In the last two decades, numerous innovative advances, strategies and protocols have been developed and optimized to improve the quality and quantity of soluble recombinant protein production in E. coli. One of the major challenges being the coelution of chaperone proteins along with desired recombinant protein of interest. The removal of chaperones is important for protein yield, structural determination, optimal activity, and desired function of the recombinant protein. In this chapter, we outline various strategies for removal of chaperone contaminants from oligomeric proteins, with the ultimate objective of ameliorating the quality and proper folding of recombinant proteins. We have discussed in detail the purification and expression of full-length protein, GNE (UDP-N-acetylglucosamine 2-epimerase/ N-acetylmannosamine kinase), as a case study for chaperone removal.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by cytoplasmic protein aggregates within motor neurons. These aggregates are linked to ALS pathogenesis. Recent evidence has suggested that stress granules may aid the formation of ALS protein aggregates. Here, we summarize current understanding of stress granules, focusing on assembly and clearance. We also assess the evidence linking alterations in stress granule formation and dynamics to ALS protein aggregates and disease pathology.

Employing biophysical and structural methods is a powerful way to elucidate mechanisms of molecular recognition in bacterial pathogenesis. Such studies invariably depend on the production of pure, folded and stable proteins. Many proteins that can be expressed recombinantly ultimately fail to meet one or more of these criteria. The cag proteins from Helicobacter pylori form a secretion system that delivers the oncoprotein, CagA, into human gastric epithelial cells through an interaction between CagL and host cell integrins, where it can cause gastric adenocarcinoma. Expression of full length CagA and CagL is problematic as CagA undergoes rapid degradation during purification and CagL is thermally unstable. Here, we describe a method for the purification of CagA that results in the production of the full length protein through coexpression with its endogenous chaperone, CagF, and its subsequent separation from its chaperone. Furthermore, we detail the production of CagL and the use of differential scanning fluorimetry to identify how CagL is thermally stabilized by reduced pH, which led to a new crystal form of CagL and novel insight to pathogenic mechanisms. The methods described here for the production of stable cag proteins can be applied to a wide range of proteins involved in bacterial pathogenesis.