Purification of Inclusion Bodies Produced in Bacteria and Yeast.

Purification of inclusion bodies (IBs) is gaining importance due to the raising of novel applications for these submicron particulate protein clusters, with potential uses in the biomedical and biotechnological fields among others. Here, we present five optimized methods to purify IBs adapting classical procedures to the material nature, as well as the requirements of the producer cell (Gram-negative bacteria, Gram-positive bacteria, or yeast) and the IB final application.

Bacterial inclusion bodies are industrially exploitable amyloids.

Understanding the structure, functionalities and biology of functional amyloids is an issue of emerging interest. Inclusion bodies, namely protein clusters formed in recombinant bacteria during protein production processes, have emerged as unanticipated, highly tunable models for the scrutiny of the physiology and architecture of functional amyloids. Based on an amyloidal skeleton combined with varying amounts of native or native-like protein forms, bacterial inclusion bodies exhibit an unusual arrangement that confers mechanical stability, biological activity and conditional protein release, being thus exploitable as versatile biomaterials.

Characterizing the spatio-temporal role of sorting nexin 17 in human papillomavirus trafficking.

The human papillomavirus (HPV) L2 capsid protein plays an essential role during the early stages of viral infection. Previous studies have shown that the interaction between HPV L2 and endosomal sorting nexin 17 (SNX17) is conserved across multiple PV types where it plays an essential role in infectious entry, suggesting an evolutionarily conserved pathway of PV trafficking. Here we show that the peak time of interaction between HPV-16 L2 and SNX17 is rather early, at 2 h post-infection.

Bacterial inclusion body purification.

Purification of bacterial inclusion bodies (IBs) is gaining importance due to the raising of novel applications for this type of submicron particulate protein clusters, with potential uses in the biomedical field among others. Here, we present two optimized methods to purify IBs adapting classical procedures to the material nature as well as the requirements of its final application.

Inclusion bodies as potential vehicles for recombinant protein delivery into epithelial cells.

Background: We present the potential of inclusion bodies (IBs) as a protein delivery method for polymeric filamentous proteins. We used as cell factory a strain of E. coli, a conventional host organism, and keratin 14 (K14) as an example of a complex protein.

Active protein aggregates produced in Escherichia coli.

Since recombinant proteins are widely used in industry and in research, the need for their low-cost production is increasing. Escherichia coli is one of the best known and most often used host organisms for economical protein production. However, upon over-expression, protein aggregates called inclusion bodies (IBs) are often formed.

Bacterial cell disruption: a crucial step in protein production.

Recombinant protein production significantly improved in the past three decades. Novel expression systems were developed, growth conditions optimised and the technology and thus monitoring and analysis significantly enhanced. However, the studies of bacterial cell disruption were more or less neglected.

Reprint of: Influence of the protein oligomericity on final yield after affinity tag removal in purification of recombinant proteins.

The new aspect concerning the applicability of histidine and other affinity tags for the purification of oligomeric proteins, with particular emphasis on cleavage efficiency and final yield, is presented in this study. The final yield depends on both the cleavage efficiency and the degree of oligomerization of the protein. Cleavage procedures that are good enough for monomeric proteins can be problematic for oligomeric proteins.

Isolation of biologically active nanomaterial (inclusion bodies) from bacterial cells.

Background: In recent years bacterial inclusion bodies (IBs) were recognised as highly pure deposits of active proteins inside bacterial cells. Such active nanoparticles are very interesting for further downstream protein isolation, as well as for many other applications in nanomedicine, cosmetic, chemical and pharmaceutical industry.To prepare large quantities of a high quality product, the whole bioprocess has to be optimised.

Engineering inclusion bodies for non denaturing extraction of functional proteins.

Background: For a long time IBs were considered to be inactive deposits of accumulated target proteins. In our previous studies, we discovered IBs containing a high percentage of correctly folded protein that can be extracted under non-denaturing conditions in biologically active form without applying any renaturation steps. In order to widen the concept of correctly folded protein inside IBs, G-CSF (granulocyte colony stimulating factor) and three additional proteins were chosen for this study: GFP (Green fluorescent protein), His7dN6TNF-alpha (Truncated form of Tumor necrosis factor alpha with an N-terminal histidine tag) and dN19 LT-alpha (Truncated form of Lymphotoxin alpha).

New properties of inclusion bodies with implications for biotechnology.

Human G-CSF (granulocyte colony-stimulating factor) is a well-known biopharmaceutical drug being mostly produced by overexpression in Escherichia coli, where it appears in the form of IBs (inclusion bodies). Following our initial findings that properties of inclusion bodies strongly depend on the growth conditions used, especially growth temperature, we compared the characteristics of the G-CSF inclusion bodies prepared at two different temperatures, namely 42 and 25 degrees C. IBs formed at higher growth temperatures have properties similar to the usually described IBs, containing mainly denatured recombinant protein and being almost completely insoluble in aqueous solutions containing mild detergents or low concentrations of denaturants.

Influence of the protein oligomericity on final yield after affinity tag removal in purification of recombinant proteins.

The new aspect concerning the applicability of histidine and other affinity tags for the purification of oligomeric proteins, with particular emphasis on cleavage efficiency and final yield, is presented in this study. The final yield depends on both the cleavage efficiency and the degree of oligomerization of the protein. Cleavage procedures that are good enough for monomeric proteins can be problematic for oligomeric proteins.