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The Influence of Structrue of Yellow Fluorescent Proteins on the Apoptotic Detection Ability of Annexin A5
Gao Mengyue1, Wang Yunke1, Huang Xianjie1, Zhang Jing1*, Hua Zichun1,2,3*
1School of Life Sciences, the State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210023, China; 2Reserach Institute of Pharmaceutical Biotechnology, Jiangsu Industrial Technology Research Institute and High-Tech Research Institute of Nanjing University at Changzhou, Changzhou 213164, China; 3Shenzhen Research Institute of Nanjing University, Shenzhen 518057, China
Abstract: At the early stage of apoptosis， phosphatidylserine (PS) will turn over from the inner cell membrane to the outer cell membrane. When calcium ions are present， Annexin A5 can combine to PS at high affinity and this is the principle of apoptosis detection. Apoptosis detection based on Annexin A5 labeled with green fluorescent probes/iodide by FCM (flow cytometry) is sensitive， efficient and specific. Annexin A5-EGFP fusion protein is a simple， reliable apoptosis detection probe and it is easy to prepare. Yellow fluorescent protein is a mutant of green fluorescent protein and its fluorescence spectra shifts to red spectra. Currently， there are three modified yellow fluorescent proteins: Citrine， Venus， Ypet and the three modified proteins’ fluorescence are brighter， more stable and mature faster. In this paper， Citrine， Venus and Ypet were selected to prepare Annexin A5 apoptosis detection probes. Annexin A5-Citrine， Annexin A5-Venus and Annexin A5-Ypet were expressed in the prokaryotic expression system with a high yield of soluble protein. Also， we explored the combination ability of the three fusion proteins and apoptotic cells and then screened out Annexin A5 labeled by yellow fluorescent proteins， which was suitable for apoptosis detection based on FCM in order to lay the foundation for further study. Three new recombinant plasmids pET28a-Annexin A5-Citrine-his， pET28a-Annexin A5-Venus-his， pET28a-Annexin A5-Ypet-his were constructed for the expression. Three recombinant proteins were purified by the Ni column affinity chromatography and their purity is about 90%. Also， this paper compared the three fusion proteins’ ability to detect apoptosis and flow cytometric analysis showed that Annexin A5-Citrine， Annexin A5-Venus， Annexin A5-Ypet could recognize and bind to apoptotic cells and their affinity with apoptotic cells was respectively 3 113.0 nmol/L， 444.3 nmol/L， 391.6 nmol/L. The affinity between the three fusion proteins and apoptotic cells was very different. The paper analyzed the amino acid sequence of Citrine， Venus and Ypet and preliminarily found the key amino acids， which determine the affinity of Annexin A5 marked with yellow fluorescent proteins and apoptotic cells.
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