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Published on: January 4, 2017
[Construction and expression of PRL-3 plasmid with C104S point mutation and CAAX deletion]
Wei-jun Li1, Xiao-fang Xing, Li-ke Qu
1Key laboratory of Carcinogenesis and Translational Research of Ministry of Education, Department of Biochemistry and Molecular Biology, Peking University School of Oncology, Beijing Cancer Hospital & Institute, Beijing 100142, China.
Objective:
To construct PRL-3 gene C104S point mutation and CAAX deletion mutants: pcDNA3-myc-PRL-3 (C104S), pEGFP-PRL-3 (C104S), pcDNA3-myc-PRL-3 (DeltaCAAX) and pEGFP-PRL-3 (DeltaCAAX), and express these plasmids in eukaryotic cells.
Methods:
Recombinant plasmids were mutated with pcDNA3-myc-PRL-3 plasmid as template and specific primers. Mutants were identified by restriction enzyme digestion and DNA sequencing. Then these recombinant plasmids were transfected into LoVo cells. The expression of fusion proteins were detected by western blotting and the localization of fusion proteins were examined by GPF fluorescence labelling.
Results:
The mutants were successfully constructed and expressed in eukaryotic cells. PRL-3 (DeltaCAAX) relocates from plasma membrane/early endosome to the cytoplasm and/or nucleus, which provides a structural insight of PRL-3 protein.
Conclusion:
Construction of eukaryotic expression recombinant plasmids of PRL-3 gene C104S point mutation and CAAX deletion mutants provide a useful tool for the study of PRL-3's role in cancer.
