همایش ملی بیوانفورماتیک ایران

صفحه اصلی / 4th international edition and 13th Iranian Conference on Bioinformatics

Towards Efficient Recombinant Protein Production: In-silico screening of signal peptides for secretory production of recombinant factor C in mammalian cells

نویسندگان :

Maryam Latifi¹ Kosar Asadi² Mansoureh Shahbazi Dastjerdeh³

1- Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran 2- Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran 3- Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran

کلمات کلیدی :

Signal Peptides،In silico Modeling،Limulus factor C،recombinant protein،computational biology

چکیده :

Background: Endotoxins (lipopolysaccharides) found in the outer membrane of gram-negative bacteria are major contributors to pyrogenic responses in contaminated pharmaceutical products and medical devices. These molecules can induce a range of effects in mammals, from mild fever to severe conditions such as septic shock and death. Consequently, there is a critical need for accurate bacterial endotoxin testing (BET). Factor C, a key protein derived from horseshoe crab blood, plays a central role in detecting endotoxins. Its recombinant production holds significant importance as it offers a sustainable and ethical alternative to the traditional preparation of Limulus Amoebocyte Lysate (LAL) assay kits, which rely on the extraction of blood from live horseshoe crabs. (Kang, Yun et al. 2024) . Mammalian cell lines, widely utilized for expressing recombinant proteins, are particularly suited for producing recombinant Factor C (rFC) because they support post-translational modifications necessary for protein stability and functionality (Kobayashi, Shiga et al. 2014) . The presence of a signal peptide is essential to direct nascent polypeptide chains to the endoplasmic reticulum, where proper folding and post-translational modifications occur (O’Neill, Mistry et al. 2023) . Signal peptides are short sequences located at the N-terminal of the protein, and they are cleaved by signal peptidases after translocation, yielding the mature, functional protein (Pool 2022) . Selecting the optimal signal peptide for recombinant protein production traditionally involves experimental screening, which is both costly and time-consuming. To address these challenges, this study conducted an in-silico evaluation of 74 secretory signal peptides to identify the most suitable candidates for the efficient secretory production of recombinant Factor C of Carcinoscorpius rotundicauda in mammalian cells. Method: Signal peptide sequences were retrieved from the UniProt database and previously published papers. The presence and location of cleavage sites within the sequences, fused to the N-terminal of the rFC protein, were predicted using SignalP 6.0. Physiochemical properties critical for protein expression, including length, net positive charge, instability index, GRAVY score, and aliphatic index, were assessed using the ProtParam server. Additionally, protein solubility and subcellular localization of protein which are essential for proper folding and activity in eukaryotic systems, were evaluated using the DeepLoc 2.1 server. Result and discussion: Out of the 74 signal peptides analyzed, 49 were excluded: one lacked a cleavage site, 17 exhibited incorrect cleavage sites, and 31 were predicted to be unstable. The remaining 33 signal peptides were ranked based on their GRAVY scores, a parameter indicative of hydrophobicity. Among these, the top five candidates—CALR, native G, Scrg1, rituximab native HC, and HSP90B1—exhibited the highest GRAVY scores, suggesting their potential suitability for the secretory production of recombinant Factor C.

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