane. The second alternative omits the signal peptide in the gene, leading to mislocalization and aggregation of the protein in the bacterial cytoplasm. These aggregates, called inclusion bodies, can be solubilized and the protein can be folded into its native form in vitro. In this chapter, we present example protocols for both strategies and discuss their advantages and disadvantages.

Fluorescent Labeling of Outer Membrane Proteins Using the SpyCatcher-SpyTag System.

The SpyCatcher-SpyTag system has become a popular and versatile tool for protein ligation. It is based on a small globular protein (SpyCatcher) that binds to a 13-residue peptide (SpyTag), which subsequently leads to the formation of a covalent isopeptide bond. Thus, the reaction is essentially irreversible. Here, we describe how the SpyCatcher-SpyTag system can be used to label surface-exposed bacterial outer membrane proteins, e.g., for topology mapping or fluorescent time-course experiments. We cover using fluorescence measurements and microscopy to measure labeling efficiency using SpyCatcher fused with superfolder GFP in this chapter.