the-multifaceted-benefits-of-protein-co-expression-in-escherichia-coli

The Multifaceted Benefits of Protein Co-expression in <em>Escherichia coli</em>

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

University of Bologna

To evaluate the outcome of cystoid macular edema treated with intravitreal injections of bevacizumab and macular grid laser photocoagulation (GLP), in patients with perfused branch retinal vein occlusion.

Bevacizumab compared with macular laser grid photocoagulation for cystoid macular edema in branch retinal vein occlusion.

Two systems for the co-expression of proteins in Escherichia coli were designed and constructed. The first system relies on the new vector, pGOOD, which is compatible with ColE1-type plasmids and sustains efficient co-expression of soluble protein complexes. The second system is based on the pGOOD1 vector (a derivative of pGOOD), useful for the production of toxic proteins, whose synthesis can be regulated by the co-expressed LacI repressor.

pGOODs: new plasmids for the co-expression of proteins in Escherichia coli.

Escherichia coli DNA polymerase III holoenzyme (HE) contains a core polymerase consisting of three subunits: α (polymerase), ε (3'-5' exonuclease), and θ. Genetic experiments suggested that θ subunit stabilizes the intrinsically labile ε subunit and, furthermore, that θ might affect the cellular amounts of Pol III core and HE. Here, we provide biochemical evidence supporting this model by analyzing the amounts of the relevant proteins. First, we show that a ΔholE strain (lacking θ subunit) displays reduced amounts of free ε. We also demonstrate the existence of a dimer of ε, which may be involved in the stabilization of the protein. Second, θ, when overexpressed, dissociates the ε dimer and significantly increases the amount of Pol III core. The stability of ε also depends on cellular chaperones, including DnaK. Here, we report that: (i) temperature shift-up of ΔdnaK strains leads to rapid depletion of ε, and (ii) overproduction of θ overcomes both the depletion of ε and the temperature sensitivity of the strain. Overall, our data suggest that ε is a critical factor in the assembly of Pol III core, and that this is role is strongly influenced by the θ subunit through its prevention of ε degradation.

Stabilization of the Escherichia coli DNA polymerase III ε subunit by the θ subunit favors in vivo assembly of the Pol III catalytic core.

Several abnormalities of the respiratory function have been reported in patients with type 1 and type 2 diabetes. These abnormalities concern lung volume, pulmonary diffusing capacity, control of ventilation, bronchomotor tone, and neuroadrenergic bronchial innervation. Many hypotheses have emerged, and characteristic histological changes have been described in the "diabetic lung", which could explain this abnormal respiratory function. Given the specific abnormalities in diabetic patients, the lung could thus be considered as a target organ in diabetes. Although the practical implications of these functional changes are mild, the presence of an associated acute or chronic pulmonary and/or cardiac disease could determine severe respiratory derangements in diabetic patients. Another clinical consequence of the pulmonary involvement in diabetes is the accelerated decline in respiratory function. The rate of decline in respiratory function in diabetics has been found to be two-to-three times faster than in normal non-smoking subjects, as reported in longitudinal studies. This finding, together with the presence of anatomical and biological changes similar to those described in the aging lung, indicates that the "diabetic lung" could even be considered a model of accelerated aging. This review describes and analyses the current insight into the relationship of diabetes and lung disease, and suggests intensifying research into the lung as a possible target organ in diabetes.

The diabetic lung--a new target organ?

A coupled enzyme assay able to monitor the kinetics of reactions catalyzed by phosphate- or pyrophosphate-releasing enzymes is presented here. The assay is based on the concerted action of inorganic pyrophosphatase (PPase), purine nucleoside phosphorylase (PNPase), and xanthine oxidase (XOD). In the presence of phosphate, PNPase catalyzes the phosphorolysis of inosine, generating hypoxanthine, which is oxidized to uric acid by XOD. The uric acid accordingly formed can be spectrophotometrically monitored at 293 nm, taking advantage of a molar extinction coefficient which is independent of pH between 6 and 9. The coupled assay was tested using DNA polymerases as a model system. The activity of Klenow enzyme was quantitatively determined, and it was found in agreement with the corresponding activity determined by traditional methods. Moreover, the continuous coupled assay was used to determine Km and Vmax of Klenow enzyme, yielding values in good agreement with previous observations. Finally, the coupled assay was also used to determine the activity of partially purified DNA polymerases, revealing its potential use to monitor purification of phosphate- or pyrophosphate-releasing enzymes.

Continuous enzyme-coupled assay of phosphate- or pyrophosphate-releasing enzymes.

The aim of this study was to verify to which extent in type 2 diabetes mellitus respiratory function and respiratory muscle efficiency decline over time in relation to the quality of glycemic control (GC).

Article	Year
Bevacizumab compared with macular laser grid photocoagulation for cystoid macular edema in branch retinal vein occlusion. Retina (Philadelphia, Pa.)\| PubMed ID: 19174717	2009
pGOODs: new plasmids for the co-expression of proteins in Escherichia coli. Biotechnology letters\| PubMed ID: 21516313	2011
Stabilization of the Escherichia coli DNA polymerase III ε subunit by the θ subunit favors in vivo assembly of the Pol III catalytic core. Archives of biochemistry and biophysics\| PubMed ID: 22546509	2012
The diabetic lung--a new target organ? The review of diabetic studies : RDS\| PubMed ID: 22972442	2012
Continuous enzyme-coupled assay of phosphate- or pyrophosphate-releasing enzymes. BioTechniques\| PubMed ID: 23030062	2012
Decline of the lung function and quality of glycemic control in type 2 diabetes mellitus. European journal of internal medicine\| PubMed ID: 25770074	2015

Emanuele Conte

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