Designed by: Dingjian Zhang, Tianle Xue   Group: iGEM23_CPU-CHINA   (2023-09-19)

Dual-Edged Harpoon

rational design of deh

Usage in short

You can use it to target and kill both Fn and CRC!

What is it?

Here is the structure of Dual-Edged Harpoon.The name,"Harpoon",is the nickname we call it.Of course, you can call it more formally as "Dual-targeted Cytotoxic peptide".

mFada B-domain

Auto cleaving linker A

LL-37 Truncated Peptide

Auto cleaving linker B

HlpA monomer


With the advancement of molecular biology, numerous active proteins and peptides have been invented or discovered for therapeutic applications. Recombinant technology, renowned for its high expression levels and straightforward operations, has been widely employed in the biopharmaceutical sector. To achieve multi-target, multi-functional active proteins, it's imperative to link and fuse two or more proteins with known functions. This method of obtaining bifunctional or multifunctional fusion proteins has emerged as one of the novel approaches for new drug development and bioproduct research. Particularly, it has been extensively utilized in the preparation of bispecific single-chain antibodies (scFv) or antibody-drug conjugates [1-5].

Many bacteria have long chain fiber-protein complexes on their surfaces, which are called pili or fimbriae. These pili are composed of individual pilus monomers that link together end-to-end in the extracellular environment, self-assembling into long chain fibers with high physical strength.

For Fusobacterium nucleatum, its pili are referred to as FadA (Fusobacterium adhesin A). The monomers that make up these pili come in two forms: ①pre-FadA, which serves as an anchoring structure, attaching the entire pilus to the bacterial inner membrane. ②mFadA (mature FadA), which can link head-to-tail and self-assemble into a long filament.[6-7]

In our project, the aim is to achieve bacteria-bacteria targeting. To accomplish this, we intend to leverage the self-assembly property of mFadA. We have fused a bacterial pilus monomer onto a membrane protein of the engineered bacterium, which we call the "fishing rod protein" . The membrane protein acts as the fishing rod, the linker serves as the fishing line, and the bacterial pilus monomer functions as the bait. By utilizing surface display techniques to display the fishing rod protein, our engineered bacteria can essentially "fish" for target bacteria, enabling precise bacteria-bacteria targeting.

Using surface display technology, pili monomers were expressed on the surface of Bifidobacterium longum. However, we quickly realized that the direct display of pili monomers was unnecessary and would lead to a range of issues, including steric hindrance, nonspecificity, and metabolic waste. Intriguingly, the self-assembly of the pili is driven by a pivotal role of a 26 amino acid-long α-helix at the N-terminus. Therefore, we contemplated using truncated pili monomers, eliminating all structures unrelated to self-assembly, and only displaying the critical 26 amino acid-long motif. We termed this structure mFadA B-domain and designed it as a Basic Part (BBa K4990002). Consequently, the Dual-targeted cytotoxic peptide utilized mFadA B-domain to target Fn.

In 2006, Tjalsma, employing the Highly accurate tandem MS method, identified a protein named Histone-like protein A (HlpA). This discovery forged a connection between Streptococcus bovis and colorectal cancer. It was posited that S. bovis binds to the colorectal cancer cell surface's heparan sulfate-proteoglycans (HSPG) through HlpA, mediating the colonization of S. bovis in colorectal cancer [8].

By 2009, Boleij substantiated that Streptococcus gallolyticus binds to the colorectal cancer cell surface through interactions between HlpA and HSPG [9].

In 2016, O'Neil elucidated the crystal structure of Hlp, revealing its crab-claw-like configuration. The claw section, rich in basic amino acids, can engage with DNA and also binds to heparin [10].

In 2018, Chun Loong Ho leveraged the binding affinity between HlpA and HSPG to engineer an Escherichia coli strain that specifically targets colorectal cancer, marking the inception of the HlpA targeting system [11].

By 2022, the iGEM22_LZU-CHINA team, harnessing synthetic biology, devised an Escherichia coli strain that targets colorectal cancer, employing the HlpA-mediated targeting of tumor cell surface HSPG [12].

In 2023, Tang designed a probiotic strain that uses HlpA for targeting and Azurin for cytotoxicity. This exhibited promising therapeutic outcomes in mouse models of colorectal cancer [13].

Figure A:Crystal structure of HlpA(O'Neil 2016) Figure B:Engineered EcN treat CRC(Chun 2018)

HlpA, as its name "Histone-like protein A" indicates, can bind to DNA like histones. Its overall shape resembles a crab claw, which allows it to non-specifically bind tightly to the minor groove of DNA using its ribbon-like β-fold region, leading to significant DNA bending, DNA compaction, and negative supercoiling. S. bovis, through an enigmatic mechanism, secretes it extracellularly, positioning it as an anchorless protein and a target for the humoral immune system during infections. By bridging bacterial lipoteichoic acid (LTA) and HSPG on colonic epithelial cells, it facilitates bacterial adherence to colon tumor cells.

