Designed by: Dingjian Zhang   Group: iGEM23_CPU-CHINA   (2023-10-11)

Tumor-Targeting Rod

Usage in short

You can use it to achieve BL-CRC adhesion.

You need to know

Acurate targeting is one important characteristics for many emerging cancer therapies, that is also what ourproject’s ambition. We designed two fusion proteins, they function like fishingrod, so we gave them the name: Fishing Rod Proteins (FRPs). The one that targetFn names Bacteria Tageting Rod(BTR), the other that target CRC cells names Tumor Tageting Rod(TTR). FRPs are displayed on engineered BL membranes, helping BLtarget to the correct place. Of course, their targets are cancer cells and Fn,rather than fish. Each FRP consists of four parts (Figure1): the signal peptide(white),helping FRPs locate on cell membrane; the membrane protein(pink), the base ofFRPs; and the linker(blue), helping FRPs become flexible; and the targeting fragments(yellow), the most pivotal role in FRPs

What is it

It consists of the GL-BP membrane protein, three repeated G4S linker peptides, and HlpA. Essentially, it involves the surface display technique to express HlpA on the BL membrane for adhesion to HSPG, thereby achieving adhesion between BL and CRC.

How it works

The HlpA monomer has been used in designing fusion proteins, as it can bind to HSPG on the surface of colorectal cancer cells. Hence, if you wish to target colorectal cancer cells with a protein, you can design a fusion protein attaching HlpA to one end. If you aim to make a cell target colorectal cancer cells, you can custom design a membrane protein containing HlpA for your chassis cell. Through surface display technology, expressing HlpA on the chassis cell surface would allow it to target colorectal cancer cells [2-4].

Examination of the DNA-binding region of Hlp revealed that this area is primarily composed of positively charged residues that form a pocket to accommodate DNA binding. The specific residues that comprise the DNA-binding pocket are depicted in Figure below. It should be noted that the side-chain electron density for Arg56, Lys60, Lys71 and Lys73 of subunit A and Arg54, Lys76 and Lys81 of subunit B was partially disordered and could not be traced in the electrondensity maps. Therefore, the side chains were modeled in idealized rotamer positions for the electrostatic surface calculations.[1]

Wetlab Characterization

The interaction of TTR and heparin.

A. Due to the prolonged storage period and the impact of the pH environment subsequent to elution, TTR undergoes degradation and linker breakage, which in turn leads to the appearance of HlpA with residual linkers (~15kDa,red squres) and their dimers (~30kDa,yellow squares). Due to TTR's dimerization, bands of TTR dimers (~100kDa,blue squares) can also be observed.

B. The structure of TTR.

C. The cleavage of the linker in TTR, forming two parts.

D. The dimeration of HlpA.

In comparison with the stripes on lanes 5-7, the molecular weight and widths of the bands on lanes 2-4 are relatively higher, suggesting that TTR may bind with heparin. This provides evidence that TTR can attach onto the sugar chains of HSPG glycoproteins located on the surface of tumor cells, therefore exhibiting the ability to target them.

The linker between GLBP and HlpA may break due to factors like high pH of the eluent and mechanical stress of the constant flow pump during purification. This results in numerous unexpected bands in TTR, which include HlpA, membrane protein fragments, and detached dimers after Linker breakage.

Using whole-cell lysate with a fluorescent protein-tagged HlpA, we co-incubated it with CRC. We observed the enrichment of HlpA on the surface of CRC, resulting in strong fluorescence. Due to the protein not being purified and being present in whole-cell lysate, it caused aggregation of cells and some impurities in the bright-field image. This experiment confirms the effective binding of HlpA to CRC.

Sequence and Features

Assembly Compatibility:
  • 10
    Illegal PstI site found at 358
  • 12
    Illegal PstI site found at 358
  • 21
    Illegal BamHI site found at 771
  • 23
    Illegal PstI site found at 358
  • 25
    Illegal PstI site found at 358
    Illegal NgoMIV site found at 1050
    Illegal NgoMIV site found at 1590
  • 1000