Part:BBa_K4803113

FCS-CD28TMD-PPVcs-KKYL

Introduction

This biobrick is created by combining Furin cleavage site (BBa_K4803012), CD28 Transmembrane domain (BBa_K4803006), PPVp cleavage site (BBa_K4803013), ER retention signal (BBa_K4803014).

Usage and Biology

This Part is a CD28 transmembrane domain(BBa_K4803006) , flanked downstream of the Furin cleavage site by an endoplasmic reticulum with an additional endoplasmic reticulum retention signal. The CD4 signal(BBa_K4803005) is transported to the ER and is retained in the ER as long as ER retention signal(BBa_K4803014) is attached[1].

This Part is designed with cleavage site for PPV Protease between the transmembrane domain and the ER retention signal; in the presence of PPV Protease, the ER retention signal is cleaved and sent to the trans-Golgi reticulum. The Furin cleavage site is cleaved by Furin in the trans-Golgi network and protein of interest (i.e. eGFP) flanked to this part is secreted.

Figure 1: Overview of Secretion system

In UTokyo 2023 Project, protease released from MESA and protease amplified by Amplification unit cleave ER trafficking signal.

Figure 2: Overview of UTokyo2023 project "SWIFT" system

This Part can promote secretion of the target protein in the presence of PPV protease by adding the target protein upstream of FCS.

Characterization

Wet Experiment

iU38 consists of CD4signal-eGFP-FCS-CD28TMD-PPVcs-KKYL, and the endoplasmic reticulum retention signal is cleaved in the presence of PPV protease.

iU39 used as the control encodes CD4signal-eGFP-FCS-CD28TMD-PPVcs-AAYL and is not retained in the endoplasmic reticulum.

Figure 3: Completed plasmid of iU-38

Figure 4: Completed plasmid of iU-39

Fluorescence of eGFP in HEK293A transfected with iU38 or iU39 was observed to confirm whether the protein attached to BBa_K4803113 is withheld in the endoplasmic reticulum.

Figure 5: Photograph taken at 40x after about 72 h in iU-38

Figure 6: Photograph taken at 40x after about 72 h in iU-39

These pictures indicate the eGFP in iU-38 is localized to one part of the cell line. Its shape suggests that it is an endoplasmic reticulum.

Modeling

This modeling compares transcriptional regulation, in which tTA is released from MESA, with secretory regulation, in which protease is released.

Figure 7: Flowchart_of_secretion_system

To verify that the model describes the reaction accurately, we compared the experimental results of previous KKYL studies with the simulation results of the models. In previous studies, SEAP was attached to KKYL, which was designed to be cut off by abscisic acid (ABA)-inducible TEV protease. For comparison with transcriptional control, SEAP secretory experiments with ABA-induced transcriptory activity were also conducted, in which SEAP activity in the medium was measured 30 min, 45 min, 60 min, 90 min, 120 min, 180 min, 240 min, and 360 min after ABA addition, and data were collected four times for transcriptional control and KKYL respectively. The data were normalized to the maximum secretory control at 360 min for transcriptional control, and we used that data[2].

We simulated with [H] = [Ta] = 3e-7 and S1 initial concentration [S1] = 5e-5 and calculated the values of [Si] after 30 min, 45 min, 60 min, 90 min, 120 min, 180 min, 240 min, and 360 min for each model of transcriptional control and secretory control. The [Si] values for transcriptional control after 360 min were set to 1 and the other data were normalized. We calculated the average of the four experimental data from each of the experiments in the previous studies, and calculated the correlation coefficient between our model values and the calculated average values for transcriptional control and secretory control, respectively. The results are shown below.

Figure 8: Comparison of experimental data and simulated data.

Transcriptional control

Correlation coefficient: 0.95

Figure 9: Comparison of experimental data and simulated data.

Secretory control

Correlation coefficient: 0.90

From these results, we conclude that the model we built is sufficient to describe the secretory module of SWIFT.

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]

References

[1] Mansouri, M., Ray, P. G., Franko, N., Xue, S., & Fussenegger, M. (2023). Design of Programmable Post-Translational Switch Control Platform for on-Demand Protein Secretion in Mammalian Cells. Nucleic Acids Research, 51(1), e1. https://doi.org/https://doi.org/10.1093/nar/gkac916

[2]Praznik, A., Fink, T., Franko, N. et al. Regulation of protein secretion through chemical regulation of endoplasmic reticulum retention signal cleavage. Nat Commun 13, 1323 (2022). https://doi.org/10.1038/s41467-022-28971-9