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Tian M.,Shanghai University of Traditional Chinese Medicine | Zeng X.-Q.,Shanghai University of Traditional Chinese Medicine | Song H.-L.,East China University of Science and Technology | Hu S.-X.,East China University of Science and Technology | And 4 more authors.
Journal of the Science of Food and Agriculture | Year: 2015

Momordica charantia (MC) has been used for treating diabetes mellitus from ancient times in Asia, Africa and South America. There are many MC accessions in local markets. Polypeptide-P as a main hypoglycemic component in MC was first studied in this experiment to illustrate the different contents in MC of different accessions and different harvesting times. RESULTS: Nineteen MC accessions collected from different regions were clustered into three groups using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) molecular markers. Content of polypeptide-P in the tested MC accessions was detected by western blot (WB) method. The WB results revealed that polypeptide-P was detected in MC accessions harvested in June and July but not in September and October. Furthermore, Polypeptide-P content corresponded well with the MC accessions. CONCLUSION: Our results suggest that the MC accessions and the harvesting times or the weather during harvest play significant roles in high content of polypeptide-P. © 2014 Society of Chemical Industry.


Zhao J.,East China University of Science and Technology | Gao P.,East China University of Science and Technology | Xiao W.,East China University of Science and Technology | Fan L.-Q.,East China University of Science and Technology | And 3 more authors.
Molecular Biology Reports | Year: 2011

Cell-penetrating peptides can carry a variety of biologically active molecules into cells. Here we have identified a novel CPP derived from the C-terminus of human extracellular superoxide dismutase (hC-SOD3) which was shown to be located throughout in the cytoplasm and nucleus by fluorescence microscopy investigation. Furthermore, when apoptin fused to hC-SOD3, it was translocated efficiently into HeLa cells resulting in antitumor activities. This study shows that hC-SOD3 has the potential to penetrate and translocate cargo molecules into cells and has no cytotoxicity at effective concentration. © Springer Science+Business Media B.V. 2010.


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.


Zhang R.,East China University of Science and Technology | Yang X.-Z.,East China University of Science and Technology | Yang X.-Z.,Zhejiang Reachall Pharmaceutical Co. | Wang J.-W.,East China University of Science and Technology | And 4 more authors.
Journal of Drug Targeting | Year: 2015

Human-derived cell penetrating peptides (CPPs) have attracted much more attentions than other CPPs which are limited by their potential toxicity and immunogenicity. Previously, we identified a novel human-originated CPP (named heparin-binding domain (HBD) in this article), which derived from the C-terminus of human extracellular superoxide dismutase, and demonstrated HBD is an efficient vector for delivering exogenous drug molecules such as apoptin into HeLa cells. In this study, we found this novel CPP showed differentiated efficiency in several tested cell lines. Heparin competitive inhibition experiment and heparanase pre-incubation experiment showed cell surface polysaccharides play an important role for the transmembrane transport. The results of endocytosis inhibitors suggested that HBD penetrates the cell membrane via a direct translocation, which is different from that of TAT, a classical clathrin-mediated endocytosis. HBD could deliver up to 90 kD protein cargoes into cells. Different conjugated modes with cargo molecules greatly affect their translocation efficiency. HBD also showed significant nuclear transport capacity when it was incubated with HeLa cells. Furthermore, the core region for HBD possessing membrane-penetrating ability was identified by deletion analyses. These results would be helpful for developing HBD as a new nuclear delivery tool for therapeutic biomolecules. © 2015 Informa UK Ltd.


Lv Q.,East China University of Science and Technology | Yang X.-Z.,East China University of Science and Technology | Yang X.-Z.,Zhejiang Reachall Pharmaceutical Co. | Fu L.-Y.,Zhejiang Reachall Pharmaceutical Co. | And 5 more authors.
Protein Expression and Purification | Year: 2015

MAP30 (Momordica Antiviral Protein 30 Kd), a single-stranded type-I ribosome inactivating protein, possesses versatile biological activities including anti-tumor abilities. However, the low efficiency penetrating into tumor cells hampers the tumoricidal effect of MAP30. This paper describes MAP30 fused with a human-derived cell penetrating peptide HBD which overcome the low uptake efficiency by tumor cells and exhibits higher anti-tumor bioactivity. MAP30 gene was cloned from the genomic DNA of Momordica charantia and the recombinant plasmid pET28b-MAP30-HBD was established and transferred into Escherichia coli BL21 (DE3). The recombinant MAP30-HBD protein (rMAP30-HBD) was expressed in a soluble form after being induced by 0.5 mM IPTG for 14 h at 15 °C. The recombinant protein was purified to greater than 95% purity with Ni-NTA affinity chromatography. The rMAP30-HBD protein not only has topological inactivation and protein translation inhibition activity but also showed significant improvements in cytotoxic activity compared to that of the rMAP30 protein without HBD in the tested tumor cell lines, and induced higher apoptosis rates in HeLa cells analyzed by Annexin V-FITC with FACS. This paper demonstrated a new method for improving MAP30 protein anti-tumor activity and might have potential applications in cancer therapy area. © 2015 Elsevier Inc. All rights reserved.


