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Updated: Jan 3, 2026
Analysis of Epididymal Protein Synthesis and Secretion
Published on: August 25, 2018
Identification of secretion domain of Neospora caninum profilin
Hamizah Suhaimi1, Jian Xu2, Tatsuya Kato3
1Laboratory of Biotechnology, Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan.
Abstract:
Profilin (PROF) is a small actin-binding protein presented in apicomplexan protozoa. It was previously reported that Neospora caninum profilin (NcPROF) is secreted into the hemolymph of silkworm larvae regardless of the lack of an identified regular secretion signal peptide. To date, which domain is required for its secretion still remains unknown. To this end, we express a fluorescent protein (mCherry) fused with NcPROF at its N-terminus or C-terminus. Both fusion proteins were expressed and secreted into the culture supernatant from Bm5 cells or hemolymph from silkworm larvae, respectively. To further narrow down the C-terminal minimal domain required for its secretion, we constructed three truncated C-terminal domain constructions, ΔN (aa41-163), ΔN1 (aa50-163), and ΔN2 (aa144-163) respectively. All three fusion proteins were detected in the culture supernatant of Bm5 cells and silkworm hemolymph. Surprisingly, a 20-aa C-terminal α-helix domain facilitates the secretion of mCherry, allowing purification of ΔN2-mCherry from silkworm larval hemolymph by affinity chromatography. Taken together, the secretion domain from NcPROF was identified, indicating that can be utilized for the secretory expression of recombinant proteins in the future.
Insights
Neospora caninum profilin (NcPROF) secretion was investigated. A 20-amino acid C-terminal alpha-helix domain was identified as crucial for NcPROF secretion, enabling recombinant protein expression.
Area of Science:
- Molecular Biology
- Parasitology
- Protein Secretion Mechanisms
Background:
- Profilin (PROF) is an actin-binding protein found in apicomplexan protozoa.
- Neospora caninum profilin (NcPROF) is secreted into silkworm hemolymph without a clear signal peptide.
- The specific domain responsible for NcPROF secretion remained unidentified.
Purpose of the Study:
- To identify the domain(s) of Neospora caninum profilin (NcPROF) required for its secretion.
- To investigate the potential of NcPROF for secretory expression of recombinant proteins.
Main Methods:
- Fusion of mCherry fluorescent protein to N-terminus and C-terminus of NcPROF.
- Expression of fusion proteins in Bm5 cells and silkworm larvae.
- Construction and expression of truncated NcPROF C-terminal domains (ΔN, ΔN1, ΔN2).
- Detection of fusion proteins in cell culture supernatant and silkworm hemolymph.
- Affinity purification of a truncated NcPROF-mCherry fusion protein.
Main Results:
- Both N-terminal and C-terminal NcPROF-mCherry fusion proteins were secreted.
- Truncated NcPROF C-terminal domains (ΔN, ΔN1, ΔN2) were also secreted.
- A 20-amino acid C-terminal alpha-helix domain was identified as sufficient for mCherry secretion.
- ΔN2-mCherry fusion protein was successfully purified from silkworm hemolymph.
Conclusions:
- The C-terminal 20-amino acid alpha-helix domain of NcPROF is essential for its secretion.
- This identified secretion domain can be leveraged for future secretory expression of recombinant proteins.
