School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, PR China.
J Drug Target. 2009 May;17(4):329-33. doi: 10.1080/10611860802582459.
Although the potential value of phenotypic/functional knockout technology with intrabody/kine in prevention and cure of some serious diseases, such as AIDS and cancer, is being regarded, there are still several technical difficulties. One of the the most critical problems is how to directly deliver the intrabody/kine proteins into endoplasm reticulum (ER). In this study, a novel recombinant protein, TAT-GFP-KDEL, was designed and constructed. In this recombinant protein, HIV-derived TAT (47-57) and an ER retention four-peptide sequence KDEL were fused at the N-terminal and C-terminal of GFP respectively. The results showed that TAT-GFP-KDEL had been successfully expressed in bacteria BL21 and its purity reached to 95%. Moreover, we observed that this recombinant protein was able to efficiently transduce into MOLT-4 cells and accurately locate at ER. This study may provide an available strategy to promote the transmembrane delivery and ER localization of protein-based intrabody/kine.
虽然利用内体/核定位序列（kine）的表型/功能敲除技术在预防和治疗艾滋病和癌症等一些严重疾病方面具有潜在的价值，但仍存在一些技术难题。其中最关键的问题之一是如何将内体/核定位序列蛋白直接递送到内质网（ER）中。本研究设计并构建了一种新型重组蛋白 TAT-GFP-KDEL。在该重组蛋白中，HIV 衍生的 TAT（47-57）和 ER 保留的四肽序列 KDEL 分别融合在 GFP 的 N 端和 C 端。结果表明，TAT-GFP-KDEL 在细菌 BL21 中成功表达，其纯度达到 95%。此外，我们观察到该重组蛋白能够有效地转导到 MOLT-4 细胞中，并准确定位于 ER。本研究可能为促进基于蛋白质的内体/核定位序列的跨膜传递和 ER 定位提供了一种可行的策略。
