Desain Kompleksi Fusi Snakin untuk Studi Kelayakan Overekspresi pada Escherichia coli

Abstract

Snakin merupakan peptida tanaman yang berpotensi dikembangkan sebagai agen antimikroba, tetapi produksi dengan ekspresi rekombinan masih terkendala oleh aktivitas antimikroba snakin yang bersifat toksik terhadap Escherichia coli (E. coli) sebagai sel inang. Penelitian ini bertujuan mendesain kompleks fusi snakin melalui pendekatan tandem repeat dan protein fusi untuk memperoleh model dengan stabilitas dan kelarutan lebih baik serta aksesibilitas sisi aktifnya tertutup. Metode ini dilakukan dengan memvariasikan protein fusi, linker, situs pemotongan protease, dan jumlah repeat snakin. Model dievaluasi berdasarkan predicted Template Modeling score (pTM), predicted Local Distance Difference Test (pLDDT), Predicted Aligned Error (PAE), indikator kelarutan, konservasi residu, daerah pocket, serta penambatan molekuler. Hasil penelitian menunjukkan, snakin asli memiliki stabilitas struktur yang baik dengan skor pTM 0,86. Model terbaik hasil modifikasi (Maltose Binding Protein-Linker-TEV Protease-Snakin) memiliki skor pTM 0,82 dan berhasil dalam mempertahankan kelarutan dengan skor muatan permukaan bersih sebesar -14,60. Hasil evaluasi situs aktif menunjukkan bahwa daerah aktif snakin pada model hasil modifikasi masih terekspos. Simpulan temuan ini menunjukkan hasil modifikasi berpotensi untuk tetap mempertahankan kelarutan, tetapi belum optimal dalam menutupi sisi aktif snakin.

Snakin is a plant antimicrobial peptide with potential to be developed as an antimicrobial agent; however, its production through recombinant expression is still limited by low solubility and toxic effects on E. coli as the host cell. This study aimed to design snakin fusion constructs using tandem repeat and fusion protein approaches to obtain models with improved stability and solubility, as well as reduced accessibility of the active site. The designs were generated by varying the fusion protein, linker, protease cleavage site, and number of snakin repeats. The models were evaluated based on pTM, pLDDT, PAE, solubility indicators, residue conservation, pocket regions, and molecular docking. Native snakin showed good structural stability with a pTM score of 0.86. The best modified model, had a pTM score of 0.82 and successfully improved solubility, with a net charge of -14.60. Conservation, pocket, and molecular docking analyses showed that the active region of snakin in the modified model remained exposed and was still able to interact with ligands. These findings indicate that the modification has the potential to maintain solubility, but is not yet optimal in covering the active site of snakin.