Results

1 Peptide Sequence Prediction

1.1. Protein De Novo Design

We use a series of protein de novo design tools driven by artificial intelligence, semi-rationalally design the mutation library, who’s prototype bases on MSH-α.

Fig.1 Result through RFdiffusion

We use a series of protein de novo design tools driven by artificial intelligence, semi-rationalally design the mutation library, who’s prototype bases on MSH-α.

Fig.2 Result through ProteinMPNN

We use a series of protein de novo design tools driven by artificial intelligence, semi-rationalally design the mutation library, who’s prototype bases on MSH-α.

Fig.3 Result through Autodock Vina

1.2. Multiple Sequence Alignment

We integrated all sequences into a global multiple sequence alignment. During this process, gaps were permitted to maximize similarity among sequences, while gap opening penalties and gap extension penalties were imposed to maintain biological plausibility.

Fig.2 Peptide Sequence Prediction Results

2 Peptide Function Validation

2.1 Cell Compatibility Test

To verify that the designed peptide sequences do not pose potential harm to tissue cells, we selected the top 8 purified peptide sequences based on their binding energy rankings. Different concentrations of peptide solutions were added to the culture medium of mouse fibroblasts (L-929) and co-cultured with the cells. The cell viability of the different groups was subsequently tested using the CCK-8 detection method. The CCK-8 assay is simple yet accurate for analyzing cell proliferation and cytotoxicity.

The CCK-8 reagent contains the water-soluble tetrazolium salt WST-8 [Chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt], which is reduced by dehydrogenases in cells under the action of the electron carrier 1-methoxy-5-methylphenazinium methyl sulfate (1-Methoxy PMS) to produce a highly water-soluble yellow formazan product. The amount of formazan produced is directly proportional to the number of living cells. This characteristic allows direct analysis of cell proliferation and toxicity.

Fig.1 Cell Viability Under Co-Cultivation with Different Peptide Sequences

2.2. Melanin Inhibition Function Validation

To verify whether the designed peptide sequences can inhibit melanin production, we selected tyrosinase-related protein 1 (TRP1), which possesses 5,6-dihydroxyindole-2-carboxylic acid oxidase activity and plays an important role in the downstream pathways of melanin synthesis. TRP1 can also be activated through the MSH receptor and its downstream signaling pathways.

To assess the effect of peptide intervention on melanin synthesis, we used immunofluorescence assays with an FITC-labeled green fluorescent secondary antibody to evaluate TRP1 expression in cells. The results demonstrated that the peptides EERVEEGRR, EAIVRYFAG, and EEIVEYFAG all reduced TRP1 expression. The fluorescence intensity of TRP1 was measured as a percentage relative to the control group, showing values of 62.1 ± 3.5%, 75.4 ± 4.9%, and 72.3 ± 2.8%, respectively. This suggests these peptides effectively downregulate TRP1 expression and may consequently inhibit melanin synthesis.

Fig.2 Expression of TRP1 Protein in Cells Under Different Peptide Intervention Conditions

3 Expression System

3.1. Plasmid Construction and Amplification

To express the required peptide sequence in engineered bacteria and apply specific modifications to its N- and C-terminals, we constructed the expression system pET-28A(+)-MelanSD-His-TC-TGF using the pET-28A(+) plasmid backbone.

We first transformed the plasmid into Escherichia coli DH5α using an improved calcium rubidium method to amplify its quantity. Subsequently, the plasmid was extracted and retransformed into Escherichia coli BL21(DE3) to construct an IPTG-inducible peptide expression system.

The results of agarose gel electrophoresis showed that the amplified bands from the transformed DH5α E. coli were consistent with the theoretical plasmid size, confirming that the pET-28A(+)-MelanSD-His-TC-TGF plasmid was successfully transformed into the bacteria and efficiently amplified.

Fig.1 The agarose gel electrophoresis results of the plasmid extracted from E. coli DH5α colonies matched the pET-28A(+)-MelanSD-His-TC-TGF plasmid

3.2 Verification of Target Peptide Expression

To detect whether the transformed BL21 (DE3) engineered bacteria could successfully express the target peptide under IPTG induction, we analyzed the total bacterial protein using SDS-PAGE and Ponceau S staining staining methods. The results showed intensified staining in the lanes corresponding to the predicted molecular weight of the Melan fusion peptide (13-16 kDa). In contrast, no intensified staining was observed at this position in uninduced, non-plasmid-transformed BL21 (DE3), or DH5α bacteria. This demonstrated that the engineered bacteria successfully expressed the protein with the expected molecular weight.

Fig.2 SDS-PAGE image

4 The Functional Validation of Recombinant Bacteria

4.1. MSH knockdown cell line

To verify whether the expression product of the recombinant bacteria can inhibit melanin production by targeting the MSH receptor and its downstream signaling pathways, we first constructed an MSH gene knockdown L-929 cell line. Specifically, we designed an siRNA that interacts with the mRNA corresponding to MSH and used a transfection reagent (Lip2000) to mix with the siRNA for cell transfection. Subsequently, total RNA was extracted from the cells, and RT-qPCR was performed to detect the mRNA expression of MSH.

The results showed that compared to the control group, the MSH expression levels in both the NC group and the siRNA group were reduced, indicating the successful establishment of the MSH knockdown cell line.

Fig.1 Quantitative analysis of PCR amplification curves

4.2. Verification of TRP1 expression

We used the whole membrane stripping and re-probing method in Western blot to simultaneously detect the expression of the reference protein and TRP-1 protein. The results showed that TRP-1 expression decreased in cells co-cultured with the filtered product of the recombinant bacterial culture supernatant. After knocking down MSH, TRP-1 expression increased, indicating that the filtered product can inhibit TRP-1 expression and is regulated by MSH.

Fig.2 Western-blot

5 Controlled bacterial clearance

5.1 Construction and transformation of suicide plasmid

To achieve controlled death of engineered bacteria, we constructed a toxin-antitoxin module regulated by an arabinose operon. When the engineered bacteria accidentally leak or reach conditions requiring termination, arabinose can replace glucose as an energy source and interact with the arabinose operon to initiate suicide gene transcription. We constructed the pBAD30-T4-lysis plasmid and transformed it into E. coli BL21 (DE3).

Fig.1 BL21(DE3) bacteria, BL21(DE3) bacteria transformed with the pET-28A(+)-MelanSD-His-TC-TGF plasmid, and bacteria transformed with both the pET-28A(+)-MelanSD-His-TC-TGF and pBAD30-T4-lysis plasmids.

5.2 Verification of Functionality

We used live/dead bacterial fluorescence staining and colony count-based bactericidal curve measurement to verify whether the suicide switch system can control bacterial apoptosis. The results showed significant death signals in bacteria 24 hours after the addition of arabinose. Colony count results indicated that the number of colonies decreased to zero after 24 hours.

Fig.2 Live/dead bacterial staining indicates that red fluorescence signals represent dead bacteria.

Fig.3 Time-series bactericidal curve