Yield, solubility and conformational quality of soluble proteins are not simultaneously favored in recombinant Escherichia coli.

Many enzymes or fluorescent proteins produced in Escherichia coli are enzymatically active or fluorescent respectively when deposited as inclusion bodies. The occurrence of insoluble but functional protein species with native-like secondary structure indicates that solubility and conformational quality of recombinant proteins are not coincident parameters, and suggests that both properties can be engineered independently. We have here proven this principle by producing elevated yields of a highly fluorescent but insoluble green fluorescent protein (GFP) in a DnaK- background, and further enhancing its solubility through adjusting the growth temperature and GFP gene expression rate. The success of such a two-step approach confirms the independent control of solubility and conformational quality, advocates for new routes towards high quality protein production and intriguingly, proves that high protein yields dramatically compromise the conformational quality of soluble versions

Learning about protein solubility from bacterial inclusion bodies.

The progressive solving of the conformation of aggregated proteins and the conceptual understanding of the biology of inclusion bodies in recombinant bacteria is providing exciting insights on protein folding and quality. Interestingly, newest data also show an unexpected functional and structural complexity of soluble recombinant protein species and picture the whole bacterial cell factory scenario as more intricate than formerly believed

The Functional quality of soluble recombinant polypeptides produced in Escherichia coli is defined by a wide conformational spectrum.

We have observed that a soluble recombinant green fluorescent protein produced in Escherichia coli occurs in a wide conformational spectrum. This results in differently fluorescent protein fractions in which morphologically diverse soluble aggregates abound. Therefore, the functional quality of soluble versions of aggregation-prone recombinant proteins is defined statistically rather than by the prevalence of a canonical native structure

Systems-level analysis of protein quality in inclusion body-forming Escherichia coli cells

Recombinant proteins produced in Escherichia coli often aggregate as amorphous masses of insoluble material known as inclusion bodies. Being quite homogeneous in their composition, inclusion bodies display amyloid-like properties such as sequence-dependent protein-protein interactions, seeding-driven deposition of their components and β-sheet intermolecular architecture. However, inclusion bodies formed by different proteins and enzymes also show important extents of native-like secondary structure and include significant proportions of properly folded, functional protein, which makes them suitable to be used in catalytic processes. Inclusion bodies are formed as a result of the incapability of the quality control cell system to cope with the non physiological amounts of misfolding-prone proteins produced upon recombinant gene expression. Multiple cellular proteins involved in the quality control, namely chaperones and proteases, participate in their formation and co-ordinately determine the amount of aggregated protein, the size of aggregates and the main structural and functional properties of the embedded polypeptides, such as their inner molecular organization. © 2009 Springer Netherlands