The TAT peptide endows PACAP with an enhanced ability to traverse bio-barriers |
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Authors: | Rongjie Yu Zhixing Zeng Xiaoling Guo HuaHua Zhang Xiaofei Liu Yong Ding Jiansu Chen |
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Institution: | 1. Bio-engineering Institute of Jinan University, Jinan University, Guangzhou, Guangdong, PR China;2. Biology Staff Room of Guangdong Medical College, Dongguan, Guangdong, PR China;3. The Ophthalmic Office in the First Affiliated Hospital of Jinan University, Jinan University, Guangzhou, Guangdong, PR China;4. Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, Guangdong, PR China |
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Abstract: | Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potential therapeutic neuropeptide. The 11-amino acid human immunodeficiency virus TAT protein transduction domain is able to deliver protein cargoes across the cell membrane and the blood–brain barrier. A novel fusion protein PACAP-TAT, containing TAT at the C-terminus of PACAP was therefore produced and studied for the ability to cross blood barriers. The gene encoding PACAP-TAT was cloned into the expression vector pKYB, and the target peptide PACAP-TAT was purified using the Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT) system. The results of cell assays showed that PACAP-TAT stimulated the cell viability of PAC1-CHO cells with the same potency as PACAP, which indicated that the fusion of TAT did not affect the ability of PACAP-TAT to activate the PACAP-specific receptor PAC1. The transfer efficiencies of PACAP-TAT and PACAP across the blood–brain barrier (BBB), blood–air barrier (BAB) and blood–testis barrier (BTB) were assayed using peptides labeled with fluorescein isothiocyanate (FITC). The results showed that PACAP-TAT traversed blood barriers with an efficiency approximately 2.5-fold greater than PACAP. Fluorescence microscopic examination showed that PACAP-TAT traversed the BBB significantly more efficiently than PACAP. Furthermore, intraperitoneal (i.p.) injection of PACAP-TAT induced a stronger inhibitory effect on food intake than PACAP (p < 0.01, PACAP-TAT vs. PACAP), which indicated that TAT helped to increase the localization of PACAP-TAT in the brain. Preparation of PACAP-TAT with the enhanced ability to cross biological barriers will improve its route of administration and expand its scope of application. |
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Keywords: | PACAP TAT Protein transduction domain Blood&ndash brain barrier Food intake |
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