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

Antimicrobial peptides are widely sourced, first discovered in the immune systems of insects. Subsequently, similar peptides have been extracted from bacteria, fungi, amphibians, higher plants, and mammals. These active peptides possess natural immune functions and serve as essential molecular barriers for host defense against invading pathogens, hence referred to as antimicrobial peptides.[14-15]

The tissue proteinase inhibitor family is unique to mammals and constitutes a type of antimicrobial peptide.LL37 is derived from one of the tissue proteinase inhibitors, human cationic antimicrobial protein 18 (hCAP18). Among them, LL-37 is the sole antimicrobial peptide found in the human body. It possesses various functions such as antimicrobial, antitoxin, immunomodulatory, and wound healing effects. Additionally, LL-37 holds potential for treating viral diseases and neoplastic conditions. Numerous research teams have investigated the activity of truncated peptides to identify the minimal antimicrobial region of LL-37. The results indicate that the smallest region of LL-37 is within amino acid positions 17 to 29 or 18 to 29. [16-17] This suggests that the C-terminal helix, consisting of 12 or 13 amino acids of LL-37, plays a role in antimicrobial, antiviral, and antitumor activities.

We name the LL-37 truncated peptide as "FK-13", since the sequence starts with "FK" amino acides. And you can also call it as "Fusobacterium Killer - 13".

What can it do?

However, not the entire structure of mFadA is involved in self-assembly. Thus, we considered removing unnecessary domains. Upon closer examination of mFadA's structure, we divided it into two domains: Domain A and Domain B. Domain A comprises two anti-parallel α-helical structures, while Domain B consists of a single anti-parallel α-helix. We believe that Domain B is the most crucial. On a microscale, it possesses the function of binding with Domain A, and on a macroscale, it exhibits the capability to target Fn (Fusobacterium nucleatum).

Therefore, by displaying the engineered bacteria with the mFadA B-domain on their surface, specific adhesion to Fn can be achieved, enabling bacteria-to-bacteria targeting.

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.[12]

Monomeric HlpA can be used in the design of fusion proteins due to its capability to bind with the HSPG present on the surface of colorectal cancer cells. Thus, if you aim to target colorectal cancer cells using proteins, you can engineer a fusion protein with HlpA tethered at one end. This bestows the fusion protein, termed as Dual-Edged Harpoon, with the ability to specifically target colorectal cancer cells [11-13].

It's worth mentioning that these HlpA monomers can spontaneously self-assemble into homodimers, exhibiting a substantial affinity between them. Consequently, in our experiments associated with the dual-targeted peptide, we observed a pronounced dimerization phenomenon.

It can be seen that if using the complete B-domain (①), there is an inability to form a complete α-helix. Therefore, the optimized DEH had the C-terminal five amino acids removed from the B-domain to ensure the stable formation of the α-helix structure at the N-terminus of DEH. As shown in ④, the added GPNG sequence once again played a role, targeting the Fn segment downward and the CRC targeting functional domain upward, effectively achieving "function domain isolation." Therefore, this optimized DEH was selected as our final targeting cytotoxic peptide.

Molecular dynamics analysis results of B-domain.| a. RMSD of B-domain protein complexes; b. Combined Rg and RMSD analysis of B-domain protein complexes. c. RMSF of the B-domain protein complex. d. Analysis of protein slewing in the space of B-domain protein complexes. e. Covariance matrix of B-domain protein complexes. f. Ramachandran diagram of the B-domain protein complex

Wetlab Characterization

1.Anticancer Activity of DEH

After completing the characterization of the anticancer activity of TTP, we considered the need for simultaneous killing of CRC cells and F.n. At that time, we constructed DEH on the basis of TTP as a dual-targeting peptide with a target head for F.n (FadA) and a target head for CRC cells (HlpA). We also further improved the linker linking the target head to FK-13 to minimize the effect of the involved part of the linker on the activity of FK-13 after breakage to ensure the killing efficiency of our FK-13. To verify the killing of sw480 by DEH, we performed an exhaustive characterization of the anticancer activity of TTP. The by Fig. 8e~f, the inhibitory effect of DEH on sw480 was significantly dose-dependent and time-dependent. And as can be seen from Figure 8a~b, the inhibition rate after 2 hours of administration showed a curve similar to S shape. And the DEH concentration-inhibition rate of the group with further prolongation of the administration time was more stable approximating the S-shaped curve. This is a promising result. As shown in Figure8c~d, we plotted the inhibition rate-concentration curves logarithmically and performed a linear fit for the 2-hour administration group with a better linear relationship between the logarithmic values of inhibition rate-concentration, and calculated the IC50 of DEH = 174.43 μg/mL. the IC50 of DEH was slightly higher than that of TTP, which was in line with our expectation. According to the flow cytometric analysis of the single administration of Fig. 8g and the two administration groups of Fig. 8h, it is known that DEH can cause severe apoptosis.

