ORIGINAL ARTICLE   

Minerva Biotechnology and Biomolecular Research 2022 March;34(1):14-21

DOI: 10.23736/S2724-542X.21.02726-9

Copyright © 2021 EDIZIONI MINERVA MEDICA

language: English

In-silico selection of the signal peptides for high-level secretory expression of aflibercept in CHO cells

Mozhdeh ZAMANI ^{1, 2}, Behnam KADKHODAEI ³ ✉, Navid NEZAFAT ^{4, 5}, Seyed V. HOSSEINI ¹

¹ Colorectal Research Center, Shiraz University of Medical Sciences, Shiraz, Iran; ² Autophagy Research Center, Shiraz University of Medical Sciences, Shiraz, Iran; ³ Department of Radiation Oncology, Shiraz University of Medical Sciences, Shiraz, Iran; ⁴ Pharmaceutical Sciences Research Center, Shiraz University of Medical Science, Shiraz, Iran; ⁵ Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran

BACKGROUND: Different strategies have been considered to enhance the production level of recombinant proteins in Chinese hamster ovary (CHO) host. Secretory production using suitable signal peptides (SPs) is one of these strategies. Aflibercept is a Fc-fusion protein, approved for treatment of macular degeneration and metastatic colorectal cancer. This in-silico study was designed to evaluate the potential efficiency of 41 different SPs in secretory production of aflibercept in CHO.
METHODS: SPs were selected based on the literature, which indicated their acceptable efficiency for different recombinant proteins secretion in CHO. The principal parameters in selection of appropriate SPs, including cleavage site of the signal peptides, important physicochemical features, aflibercept subcellular localization and solubility likelihood were predicted using SignalP 5.0, Protparam and DeepLoc-1.0 servers, respectively.
RESULTS: Three SPs among all evaluated ones, including A2, TPA and SP2 were excluded due to their weak cleavage scores. Eleven SPs, including SP5, SP9, SP13, F, H, N, K, 8Ksp, IgK H, R and S were excluded owing to their inadequate stability. VEGFR1 SP was also excluded because of its negative net charge. Finally, T, SP11, SYN1 and SP8 showed best properties for secretory production of aflibercept, respectively. 3D structures of aflibercept fused to these SPs were predicted using homology modeling.
CONCLUSIONS: T, SP11, SYN1 and SP8 were identified as the best theoretical SP candidates for secretory production of aflibercept, respectively. Given the importance of other factors like host features and cultivation condition optimization along with suitable SPs, further experimental studies are essential to confirm these bioinformatics results.

KEY WORDS: Protein sorting signals; Aflibercept; CHO cells; Computational biology