Biomedical subjects

Andrea Vera

Publications and source records attributed to Andrea Vera.

7 recordsLinked to original sources

PubMed2007

The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures.

Protein aggregation is a major bottleneck during the bacterial production of recombinant proteins. In general, the induction of gene expression at sub-optimal growth temperatures improves the solubility of aggregation-prone polypeptides and minimizes inclusion body (IB) formation. However, the effect of low temperatures on the quality of the recombinant protein, especially within the insoluble cell fraction, has been hardly ever explored. In this work, we have examined the conformational status of a recombinant GFP protein when produced in Escherichia coli below 37 degrees C. As expected, the fraction of aggregated protein largely decreased at lower temperatures, while the conformational quality of both soluble and aggregated GFP, as reflected by its specific fluorescence emission, progressively improved. This observation indicates that physicochemical conditions governing protein folding affect concurrently the quality of the soluble and the aggregated forms of a misfolding-prone protein, and that protein misfolding and aggregation are clearly not coincident events.

Cold Temperature↗

PubMed2006

Insertional protein engineering for analytical molecular sensing.

The quantitative detection of low analyte concentrations in complex samples is becoming an urgent need in biomedical, food and environmental fields. Biosensors, being hybrid devices composed by a biological receptor and a signal transducer, represent valuable alternatives to non biological analytical instruments because of the high specificity of the biomolecular recognition. The vast range of existing protein ligands enable those macromolecules to be used as efficient receptors to cover a diversity of applications. In addition, appropriate protein engineering approaches enable further improvement of the receptor functioning such as enhancing affinity or specificity in the ligand binding. Recently, several protein-only sensors are being developed, in which either both the receptor and signal transducer are parts of the same protein, or that use the whole cell where the protein is produced as transducer. In both cases, as no further chemical coupling is required, the production process is very convenient. However, protein platforms, being rather rigid, restrict the proper signal transduction that necessarily occurs through ligand-induced conformational changes. In this context, insertional protein engineering offers the possibility to develop new devices, efficiently responding to ligand interaction by dramatic conformational changes, in which the specificity and magnitude of the sensing response can be adjusted up to a convenient level for specific analyte species. In this report we will discuss the major engineering approaches taken for the designing of such instruments as well as the relevant examples of resulting protein-only biosensors.

Journal Article↗

PubMed2005

Lon and ClpP proteases participate in the physiological disintegration of bacterial inclusion bodies.

Aggregated protein is solubilized by the combined activity of chaperones ClpB, DnaK and small heat-shock proteins, and this could account, at least partially, for the physiological disintegration of bacterial inclusion bodies. In vivo, the involvement of proteases in this process had been suspected but not investigated. By using an aggregation prone beta-galactosidase fusion protein produced in Escherichia coli, we show in this study that the main ATP-dependent proteases Lon and ClpP participate in the physiological disintegration of cytoplasmic inclusion bodies, their absence minimizing the protein removal up to 40%. However, the role of these proteases is clearly distinguishable especially regarding the fate of solubilized protein. While Lon appears as a minor contributor in the disintegration process, ClpP directs an important attack on the released or releasable protein even not being irreversibly misfolded. ClpP is then observed as a wide-spectrum, main processor of aggregation-prone proteins and also of polypeptides physiologically released from inclusion bodies, even when occurring as soluble versions with a conformation compatible with their enzymatic activity.

Endopeptidase Clp↗

PubMed2005

Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins.

BACKGROUND: Many enzymes of industrial interest are not in the market since they are bio-produced as bacterial inclusion bodies, believed to be biologically inert aggregates of insoluble protein. RESULTS: By using two structurally and functionally different model enzymes and two fluorescent proteins we show that physiological aggregation in bacteria might only result in a moderate loss of biological activity and that inclusion bodies can be used in reaction mixtures for efficient catalysis. CONCLUSION: This observation offers promising possibilities for the exploration of inclusion bodies as catalysts for industrial purposes, without any previous protein-refolding step.

Journal Article↗

PubMed2005

Amyloid-like properties of bacterial inclusion bodies.

Bacterial inclusion bodies are major bottlenecks in protein production, narrowing the spectrum of relevant polypeptides obtained by recombinant DNA. While regarded as amorphous deposits formed by passive and rather unspecific precipitation of unfolded chains, we prove here that they are instead organized aggregates sharing important structural and biological features with amyloids. By using an Escherichia coli beta-galactosidase variant, we show that aggregation does not necessarily require unfolded polypeptide chains but rather depends on specific interactions between solvent-exposed hydrophobic stretches in partially structured species. In addition, purified inclusion bodies are efficient and highly selective nucleation seeds, promoting deposition of soluble homologous but not heterologous polypeptides in a dose-dependent manner. Finally, inclusion bodies bind amyloid-diagnostic dyes, which, jointly with Fourier transform infra red spectroscopy data, indicates a high level of organized intermolecular beta-sheet structure. The evidences of amyloid-like structure of bacterial inclusion bodies, irrespective of potential applications in bioprocess engineering, prompts the use of bacterial models to explore the molecular determinants of protein aggregation by means of simple biological systems.

Amyloid↗

PubMed2005

Engineering the E. coli beta-galactosidase for the screening of antiviral protease inhibitors.

Site-specific proteolysis is essential in many fundamental cellular and viral processes. It has been previously shown that the Escherichia coli beta-galactosidase can be useful for the high-throughput screening of human immunodeficiency virus type 1 protease inhibitors. Here, by using crystallographic and functional data of the bacterial enzyme, we have identified a new accommodation site between amino acids 581 and 582, in a solvent-exposed and flexible beta-turn of domain III. The placement of the model peptide reproducing the matrix-capsid (p17/p24) gag cleavage sequence renders a highly active and efficiently digested chimeric construct. The use of this insertion site, that increases the cleavage potential of this reporter enzyme, can improve the sensitivity and dynamic range of the antiviral drug assay. This simple and highly specific analytical test may also be extended to the screening of other specific protease inhibitors by a convenient colorimetric assay.

Amino Acid Substitution↗

PubMed2004

The impact of dnaKJ overexpression on recombinant protein solubility results from antagonistic effects on the control of protein quality.

We have produced increasing levels of DnaK and its co-chaperone DnaJ along with the model VP1LAC misfolding-prone protein, to explore the role of DnaK on the management of Escherichia coli inclusion bodies. While relative solubility of VP1LAC is progressively enhanced, the heat-shock response is down-regulated as revealed by decreasing levels of GroEL. This is accompanied by an increasing yield of VP1LAC and a non-regular evolution of its insoluble fraction, at moderate levels of DnaK resulting in more abundant inclusion bodies. Also, the impact of chaperone co-expression is much more pronounced in wild type cells than in a DnaK- mutant, probably due to the different background of heat shock proteins in these cells. The involvement of DnaK in the supervision of misfolding proteins is then pictured as a dynamic balance between its immediate holding and folding activities, and the side-effect downregulation of the heat shock response though the limitation of other chaperone and proteases activities.

Dimerization↗