Part:BBa_K4275032

PfuFerritin-Nb3

PfuFerritin--Nb3 is combination of a ferritin protein and the nanobodies displayed on the surface. Ferritin is a spherical iron storage protein composed of a self-assembled 24- subunit protein shell and it is commonly used in blood tests for iron levels in humans. Due to its large numbers of salt bridges and hydrogen bonds, ferritin is extremely heat-resistant and exhibits excellent biocompatibility in vivo. Due to its ability to self-assemble and because of its unique architecture, ferritin has been used to display peptides as well as larger proteins[1].

Usage and Biology

PfuFerritin-Nb3 can be used to increase binding affinity, thermal resistance and extending blood half-life in vivo of nanobodies. And because PfuFerritin has high heat stability it tends to be suitable for scale up experiments and translational studies[1]. Moreover, its long half-life also means that there are less modifications required to the protein which may affect the functional properties of the protein.

Characterization

Magnetic recycling

A novel design feature of our engineered E.coli was the inclusion of intracellular ferritin expression. The Fe2+ ions in ferritin allows the E.coli cells displaying cellulosome complex to be attracted by strong magnets, therefore enabling the magnetic recycling of cellulosomes to be reused.

We’ve constructed three ferritin plasmids: the ferritin wild type (Pfuferritin), the existing part (BBa_K1189065), and the wild type ferritin fused with Nb3 (Pfuferritin-Nb3) (Fig.1A). All three vectors were transformed and cultured for IPTG-inducible expression. The target proteins of all three ferritins were detected in the whole cell and supernatant samples of SDS-page (Fig.1B).

To further prove that the host E.coli cells carrying ferritin is magnetically attractive, we transformed prha-mRFP1 plasmids into competent cells containing Pfuferritin and BBa_K1189065 respectively. The co-expression of ferritin and RFP allowed magnetic recycling results to be better visualized. In both ferritin wild type and ferritin part, aggregation of red fluorescence were found near the strong magnets, proving the feasibility of magnetic recycling system (Fig.1C).

Figure 1: Fig.1 Ferritin expression and magnetic recycling. (A) Genetic circuit construction for three types of ferritin: ferritin wild type (PfuFerritin), IGEM existing ferritin part (BBa_K1189025), and ferritin-Nb3 (PfuFerritin-Nb3) adapted for surface display system. (B) SDS-page analysis of PfuFerritin-Nb3, BBa_K1189025, Ferritin wild type (WT) respectively. (C) Magnetic recycling was conducted with Ferritin control group, RFP control group, BBa_K1189025-RFP, and Ferritin WT-RFP. Apparent aggregation of RFP fluorescence shown in cells co-expressing ferritin and RFP verified the ability for ferritin to be attracted by strong magnets.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 547
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Fan, Kelong et al. “Fenobody: A Ferritin-Displayed Nanobody with High Apparent Affinity and Half-Life Extension.” Analytical chemistry vol. 90,9 (2018): 5671-5677. doi:10.1021/acs.analchem.7b05217