Part:BBa_K4080006

eCPX (Enhanced Circularly Permuted Outer Membrane Protein X)

Enhanced CPX (eCPX) is a circularly permuted form of the OmpX protein that has been optimized to display unconstrained (15mer) peptides in E. coli [1]. eCPX is an outer membrane display scaffold that yields a more robust display scaffold to present diverse peptides as C- and N-termini fusions on the surface of bacteria. eCPX has a display rate that substantially improves relative to CPX and OmpX. This part can be used to improve the display of diverse peptides by reducing the impact of the passenger peptide upon the surface localization efficiency [2].

Introduction

UBrawijaya iGEM 2021 project CHOP system is an outer membrane vesicle based protein overexpression system that includes three goals towards producing a blueprint for an ideal protein production system. This project can be customized because the system can be adapted with various proteins of interest, the overexpression system that can increase protein yield, and the clean harvest with simplified purification steps that is equipped with a protease enzyme expression mechanism in the concentrated system - from vesicles and cell pellets.

This part is used to improve the display rate of the targeted protein (in this experiment we used chromoprotein, amilCP). eCPX is also known to improve the robustness of bacterial surfaces via CPX. The biterminal display scaffold eCPX will be localized targeted protein on the outer membrane of the E.coli host to simpler the downstream process of overexpressed protein [3].

Usage and Biology

eCPx is a bacterial cell-surface display scaffold and yields a circularly permuted transmembrane protein that has both termini present on the exterior of the cell and inserts into the outer membrane. The evolution of eCPX provides several important new capabilities for peptide engineering. This novel protein scaffold substantially improves the robustness of bacterial surface, increased the display rate of various unrelated peptide fusions, on either the N- or C- terminus when compared to that achieved using the parental CPX, and improves library screening efficiency by promoting the recovery of peptides that are otherwise difficult to display [2].

References

[1] Adams, B.L., Hurley, M.M., Jahnke, J.P. et al, “Functional and Selective Bacterial Interfaces Using Cross-Scaffold Gold Binding Peptides,” JOM, no.67, Pp. 2483–2493, Nov.2015.

[2] Kenrick, Sophia A., and Patrick S. Daugherty, "Bacterial display enables efficient and quantitative peptide affinity maturation," Protein Engineering, Design & Selection 23, no. 1.Pp. 9-17. Jan.2010.

[3] Rice, Jeffrey J., and Patrick S. Daugherty, "Directed evolution of a biterminal bacterial display scaffold enhances the display of diverse peptides," Protein Engineering, Design & Selection 21, no. 7. Pp. 435-442, Jul.2008.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]