Andrea Vera

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

BackgroundMany 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.ResultsBy 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.ConclusionThis observation offers promising possibilities for the exploration of inclusion bodies as catalysts for industrial purposes, without any previous protein-refolding step.

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.

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.

Analysis of recombinant protein toxicity in E. coli through a phage λ-based genetic screening system

The aspartic protease from the human immunodeficiency virus type 1 (HIV-1) is highly toxic to E. coli, thus impairing its yield in production processes. Proteolytic cleavage of essential cellular proteins is probably a major contributor to the bacteriocidal effect but this has not been proven. Through an adapted high-throughput λ-based screening system, we have analyzed a set of HIV-1 protease mutants with distinguishable catalytic properties and we show that inactive enzymes are as toxic to E. coli cells as the wild-type enzyme. Together with additional data from directed molecular evolution approaches, these results indicate that the toxicity of the viral protease is not linked to its proteolytic activity. Our study also reveals that the λ-based screening system is a robust new tool for the genetic analysis of highly toxic recombinant products in E. coli.

DnaK-J are limiting for proper recombinant protein folding only at low production rates and when the physiological heat-shock stress response is not triggered

Meeting: a comparative view on host physiology The organisers would like to thank Novozymes Delta Ltd who generously supported the meeting. Meeting

Low growth temperatures improve the conformational quality of aggregation prone recombinant proteins in both soluble and insoluble E. coli cell fractions

a comparative view on host physiology The organisers would like to thank Novozymes Delta Ltd who generously supported the meeting. Meeting