Figure7.FK-13 with linkerA and linkerB

According to Figs. 8i~j, it can be seen that the anticancer activity of DEH was mainly realized by inducing apoptosis. And there was a significant correlation between the number of late apoptotic cells and the number of administration. As FK-13 binding linker breakage will leave positively charged amino acid residues at the C-terminal and N-terminal of FK-13, which will further enhance the anticancer activity of FK-13. Since DEH can cause sw480 necrosis, however, the percentage of necrotic cells is slightly lower because they tend to be suspended in the culture medium and are not easy to collect. However, overall, DEH can induce apoptosis well.

In addition we also detected the apoptosis of adherent cells using TUNEL staining, and those that were stained green were apoptotic cells (Figure 8k).

The above experiments proved that our new dual-targeting peptide, DEH, has good anticancer activity, which can effectively inhibit the activity of sw480 and also induce apoptosis and cell necrosis.

Figure 8. Detection of anticancer activity of DEH and it induces cell death mode | a.Absorbance at 450 nm versus time of administration and concentration of administration. b. Inhibition ratio versus time of administration and concentration of administration. c. Inhibition ratio versus concentration of administration logarithmically. d. Inhibition ratio versus concentration of administration logarithmically of 2 hours after DEH administration and its fitted straight line. e. Heat map of absorbance at 450 nm versus time of administration and concentration of administration. f. Heat map of Inhibition ratio versus time of administration and concentration of administration. g. Flow cytometric analysis of the single-dose administration group. h. Flow cytometric analysis of the two-dose administration group. i. Comparison of the percentage of necrotic, late apoptotic, early apoptotic, and normal cells in the flow cytometric analysis results of the single-dose administration group. j. Comparison of the percentage of necrotic, late apoptotic, early apoptotic, and normal cells in the flow cytometric analysis results of the two-dose administration group. k. TUNEL staining detected apoptosis in adherent cells. (The data were analyzed using student’s T-test; *: P<0.05; **: P<0.01; ***: P<0.001).

We also counted the percentage of cells with early apoptosis, late apoptosis, and necrosis in the fine death induced by LL-37 versus TTP and DEH (e.g., Fig. 9a).

To better characterize our modified peptides, we calculated the IC50 of LL-37, FK-13, TTP, DEH, and two positive drugs paclitaxel and 5-FU against sw480, respectively (as shown in Figure 9b).

According to the above experiments, our rationally modified FK-13 and innovatively designed TTP and DEH can achieve the function of killing cancer cells by inducing apoptosis and cell necrosis.

Figure 9. Detection of anticancer activity of DEH and it induces cell death mode | a. Percentage of cells with early apoptosis, late apoptosis, and necrosis in fine death induced by LL-37 vs. TTP and DEH. b. the IC50 of LL-37, FK-13, TTP, DEH, and paclitaxel and 5-FU against sw480, respectively. A&B. An overview of the two cell death mode.

Here are the results of all our flow cytometry assays.

2.Antimicrobial Activities preliminary analysis

Our main methods for verifying antimicrobial activity were the Gel-red staining method and inhibition zone method(Oxford cup). Since the laboratory is not equipped with an anaerobic incubator, most of the inhibition zone experiments failed. However, we have completed the qualitative verification of the antimicrobial activities of FK13,BTP and DEH using the Gel-red staining method. So we can come to an conclusion that the designed antimicrobial peptides have the ability to kill Fn.

Figure 10. Antimicrobial activities of designed antimicrobial peptieds | a. Gel-red staining of Fn after antibiotics or cytotoxic peptides’ treatment for 10h. Str: streptomycin, CM: chloramphenicol, AMP: ampicillin. The concentrations of antibiotics or designed antimicrobial peptieds from tube 1-8 were 0.1mg/mL ,0.5mg/mL, 2.5mg/mL, 12.5mg/mL, 62.5mg/mL, 150mg/mL, 300mg/mL, 500mg/mL.

Dimerazation and Cleavage Test

SDS-PAGE of total protein. A. The total protein SDS-PAGE results. B. The protein fragment that may form due to the linkers’ cleavage and the dimerization of HlpA.

During the experiment, it was discovered that proteins containing HlpA such as DEH, TTR, and TTP consistently display characteristic bands across specific molecular weight regions. This cannot be attributed to chance occurrences. After thorough investigation and experimental simulations, it is believed that this is caused by the linkers’ being cleaved and the dimerization ability of HlpA.

Validation of Cancer-targeting Ability

Interaction of DEH and heparin.

