Part:BBa_K4960021

Engineered Mitochondrial Uncoupler Pdp1NTD-EGFP-UCP1

Usage and Biology

Noting the increasing demand for weight-loss drugs and the potential value and prospect of UCP1 (uncoupling protein 1) as a target for the treatment of obesity, [1] we conducted a series of design and experimental work on a modified UCP1 delivery strategy based on PVCs. Prior to this, we designed the payload in PVCs, a protein in which Pdp1NTD plays a key role in delivering protein loading into PVC, and we overexpressed UCP1 in the HEK293 cell line and added EGFP (Enhanced Green Fluorescent Protein) to see if it could target and work on the inner mitochondrial membrane.[1].
we conducted a series of design and experimental work on a modified UCP1 delivery strategy based on PVCs. Prior to this, we designed the payload in PVCs, a protein in which Pdp1NTD plays a key role in delivering protein loading into PVC, and we overexpressed UCP1 in the HEK293 cell line and added EGFP (Enhanced Green Fluorescent Protein) to see if it could target and work on the inner mitochondrial membrane.

Special Design

In order to prevent UCP1 from forming inclusion bodies after expression in Escherichia coli and facilitate the localization of UCP1 after transfection, we introduced EGFP protein for fusion expression with UCP1. We designed a flexible peptide linker (GGSGG) to link Pdp1NTD, UCP1 and EGFP to form a fusion protein. However, based on the text result of 砖号, we switched the order of EGFP and UCP1 to ensure that the n-terminal sequence of UCP1 and the Pdp1NTD will no longer interact. (Figure 1)
Figure 1. Updated schematic diagram of design ideas. In the process of designing part, we switched the original sequence of EGFP and UCP1, and carried out the same experimental treatment as a new group of experimental groups, hoping to solve the problems encountered before.

Sequence and Feature

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1258
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 235
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Functional test

This part is testing through cell experiments, that is, the SEP-UCP1-EGFP fusion protein was overexpressed on HEK293T cell lines using pcDNA3.1 as the carrier (pNC087). The purpose is to verify that the fusion protein can enter the inner membrane of mammalian mitochondria and play a normal uncoupling role to interfere with the energy metabolism of cells.

Method

To assess the function of the fusion protein, we transfected HEK-293T cells with pNC087, which encodes a PCMV promoter-driven Pdp1NTD-UCP1-EGFP protein expression cassette. Cells were imaged at 48 h post transcription to identify the cellular localization of the fusion protein. Cellular glucose consumption was then evaluated by measuring the remaining glucose levels in the cell culture medium. Results showed that instead of localizing in the mitochondria, the Pdp1NTD-UCP1-EGFP protein was localized all over the cytoplasm and nucleus (Fig. 2a). Consistent with the failed mitochondrial localization, glucose levels in the pNC087-transfected cells showed no significant difference compared to the control group transfected with pcDNA3.1(+) vector only (Fig. 2b). Figure 2a. Localization of Overexpressed Pdp1NTD-UCP1-EGFP fusion protein Observed by Wide Field Microscopy. Figure 2b. Bar Chart Illustrating Glucose Consumption in pNC087 Transfected HEK-293T Cells.

Functional text

This part is testing through cell experiments, that is, the Pdp1NTD-EGFP-UCP1 fusion protein was overexpressed on HEK293T cell lines using pcDNA3.1 as the carrier. Similar to the 砖号, we transfected HEK-293T cells with pNCXXX, a Pdp1NTD-EGFP-UCP1 expressing plasmid, and evaluated the cellular localization and function of the fusion protein at 48 h post transfection. As expected, both wide-field fluorescent imaging (Fig. 2a) and live-cell confocal imaging (Fig. 2b) showed a highly specific colocalization of Pdp1NTD-EGFP-UCP1 signal with mitochondria (labeled by MTS-mcherry). Moreover, cells transfected with pNCXXX showed a significantly higher glucose consumption compared to the control cells transfected with pcDNA3.1(+) vector (Fig. 2c), suggesting a significantly improved energy consumption in these cells.

Figure 2a. Localization of Overexpressed pNCxxx Observed by Wide-Field Microscopy. Figure 2b. Figure 2c. Bar Chart Illustrating Glucose Consumption in pNCXXX Transfected HEK-293T Cells.

References

[1] Kolonin MG, Saha PK, Chan L, Pasqualini R, Arap W. Reversal of obesity by targeted ablation of adipose tissue. Nat Med. 2004 Jun;10(6):625-32.