GroEL–GroES assisted folding of multiple recombinant proteins simultaneously over-expressed in Escherichia coli

Folding of aggregation prone recombinant proteins through co-expression of chaperonin GroEL and GroES has been a popular practice in the effort to optimize preparation of functional protein in Escherichia coli. Considering the demand for functional recombinant protein products, it is desirable to apply the chaperone assisted protein folding strategy for enhancing the yield of properly folded protein. Toward the same direction, it is also worth attempting folding of multiple recombinant proteins simultaneously over-expressed in E. coli through the assistance of co-expressed GroEL–ES. The genesis of this thinking was originated from the fact that cellular GroEL and GroES assist in the folding of several endogenous proteins expressed in the bacterial cell. Here we present the experimental findings from our study on co-expressed GroEL–GroES assisted folding of simultaneously over-expressed proteins maltodextrin glucosidase (MalZ) and yeast mitochondrial aconitase (mAco). Both proteins mentioned here are relatively larger and aggregation prone, mostly form inclusion bodies, and undergo GroEL–ES assisted folding in E. coli cells during over-expression. It has been reported that the relative yield of properly folded functional forms of MalZ and mAco with the exogenous GroEL–ES assistance were comparable with the results when these proteins were overexpressed alone. This observation is quite promising and highlights the fact that GroEL and GroES can assist in the folding of multiple substrate proteins simultaneously when over-expressed in E. coli. This method might be a potential tool for enhanced production of multiple functional recombinant proteins simultaneously in E. coli.     

Pathogen–pathogen interactions: a comparative study of Escherichia coli interactions with the clinical and environmental isolates of Acanthamoeba

In this study, we compared the interactions of invasive and non-invasive strains of E. coli with clinical and environmental isolates of Acanthamoeba. The environmental isolate of Acanthamoeba exhibited significantly higher association with E. coli compared with the clinical isolates of Acanthamoeba. The ratio of E. coli per amoebae was more than 8-fold higher in the environmental isolate compared with the clinical isolates of Acanthamoeba. Interestingly, non-pathogenic environmental Acanthamoeba showed uptake and/or survival of the non-invasive E. coli. In contrast, clinical isolates of Acanthamoeba did not support uptake and/or survival of non-invasive E. coli. Using several mutants derived from K1, we demonstrated that outer membrane protein A (OmpA) and lipopolysaccharide (LPS) are crucial bacterial determinants responsible for E. coli K1 interactions and in the intracellular survival of E. coli in Acanthamoeba. The use of Acanthamoeba as a model to study E. coli K1 pathogenesis and to understand bacterial immune evasion strategies is discussed further.     

Cholesterol transport in steroid biosynthesis: Role of protein–protein interactions and implications in disease states

The transfer of cholesterol from the outer to the inner mitochondrial membrane is the rate-limiting step in hormone-induced steroid formation. To ensure that this step is achieved efficiently, free cholesterol must accumulate in excess at the outer mitochondrial membrane and then be transferred to the inner membrane. This is accomplished through a series of steps that involve various intracellular organelles, including lysosomes and lipid droplets, and proteins such as the translocator protein (18 kDa, TSPO) and steroidogenic acute regulatory (StAR) proteins. TSPO, previously known as the peripheral-type benzodiazepine receptor, is a high-affinity drug- and cholesterol-binding mitochondrial protein. StAR is a hormone-induced mitochondria-targeted protein that has been shown to initiate cholesterol transfer into mitochondria. Through the assistance of proteins such as the cAMP-dependent protein kinase regulatory subunit Iα (PKA-RIα) and the PKA-RIα- and TSPO-associated acyl-coenzyme A binding domain containing 3 (ACBD3) protein, PAP7, cholesterol is transferred to and docked at the outer mitochondrial membrane. The TSPO-dependent import of StAR into mitochondria, and the association of TSPO with the outer/inner mitochondrial membrane contact sites, drives the intramitochondrial cholesterol transfer and subsequent steroid formation. The focus of this review is on (i) the intracellular pathways and protein–protein interactions involved in cholesterol transport and steroid biosynthesis and (ii) the roles and interactions of these proteins in endocrine pathologies and neurological diseases where steroid synthesis plays a critical role.     

Proteolytic degradation of fused protein A-β-galactosidase in Escherichia coli

The stability of the fusion protein staphylococcal protein A-E. coli β-galactosidase (SpA-βgal) produced in E. coli has been studied both in cell disintegrate and in purified preparations. SpA-βgal was degraded by a proteolytic cleavage between the two functional parts of the molecule, resulting in one β-galactosidase tetramer and four protein A molecules. Intermediates were detected, namely β-galactosidase containing three, two and one protein A. The β-galactosidase was stable with respect to enzyme activity and molecular weight, while protein A was further degraded. In cell disintegrate the half-life of SpA-βgal was found to be 6 h at 20°C and 1.5 h at 37°C. The protease responsible for initial proteolytic cleavage of SpA-βgal was shown to be cell debris associated.     

