Designed by: Dingjian Zhang   Group: iGEM23_CPU-CHINA   (2023-09-13)


Usage in short

You can use it to target coloretal cancer!

Sequence and Features

Assembly Compatibility:
  • 10
  • 12
  • 21
  • 23
  • 25
    Illegal NgoMIV site found at 231
  • 1000

What you need to know!

In 2006, Tjalsma employed the Highly accurate tandem MS method to identify a protein named Histone-like protein A (HlpA). This discovery highlighted a bridge between Streptococcus bovis and colorectal cancer. It is postulated that S. bovis establishes a bond with colorectal cancer cell surfaces via heparan sulfate-proteoglycans (HSPG) mediated by HlpA [1].

By 2009, Boleij confirmed that Streptococcus gallolyticus binds to the surface of colorectal cancer cells through HlpA interacting with HSPG [2].

In 2016, O'Neil unveiled the crystal structure of Hlp, revealing its crab-claw-like architecture. The claw section's basic amino acids can bind with DNA and simultaneously with heparin [3].

In 2018, Chun Loong Ho harnessed the binding of HlpA to HSPG to construct an engineered Escherichia coli that specifically targets colorectal cancer, thus marking the commencement of the HlpA targeting system [4].

In 2022, the iGEM22_LZU-CHINA team, utilizing synthetic biology, developed an Escherichia coli that targets colorectal cancer, employing the method of HlpA targeting tumor cell surface HSPG [5].

By 2023, Tang engineered a probiotic using HlpA targeting and Azurin-induced cytotoxicity, demonstrating promising therapeutic efficacy in colorectal cancer mice models [6].

Figure A:Crystal structure of HlpA(O'Neil 2016) Figure B:Engineered EcN treat CRC(Chun 2018)
HlpA, as suggested by its name "Histone-like protein A", binds to DNA similar to histones. Its overall morphology resembles a crab claw, allowing it to non-specifically and tightly bind to the minor groove of DNA using its ribbon-like β-fold region. This results in significant DNA bending, DNA compaction, and negative supercoiling. S. bovis, through an unknown mechanism, secretes it extracellularly, acting as an anchorless protein. It becomes a target for the humoral immune system during infections. By linking bacterial lipoteichoic acid (LTA) and HSPG on colon epithelial cells, it mediates bacterial adhesion to colon tumor cells.

Therefore, HlpA possesses dual capabilities: 1. Targeting colorectal cancer and 2. Non-specifically binding to DNA chains.

What is it?

Here is the structure of HlpA monomer and homodimer.

Two helical segments from each monomeric subunit constitute an α-helical ‘body’ with two protruding β-ribbon ‘arms’ , which extend to bind the DNA helix. These DNA-binding β-ribbons are largely disordered in the absence of DNA.

What can it do?

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 [4-6].

Furthermore, HlpA bears a resemblance to the HU protein of Escherichia coli. The E. coli HU heterodimer non-specifically binds to double-stranded DNA, having a higher affinity for distorted DNA structures, which induces significant DNA bending, DNA compaction, and the formation of negative supercoils. HlpA's binding to DNA is non-specific and displays a preference for AT-rich DNA [3].

Lastly, it's worth noting that these HlpA monomers can self-assemble into homodimers. Their interconnection exhibits immense affinity. Therefore, in our dual-targeting peptide-related experiments, a significant dimerization phenomenon was observed.

How does it work?

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.[3]

we tested the interaction between HlpA and HSPG at cellular level.The entire cellular lysate obtained from cells that expressed the HlpA-EGFP fusion protein was utilised. To remove adhesion and allow full interaction between the tumor cells and fusion protein, trypsin was applied to the cells. The trypsin was washed off with PBS and then co-incubation for a period of 6 hours. Following the incubation, the sample was resuspended in PBS and centrifuged thrice to eliminate weakly-bound fusion proteins from the solution. The results indicate that the HlpA-EGFP fusion protein accumulates abundantly and intensely on the surface of tumour cells, emitting a green fluorescence.

Co-incubation of HlpA-EGFP and sw480.


[1]Tjalsma H, Schöller‐Guinard M, Lasonder E, et al. Profiling the humoral immune response in colon cancer patients: diagnostic antigens from Streptococcus bovis[J]. International journal of cancer, 2006, 119(9): 2127-2135.

[2]Boleij A, Schaeps R M J, de Kleijn S, et al. Surface-exposed histone-like protein a modulates adherence of Streptococcus gallolyticus to colon adenocarcinoma cells[J]. Infection and immunity, 2009, 77(12): 5519-5527.

[3]O'Neil P, Lovell S, Mehzabeen N, et al. Crystal structure of histone-like protein from Streptococcus mutans refined to 1.9 Å resolution[J]. Acta Crystallographica Section F: Structural Biology Communications, 2016, 72(4): 257-262.

[4]Ho C L, Tan H Q, Chua K J, et al. Engineered commensal microbes for diet-mediated colorectal-cancer chemoprevention[J]. Nature biomedical engineering, 2018, 2(1): 27-37.


[6]Tang, H., Zhou, T., Jin, W., Zong, S., Mamtimin, T., Salama, E. S., ... & Li, X. (2023). Tumor-targeting engi