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Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii
Published on: July 12, 2013
Molecular cloning of a rhoptry protein (ROP6) secreted from Toxoplasma gondii
Hye-Jin Ahn1, Sehra Kim, Ho-Woo Nam
1Department of Parasitology and the Catholic Institute of Parasitic Diseases, College of Medicine, Catholic University of Korea, Seoul, Korea.
Abstract:
Monoclonal antibody (mAb) Tg786 against Toxoplasma gondii has been found to detect a 42-kDa rhoptry protein (ROP6) which showed protease activity and host cell binding characteristics after secretion. Using the mAb, a colony containing a 3o-UTR was probed in a T. gondii cDNA expression library. A full length cDNA sequence of the rhoptry protein was completed after 5o-RACE, which consisted of 1,908 bp with a 1,443 bp ORF. The deduced amino acid sequence of ROP6 consisted of a polypeptide of 480 amino acids without significant homology to any other known proteins. This sequence contains an amino terminal stop transfer sequence downstream of a short neutral sequence, hydrophilic middle sequence, and hydrophobic carboxy terminus. It is suggested that the ROP6 is inserted into the rhoptry membrane with both N- and C-termini.
Insights
Researchers identified Toxoplasma gondii rhoptry protein 6 (ROP6), a 42-kDa protein with protease activity. This study characterized the ROP6 gene and its protein sequence, revealing its unique structure and membrane insertion mechanism.
Area of Science:
- Parasitology
- Molecular Biology
- Protein Biochemistry
Background:
- Toxoplasma gondii rhoptry proteins are crucial for host cell invasion.
- Monoclonal antibody (mAb) Tg786 detects a 42-kDa rhoptry protein, ROP6, exhibiting protease activity and host cell binding.
- The precise function and genetic basis of ROP6 were previously uncharacterized.
Purpose of the Study:
- To clone and sequence the full-length cDNA of the Toxoplasma gondii rhoptry protein 6 (ROP6).
- To analyze the deduced amino acid sequence of ROP6 for functional insights.
- To elucidate the membrane insertion mechanism of ROP6.
Main Methods:
- Screening of a T. gondii cDNA expression library using mAb Tg786.
- 5'-RACE (Rapid Amplification of cDNA Ends) to obtain the full-length cDNA sequence.
- Bioinformatic analysis of the deduced amino acid sequence.
Main Results:
- A full-length cDNA sequence of 1,908 bp was obtained, containing a 1,443 bp open reading frame (ORF).
- The deduced ROP6 protein consists of 480 amino acids with no significant homology to known proteins.
- ROP6 possesses predicted structural features including an amino-terminal stop transfer sequence, suggesting a specific membrane insertion pathway.
Conclusions:
- The ROP6 gene has been successfully cloned and sequenced.
- The ROP6 protein is a novel rhoptry protein with a unique amino acid sequence and predicted structure.
- ROP6 is likely inserted into the rhoptry membrane via its N- and C-termini.
