Molecular, Enzymatic, and Cellular Characterization of Soluble Adenylyl Cyclase From Aquatic Animals.

The enzyme soluble adenylyl cyclase (sAC) is the most recently identified source of the messenger molecule cyclic adenosine monophosphate. sAC is evolutionarily conserved from cyanobacteria to human, is directly stimulated by [Formula: see text] ions, and can act as a sensor of environmental and metabolic CO2, pH, and [Formula: see text] levels. sAC genes tend to have multiple alternative promoters, undergo extensive alternative splicing, be translated into low mRNA levels, and the numerous sAC protein isoforms may be present in various subcellular localizations. In aquatic organisms, sAC has been shown to mediate various functions including intracellular pH regulation in coral, blood acid/base regulation in shark, heart beat rate in hagfish, and NaCl absorption in fish intestine. Furthermore, sAC is present in multiple other species and tissues, and sAC protein and enzymatic activity have been reported in the cytoplasm, the nucleus, and other subcellular compartments, suggesting even more diverse physiological roles. Although the methods and experimental tools used to study sAC are conventional, the complexity of sAC genes and proteins requires special considerations that are discussed in this chapter

Interplays between copper and Mycobacterium tuberculosis GroEL1

The recalcitrance of pathogenic Mycobacterium tuberculosis, the agent of tuberculosis, to eradication is due to various factors allowing bacteria to escape from stress situations. The mycobacterial chaperone GroEL1, overproduced after macrophage entry and under oxidative stress, could be one of these key players. We previously reported that GroEL1 is necessary for the biosynthesis of phthiocerol dimycocerosate, a virulence-associated lipid and for reducing antibiotic susceptibility. In the present study, we showed that GroEL1, bearing a unique C-terminal histidine-rich region, is required for copper tolerance during Mycobacterium bovis BCG biofilm growth. Mass spectrometry analysis demonstrated that GroEL1 displays high affinity for copper ions, especially at its C-terminal histidine-rich region. Furthermore, the binding of copper protects GroEL1 from destabilization and increases GroEL1 ATPase activity. Altogether, these findings suggest that GroEL1 could counteract copper toxicity, notably in the macrophage phagosome, and further emphasizes that M. tuberculosis GroEL1 could be an interesting antitubercular target

Practical protocols for production of very high yields of recombinant proteins using Escherichia coli

The gram-negative bacterium Escherichia coli offers a mean for rapid, high yield, and economical production of recombinant proteins. However, high-level production of functional eukaryotic proteins in E. coli may not be a routine matter, sometimes it is quite challenging. Techniques to optimize heterologous protein overproduction in E. coli have been explored for host strain selection, plasmid copy numbers, promoter selection, mRNA stability, and codon usage, significantly enhancing the yields of the foreign eukaryotic proteins. We have been working on optimizations of bacterial expression conditions and media with a focus on achieving very high cell density for high-level production of eukaryotic proteins. Two high-cell-density bacterial expression methods have been explored, including an autoinduction introduced by Studier (Protein Expr Purif 2005;41:207–234) recently and a high-cell-density IPTG-induction method described in this study, to achieve a cell-density OD600 of 10–20 in the normal laboratory setting using a regular incubator shaker. Several practical protocols have been implemented with these high-cell-density expression methods to ensure a very high yield of recombinant protein production. With our methods and protocols, we routinely obtain 14–25 mg of NMR triple-labeled proteins and 17–34 mg of unlabeled proteins from a 50-mL cell culture for all seven proteins we tested. Such a high protein yield used the same DNA constructs, bacterial strains, and a regular incubator shaker and no fermentor is necessary. More importantly, these methods allow us to consistently obtain such a high yield of recombinant proteins using E. coli expression