Bio-engineering Institute of Jinan University, Jinan University, Guangzhou, Guangdong, PR China.
Neurosci Lett. 2012 Oct 3;527(1):1-5. doi: 10.1016/j.neulet.2012.08.005. Epub 2012 Aug 23.
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.
垂体腺苷酸环化酶激活肽（PACAP）是一种有潜力的治疗性神经肽。11 个氨基酸的人类免疫缺陷病毒 TAT 蛋白转导结构域能够将蛋白货物穿过细胞膜和血脑屏障。因此，制备了一种新型融合蛋白 PACAP-TAT，其 C 末端包含 TAT，用于研究其穿过血脑屏障的能力。将编码 PACAP-TAT 的基因克隆到表达载体 pKYB 中，并使用内含肽介导的纯化与亲和几丁质结合标签（IMPACT）系统纯化目标肽 PACAP-TAT。细胞实验结果表明，PACAP-TAT 以与 PACAP 相同的效力刺激 PAC1-CHO 细胞的细胞活力，这表明 TAT 的融合并未影响 PACAP-TAT 激活 PACAP 特异性受体 PAC1 的能力。使用异硫氰酸荧光素（FITC）标记的肽测定 PACAP-TAT 和 PACAP 穿过血脑屏障（BBB）、血气屏障（BAB）和血睾屏障（BTB）的转移效率。结果表明，PACAP-TAT 穿过血脑屏障的效率约比 PACAP 高 2.5 倍。荧光显微镜检查显示，PACAP-TAT 穿过 BBB 的效率明显高于 PACAP。此外，腹腔内（i.p.）注射 PACAP-TAT 对食物摄入的抑制作用强于 PACAP（p<0.01，PACAP-TAT 与 PACAP），这表明 TAT 有助于增加 PACAP-TAT 在大脑中的定位。制备具有增强穿过生物屏障能力的 PACAP-TAT 将改善其给药途径并扩大其应用范围。
