Secretion of Folded Recombinant Proteins for Enhancing the Production of Complex Proteins in Bacteria

Life Sciences : Materials and Compounds

Available for licensing

Inventors

  • George Georgiou, Ph.D. , Chemical Engineering
  • Lluis Masip, B.S. , Chemical Engineering
  • Laura Segatori, B.S. , Chemical Engineering
  • Matthew DeLisa, Ph.D. , Chemical Engineering

Background/unmet need

Bacteria are a common source of recombinant proteins for a variety of research, therapeutic, and vaccine applications. Recombinant proteins accumulating intracellularly often form insoluble inclusion bodies. Extraction of properly folded recombinant proteins from inclusion bodies is a complicated, cumbersome, and expensive process. The formation of unwanted disulfide couplings in refolded proteins greatly complicates the process of necessary disulfide bond formation in recombinant products. Therefore, researchers have sought alternative means of protein expression.

Secretion of protein from bacteria offers an attractive alternative. However, the bacterial periplasmic space is devoid of chaperone activities. Many proteins do not fold well within the bacterial periplasmic space in the absence of these activities and form inclusion bodies. As a result, the diversity of protein expression libraries is limited by the restrictions imposed by secretory or cytoplasmic expression. Bypassing these limitations will likely expand the diversity of polypeptide sequences in combinatorial libraries and thus, the ability to isolate novel binding proteins and enzymes.

Invention Description

The invention provides methods for using the Twin Arginine Translocation (TAT) pathway in bacteria to produce heterologous polypeptides that have multiple disulfide bonds. Further, a dramatic increase in the production of useful complex proteins can be obtained in cells that have been engineered to co-produce high levels of disulfide isomorases. The dual application of the TAT pathway and expression of disulfide isomerase greatly broadens the array of proteins that can be produced in a properly folded and functional form by bacterial systems, opening vast new production and discovery opportunities for researchers.

Benefits/Advantages

    New opportunities for bacterial expression of complex proteins

Features

  • New methods of screening polypeptide libraries
  • Improved production of recombinant proteins

IP Status

  • 1 foreign patent application filed
  • 9 foreign patents issued