Lin B.,East China University of Science and Technology | Yang X.-Z.,Zhejiang Reachall Pharmaceutical Co. | Cao X.-W.,East China University of Science and Technology | Zhang T.-Z.,Zhejiang Reachall Pharmaceutical Co. | And 3 more authors.
Biotechnology Letters | Year: 2016

Objective: To evaluate the anti-tumor effects of trichosanthin after fusion with a cell penetrating peptide, heparin-binding peptide (HBP), derived from human heparin-binding EGF-like growth factor (HB-EGF). Results: The fusion protein of trichosanthin-HBP was expressed in Escherichia coli BL21 and purified by Ni–NTA affinity chromatography. The HBP domain had no influence on the topological inactivation activity and N-glycosidase activity of trichosanthin. Trichosanthin-HBP significantly inhibited the growth of tested cancer cells which are impervious to trichosanthin. Tumor cell apoptosis and both the mitochondrial- and death receptor-mediated apoptotic signaling pathways induced by trichosanthin-HBP were more significant than those induced by trichosanthin in HeLa cells. Conclusion: HBP is an efficient intracellular delivery vehicle for trichosanthin and makes trichosanthin-HBP become a promising agent for cancer therapy. © 2016 Springer Science+Business Media Dordrecht


Patent
Zhejiang Reachall Pharmaceutical Co. | Date: 2011-10-26

Provided are fusion proteins of apoptin-protein transduction domain of carboxyl-terminus of EC-SOD, their encoding genes, the recombinant vectors containing said encoding genes, transformants containing said recombinant vectors, as well as the uses and preparation methods thereof. Especially provided are fusion proteins which fuse protein transduction domain of carboxyl-terminus of EC-SOD in the amino acid sequence of SEQ ID NO:1 or its mutants and apoptin in the amino acid sequence of SEQ ID NO:2 or its mutants, said fusion proteins have strong ability of inducing apoptosis of tumor cells, and can be used for manufacturing of medicament for the treatment of tumors.


PubMed | East China University of Science and Technology, Zhejiang Reachall Pharmaceutical Co. and Shanghai University of Traditional Chinese Medicine
Type: | Journal: Protein expression and purification | Year: 2015

MAP30 (Momordica Antiviral Protein 30 Kd), a single-stranded type-I ribosome inactivating protein, possesses versatile biological activities including anti-tumor abilities. However, the low efficiency penetrating into tumor cells hampers the tumoricidal effect of MAP30. This paper describes MAP30 fused with a human-derived cell penetrating peptide HBD which overcome the low uptake efficiency by tumor cells and exhibits higher anti-tumor bioactivity. MAP30 gene was cloned from the genomic DNA of Momordica charantia and the recombinant plasmid pET28b-MAP30-HBD was established and transferred into Escherichia coli BL21 (DE3). The recombinant MAP30-HBD protein (rMAP30-HBD) was expressed in a soluble form after being induced by 0.5mM IPTG for 14h at 15C. The recombinant protein was purified to greater than 95% purity with Ni-NTA affinity chromatography. The rMAP30-HBD protein not only has topological inactivation and protein translation inhibition activity but also showed significant improvements in cytotoxic activity compared to that of the rMAP30 protein without HBD in the tested tumor cell lines, and induced higher apoptosis rates in HeLa cells analyzed by Annexin V-FITC with FACS. This paper demonstrated a new method for improving MAP30 protein anti-tumor activity and might have potential applications in cancer therapy area.


PubMed | East China University of Science and Technology and Zhejiang Reachall Pharmaceutical Co.
Type: Journal Article | Journal: Journal of peptide science : an official publication of the European Peptide Society | Year: 2016

Cell-penetrating peptides (CPPs) have been shown to be potential drug carriers for cancer therapy. The inherently low immunogenicity and cytotoxicity of human-derived CPPs make them more suitable for intracellular drug delivery compared to other delivery vehicles. In this work, the protein transduction ability of a novel CPP (termed HBP) derived from the heparin-binding domain of HB-EGF was evaluated. Our data shows, for the first time, that HBP possesses similar properties to typical CPPs and is a potent drug delivery vector for improving the antitumor activity of impermeable MAP30. The intrinsic bioactivities of recombinant MAP30-HBP were well preserved compared to those of free MAP30. Furthermore, HBP conjugated to the C-terminus of MAP30 promoted the cellular uptake of recombinant MAP30-HBP. Moreover, the fusion of HBP to MAP30 gave rise to significantly enhanced cytotoxic effects in all of the tumor cell lines tested. In HeLa cells, this cytotoxicity was mainly caused by the induction of cell apoptosis. Further investigation revealed that HBP enhanced MAP30-induced apoptosis through the activation of the mitochondrial- and death receptor-mediated signaling pathways. In addition, the MAP30-HBP fusion protein caused more HeLa cells to become arrested in S phase compared to MAP30 alone. These results highlight the MAP30-HBP fusion protein as a promising drug candidate for cancer therapy and demonstrate HBP, a novel CPP derived from human HB-EGF, as a new potential vector for antitumor drug delivery. Copyright 2016 European Peptide Society and John Wiley & Sons, Ltd.


PubMed | East China University of Science and Technology and Zhejiang Reachall Pharmaceutical Co.
Type: Journal Article | Journal: Biotechnology letters | Year: 2016

To evaluate the anti-tumor effects of trichosanthin after fusion with a cell penetrating peptide, heparin-binding peptide (HBP), derived from human heparin-binding EGF-like growth factor (HB-EGF).The fusion protein of trichosanthin-HBP was expressed in Escherichia coli BL21 and purified by Ni-NTA affinity chromatography. The HBP domain had no influence on the topological inactivation activity and N-glycosidase activity of trichosanthin. Trichosanthin-HBP significantly inhibited the growth of tested cancer cells which are impervious to trichosanthin. Tumor cell apoptosis and both the mitochondrial- and death receptor-mediated apoptotic signaling pathways induced by trichosanthin-HBP were more significant than those induced by trichosanthin in HeLa cells.HBP is an efficient intracellular delivery vehicle for trichosanthin and makes trichosanthin-HBP become a promising agent for cancer therapy.

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