Peptides and peptidomimetics as regulators of protein–protein interactions

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Thermodynamics of co-translational folding and ribosome–nascent chain interactions

Proteins can begin the conformational search for their native structure in parallel with biosynthesis on the ribosome, in a process termed co-translational folding. In contrast to the reversible folding of isolated domains, as a nascent chain emerges from the ribosome exit tunnel during translation the free energy landscape it explores also evolves as a function of chain length. While this presents a substantially more complex measurement problem, this review will outline the progress that has been made recently in understanding, quantitatively, the process by which a nascent chain attains its full native stability, as well as the mechanisms through which interactions with the nearby ribosome surface can perturb or modulate this process.     

Assessment and significance of protein–protein interactions during development of protein biopharmaceuticals

Early development of protein biotherapeutics using recombinant DNA technology involved progress in the areas of cloning, screening, expression and recovery/purification. As the biotechnology industry matured, resulting in marketed products, a greater emphasis was placed on development of formulations and delivery systems requiring a better understanding of the chemical and physical properties of newly developed protein drugs. Biophysical techniques such as analytical ultracentrifugation, dynamic and static light scattering, and circular dichroism were used to study protein–protein interactions during various stages of development of protein therapeutics. These studies included investigation of protein self-association in many of the early development projects including analysis of highly glycosylated proteins expressed in mammalian CHO cell cultures. Assessment of protein–protein interactions during development of an IgG1 monoclonal antibody that binds to IgE were important in understanding the pharmacokinetics and dosing for this important biotherapeutic used to treat severe allergic IgE-mediated asthma. These studies were extended to the investigation of monoclonal antibody–antigen interactions in human serum using the fluorescent detection system of the analytical ultracentrifuge. Analysis by sedimentation velocity analytical ultracentrifugation was also used to investigate competitive binding to monoclonal antibody targets. Recent development of high concentration protein formulations for subcutaneous administration of therapeutics posed challenges, which resulted in the use of dynamic and static light scattering, and preparative analytical ultracentrifugation to understand the self-association and rheological properties of concentrated monoclonal antibody solutions.     

Direct molecular fishing in molecular partners investigation in protein–protein and protein–peptide interactions

An original experimental method of direct molecular fishing has been developed for identification of potential partners of protein–protein and protein–peptide interactions. It is based on combination of surface plasmon resonance technology (SPR), size exclusion and affinity chromatography and mass spectrometric identification of proteins (LC-MS/MS). Previously, we demonstrated applicability of this method for protein interactomics using experimental model system, as well as in the pilot study in the frame of the Human Proteome Project (HPP). In the present paper, this method was successfully applied to identify possible molecular partners of 7 target proteins encoded by genes of 18 chromosome (also in the frame of the HPP). Fishing on the affinity sorbents with immobilized target proteins as ligands was carried out using total lysate of human liver tissue as well as pooled sets of fractions (individual for each bait-protein) obtained by means of a combination of size exclusion chromatography and SPR analysis for the presence of potential prey-proteins in each fraction. As a result we obtained lists of possible molecular partners of all 7 proteins and performed a comparative evaluation of direct fishing specificity for these target proteins. Direct molecular fishing was also successfully used for search of potential protein partners interacting with different isoforms of amyloid-beta peptide, playing a key role in the development of Alzheimer’s disease. The synthetic peptides that are analogues of the metal-binding domain isoforms of beta-amyloid were used as molecular baits and the fishing was performed in various fractions of immortalized human neural cells. As a result, 13 potential partner proteins were identified in the cytosol fraction of the cells by fishing on amyloid-beta peptide (1-16).     

Role of hydrophobic interactions and salt-bridges in β-hairpin folding

β-Hairpins are the simplest form of β-sheets which, due to the presence of long-range interactions, can be considered as tertiary structures. Molecular dynamics simulation is a powerful tool that can unravel whole pathways of protein folding/unfolding at atomic resolution. We have performed several molecular dynamics simulations, to a total of over 250 ns, of a β-hairpin peptide in water using GROMACS. We show that hydrophobic interactions are necessary for initiating the folding of the peptide. Once formed, the peptide is stabilized by hydrogen bonds and disruption of hydrophobic interactions in the folded peptide does not denature the structure. In the absence of hydrophobic interactions, the peptide fails to fold. However, the introduction of a salt-bridge compensates for the loss of hydrophobic interactions to a certain extent. Figure Model of b-hairpin folding: Folding is initiated by hydrophobic interactions (Brown circles). The folded structure, once formed, is stabilized by hydrogen bonds (red lines) and is unaffected by loss of hydrophobic contacts