960 results about "Inclusion bodies" patented technology

Peptide tags, referred to here as inclusion body tags, are disclosed and are useful for the generation of insoluble fusion peptides. The fusion peptides comprise at least one inclusion body tag operably linked to a peptide of interest. Expression of the fusion peptide in a host cell results in a product that is insoluble and contained within inclusion bodies in the cell and / or cell lysate. The inclusion bodies may then be purified and the protein of interest may be isolated after cleavage from the inclusion body tag.

A harvesting device for capturing a biological product directly by binding the secreted biological product with a resin, discarding the nutrient medium and eluting the biological product as a concentrated solution, eliminating the steps of sterile filtration and volume reduction, thus allowing one to combine the steps of recombinant expression and separation of a biological product. The method allows loading of resin for column-purification, eliminating all steps of perfusion process and maintaining a sink condition of a toxic product in nutrient medium to optimize productivity of host cells. The instant invention also allows harvesting of solubilized inclusion bodies after the cells have been lysed and refolding of proteins inside the bioreactor.

Described herein are methods for the solubilization and proteolytic cleavage of fusion protein aggregates, including autocatalytic fusion proteins, at pressures greater than atmospheric pressure to yield soluble target polypeptides.

Botulinum neurotoxins, the most potent of all toxins, induce lethal neuromuscular paralysis by inhibiting exocytosis at the neuromuscular junction. The light chains (LC) of these dichain neurotoxins are a new class of zinc-endopeptidases that specifically cleave the synaptosomal proteins, SNAP-25, VAMP, or syntaxin at discrete sites. The present invention relates to the construction, expression, purification, and use of synthetic or recombinant botulinum neutoroxin genes. For example, a synthetic gene for the LC of the botulinum neurotoxin serotype A (BoNT / A) was constructed and overexpressed in Escherichia coli. The gene product was purified from inclusion bodies. The methods of the invention can provide 1.1 g of the LC per liter of culture. The LC product was stable in solution at 4° C. for at least 6 months. This rBoNT / A LC was proteolytically active, specifically cleaving the Glu-Arg bond in a 17-residue synthetic peptide of SNAP-25, the reported cleavage site of BoNT / A. Its calculated catalytic efficiency kcat / Km was higher than that reported for the native BoNT / A dichain. Treating the rBoNT / A LC with mercuric compounds completely abolished its activity, most probably by modifying the cysteine-164 residue located in the vicinity of the active site. About 70% activity of the LC was restored by adding Zn2+ to a Zn2+-free, apo-LC preparation. The LC was nontoxic to mice and failed to elicit neutralizing epitope(s) when the animals were vaccinated with this protein. In addition, injecting rBoNT / A LC into sea urchin eggs inhibited exocytosis-dependent plasma membrane resealing.