Ubiquitin fusion technology: bioprocessing of peptides

Biotechnol Prog. 1997 Jul-Aug;13(4):374-9. doi: 10.1021/bp970040f.

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Escherichia coli / genetics*
  • Fermentation
  • Recombinant Fusion Proteins / biosynthesis*
  • Ubiquitins / biosynthesis*

Substances

  • Recombinant Fusion Proteins
  • Ubiquitins