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Du X.,East China University of Science and Technology | Gu H.-Y.,Zhejiang Reachall Pharmaceutical Co. | Li Y.-D.,Zhejiang Reachall Pharmaceutical Co. | Wu Z.,Zhejiang Reachall Pharmaceutical Co. | Zhao J.,East China University of Science and Technology
Huadong Ligong Daxue Xuebao/Journal of East China University of Science and Technology | Year: 2014

To study the transmembrane activity of Heparin-Binding Domain (HBD) derived from human fused at N-terminal of heterologous protein, the expression plasmid pET28b-HBD-EGFP-Histag was constructed by genetically fusing HBD to the N-terminal of enhanced green fluorescent protein (EGFP). The fusion protein HBD-EGFP was expressed in E.coli and purified by Ni2+-NTA affinity chromatography. The results show that green fluorescence can be observed in HeLa cells using laser scanning confocal microscope when HeLa cells were co-cultured with HBD-EGFP. Flow-cytometry results show that the transmembrane efficiency of HBD fused at the N-terminal of EGFP is improved about 10 times compared with the HBD fused at the C-terminal of EGFP. These results demonstrated that HBD fused to N-terminal of heteologous protein exhibited tansmembrane activity. The HBD fused at the N-terminal of EGFP had a higher efficiency than the HBD fused at the C-terminal of EGFP. These results will provide theoretical foundation for the efficient application of HBD in the field of the delivery of anticancer drugs.


Nguyen L.T.,East China University of Science and Technology | Yang X.-Z.,East China University of Science and Technology | Yang X.-Z.,Zhejiang Reachall Pharmaceutical Co. | Du X.,East China University of Science and Technology | And 7 more authors.
Amino Acids | Year: 2015

Cell-penetrating peptides (CPPs) are well known as intracellular delivery vectors. However, unsatisfactory delivery efficiency and poor specificity are challenging barriers to CPP applications at the clinical trial stage. Here, we showed that S3, an EGFR-binding domain derived from vaccinia virus growth factor, when fused to a CPP such as HBD or TAT can substantially enhance its internalization efficiency and tumor selectivity. The uptake of S3-HBD (S3H) recombinant molecule by tumor cells was nearly 80 folds increased compared to HBD alone. By contrast, the uptake of S3H by non-neoplastic cells still remained at a low level. The specific recognition between S3 and its receptor, EGFR, as well as between HBD and heparan sulfate proteoglycans on the cell surface was essential for these improvements, suggesting a syngeneic effect between the two functional domains in conjugation. This syngeneic effect is likely similar to that of the heparin-binding epidermal growth factor, which is highly abundant particularly in metastatic tumors. The process that S3H entered cells was dependent on time, dosage, and energy, via macropinocytosis pathway. With excellent cell-penetrating efficacy and a novel tumor-targeting ability, S3H appears as a promising candidate vector for targeted anti-cancer drug delivery. © 2015 Springer-Verlag Wien.

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