Infectious Signaling Network Research Center and Research Institute for Medical Sciences, Department of Physiology, School of Medicine, Chungnam National University, Daejeon 301-747, Republic of Korea.
Biochem Biophys Res Commun. 2011 Jul 1;410(2):312-6. doi: 10.1016/j.bbrc.2011.05.139. Epub 2011 May 30.
Cellular protein delivery is an emerging technique by which exogenous recombinant proteins are delivered into mammalian cells across the membrane. We have developed an Escherichia coli expression vector including human specific gene sequences for protein cellular delivery. The plasmid was generated by ligation the nucleotides 770-817 of the homeobox A5 mRNA sequence which was matched with protein transduction domain (PTD) of homeodomain protein A5 (HOXA5) into pET expression vector. The cellular uptake of HOXA5-PTD-EGFP was detected in 1min and its transduction reached a maximum at 1h within cell lysates. The cellular uptake of HOXA5-EGFP at 37°C was greater than in 4°C. For study for the functional role of human HOXA5-PTD, we purified HOXA5-APE1/Ref-1 and applied it on monocyte adhesion. Pretreatment with HOXA5-APE1/Ref-1 (100nM) inhibited TNF-α-induced monocyte adhesion to endothelial cells, compared with HOXA5-EGFP. Taken together, our data suggested that human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins.
细胞内蛋白质递送是一种新兴技术，通过该技术可以将外源性重组蛋白跨膜递送至哺乳动物细胞内。我们开发了一种包含人类特异性基因序列的大肠杆菌表达载体，用于蛋白质的细胞内递送。该质粒通过将同源盒 A5 mRNA 序列的 770-817 个核苷酸与同源域蛋白 A5（HOXA5）的蛋白转导结构域（PTD）连接到 pET 表达载体中而生成。在细胞裂解物中，HOXA5-PTD-EGFP 的细胞摄取在 1 分钟内被检测到，其转导在 1 小时内达到最大值。在 37°C 下的细胞摄取大于 4°C 下的摄取。为了研究人类 HOXA5-PTD 的功能作用，我们纯化了 HOXA5-APE1/Ref-1 并将其应用于单核细胞黏附。与 HOXA5-EGFP 相比，用 HOXA5-APE1/Ref-1（100nM）预处理可抑制 TNF-α诱导的单核细胞黏附内皮细胞。总之，我们的数据表明，人类 HOXA5-PTD 载体为揭示蛋白质的细胞功能或生成包含人类 PTD 的蛋白质提供了强大的研究工具。