A. DEH monomers are typically observed at 17kDa (yellow squares) while dimers are observed at 35kDa (red squares). Breakage of the linker and dimerization of HlpA may form a DEH-HlpA complex with a molecular weight of approximately 28kDa (blue squares).

B. The structure of DEH monomer (~15kDa, or ~17kDa with purification tag)

C. The structure of DEH dimer (~30kDa, or ~35kDa with purification tag)

D. The dimerization of HlpA and the interaction between the dimer and heparin.

The molecular weight of lanes 5~8 is relatively larger compared to lanes 2-4, suggesting that DEH can interact with heparin. This demonstrates DEH's capacity to attach to the sugar moieties of HSPG glycoproteins that are extensively expressed on the tumor cell surface, thereby permitting effective targeting of tumor cells.

PD of DEH and TTR

The corresponding molecular weights of DEH (~15kDa,red square on the left) and TTR (~55Da, red squares on the right) show clear bands.The cleavage of the leanker leads to the bands of HlpA with resicual linkers(~12kDa, blue square)

To further confirm the cancer targeting capacity, we utilized magnetic beads containing heparin to attract DEH and TTR in association with HlpA, and subsequently conducted SDS-PAGE after elution, proving that heparin on the surface of heparin magnetic beads can interact with and enrich for HlpA, indicating that proteins with HlpA can interact with heparin. This means that proteins with HlpA have the ability to bind to the sugar chains of HSPG glycoproteins, which are highly expressed on the tumour surface, and can target tumour cells.


[1] Tao, L., Gao, M., & Zhou, H. (2015). Research progress on novel antibody-chemotherapy drug conjugates. Journal of Pharmaceutical Biotechnology, 22(3), 253-258.

[2] Gustavsson M,Lehtio J,Denman S,et al. Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in pichia pastoris[J]. Protein Eng,2001,14(9):711-715.

[3] Wang SH,Zheng CJ,Liu Y,et al. Construction of multiform scFv antibodies using linker peptide[J]. J Genetics and Genomics,2008,35:313-316.

[4] Zhang JH,Yun J,Shang ZG,et al. Design and optimization of a linker for fusion protein construction[J]. Progr Natur Sci,2009,19: 1197-1200.

[5] Shan D,Press OW,Tsu TT,et al. Characterization of scFv-Ig constructs generated from the Anti-CD20 mAb 1F5 using linker peptides of varying lengths[J].J Immunology,2014,162:6589-6595.

[6]Nithianantham, S., Xu, M., Yamada, M., Ikegami, A., Shoham, M., & Han, Y. W. (2009). Crystal structure of FadA adhesin from Fusobacterium nucleatum reveals a novel oligomerization motif, the leucine chain. Journal of Biological Chemistry, 284(6), 3865-3872.

[7]Témoin, S., Wu, K. L., Wu, V., Shoham, M., & Han, Y. W. (2012). Signal peptide of FadA adhesin from Fusobacterium nucleatum plays a novel structural role by modulating the filament’s length and width. FEBS letters, 586(1), 1-6.

[8]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.

[9]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.

[10]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.

[11]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.


[13]Tang, H., Zhou, T., Jin, W., Zong, S., Mamtimin, T., Salama, E. S., ... & Li, X. (2023). Tumor-targeting engineered probiotic Escherichia coli Nissle 1917 inhibits colorectal tumorigenesis and modulates gut microbiota homeostasis in mice. Life Sciences, 324, 121709.

[14]de Breij, A., Riool, M., Cordfunke, R. A., Malanovic, N., de Boer, L., Koning, R. I., ... & Nibbering, P. H. (2018). The antimicrobial peptide SAAP-148 combats drug-resistant bacteria and biofilms. Science translational medicine, 10(423), eaan4044.

[15]Zhang, M., Liang, W., Gong, W., Yoshimura, T., Chen, K., & Wang, J. M. (2019). The critical role of the antimicrobial peptide LL-37/CRAMP in protection of colon microbiota balance, mucosal homeostasis, anti-inflammatory responses, and resistance to carcinogenesis. Critical Reviews™ in Immunology, 39(2).

[16]Fabisiak, A., Murawska, N., & Fichna, J. (2016). LL-37: Cathelicidin-related antimicrobial peptide with pleiotropic activity. Pharmacological Reports, 68(4), 802-808.

[17]Chieosilapatham, P., Ikeda, S., Ogawa, H., & Niyonsaba, F. (2018). Tissue-specific regulation of innate immune responses by human cathelicidin LL-37. Current Pharmaceutical Design, 24(10), 1079-1091. Sequence and Features

Assembly Compatibility:
  • 10
    Illegal PstI site found at 31
  • 12
    Illegal PstI site found at 31
  • 21
  • 23
    Illegal PstI site found at 31
  • 25
    Illegal PstI site found at 31
    Illegal NgoMIV site found at 378
  • 1000