title: Ubiquitin fusion technology: bioprocessing of peptides.
abstract: Ubiquitin fusion technology represents an emerging method for economically producing peptides and small proteins in the bacterium Escherichia coli. Our focus is on peptide production where the need for cost-effective, scaleable processes has recently been highlighted by Kelley (1996). There are two principal features: (1) the expression system consists of a suitable E. coli host strain paired with a plasmid that encodes the ubiquitin fusion and (2) an ubiquitin-specific protease, UCH-L3, which cleaves only C-terminal extensions from ubiquitin. In this work, multigram yields were obtained of four ubiquitin fusions derived from cell paste generated in single 10-L fermentations. All were expressed intracellularly and remained soluble at extremely high levels of expression. Bacterial freeze--thaw lysates contained over 95% pure ubiquitin fusion protein. All four fusions were efficiently cleaved to ubiquitin and the peptide products. In one case, the final yield of peptide was 1.08 g from 3 L of low cell density bacterial culture. The combination of exceptional overexpression of the ubiquitin--peptide fusion proteins and a robust and specific protease are unique advantages contributing to a cost-effective, scaleable, and generic bioprocess for peptide production.
authors.affiliation: Proteinix Company, Gaithersburg, Maryland 20877, USA.
authors.forename: A
authors.initials: A
authors.lastname: Pilon
authors.forename: P
authors.initials: P
authors.lastname: Yost
authors.forename: T E
authors.initials: TE
authors.lastname: Chase
authors.forename: G
authors.initials: G
authors.lastname: Lohnas
authors.forename: T
authors.initials: T
authors.lastname: Burkett
authors.forename: S
authors.initials: S
authors.lastname: Roberts
authors.forename: W E
authors.initials: WE
authors.lastname: Bentley
mesh_terms: D004926:Escherichia coli / Q000235:genetics*; D005285:Fermentation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D014452:Ubiquitins / Q000096:biosynthesis*
chemical_list: D011993:Recombinant Fusion Proteins; D014452:Ubiquitins