Trichosanthin is a valuable protein in medical applications. An NMR analytical of the trichosanthin solution conformation is made to study the structural-functional relationship of this protein. For preparing a sampl...Trichosanthin is a valuable protein in medical applications. An NMR analytical of the trichosanthin solution conformation is made to study the structural-functional relationship of this protein. For preparing a sample labled with 15 N, the tcs gene was cloned into the expression vector pQE 30, and produced a high level expression of trichosanthin in the E coli strain M15. The amount of fusion TCS synthesized in E coli was about 6 4% of the total cellular protein. The fusion protein was purified by using affinity chromatography with Ni NTA resin.展开更多
An essential step for cancer vaccination is to break the immunosuppression and elicit a tumor-specific immunity.A major hurdle against cancer therapeutic vaccination is the insufficient immune stimulation of the cance...An essential step for cancer vaccination is to break the immunosuppression and elicit a tumor-specific immunity.A major hurdle against cancer therapeutic vaccination is the insufficient immune stimulation of the cancer vaccines and lack of a safe and efficient adjuvant for human use.We discovered a novel cancer immunostimulant,trichosanthin(TCS),that is a clinically used protein drug in China,and developed a well-adaptable protein-engineering method for making recombinant protein vaccines by fusion of an antigenic peptide,TCS,and a cell-penetrating peptide(CPP),termed an"allin-one"vaccine,for transcutaneous cancer immunization.The TCS adjuvant effect on antigen presentation was investigated and the antitumor immunity of the vaccines was investigated using the different tumor models.The vaccines were prepared via a facile recombinant method.The vaccines induced the maturation of DCs that subsequently primed CD8^(+)T cells.The TCS-based immunostimulation was associated with the STING pathway.The general applicability of this genetic engineering strategy was demonstrated with various tumor antigens(i.e.,legumain and TRP2 antigenic peptides)and tumor models(i.e.,colon tumor and melanoma).These findings represent a useful protocol for developing cancer vaccines at low cost and time-saving,and demonstrates the adjuvant application of TCSdan old drug for a new application.展开更多
β-Trichosanthin, a type 1 ribosome-inactivating protein （RIP） isolated from the root tuber of Trichosanthe kirilowii Maxim, is an isoform of trichosanthin. Here we report its crystallization in two crystal forms us...β-Trichosanthin, a type 1 ribosome-inactivating protein （RIP） isolated from the root tuber of Trichosanthe kirilowii Maxim, is an isoform of trichosanthin. Here we report its crystallization in two crystal forms using the hanging-drop vapor-diffusion method. The form A and form B crystals belong to the orthorhombic space group P212121 and monoclinic space group P21, respectively. X-ray data have been collected to 1.6 and 1.2 A resolution for form A and form B crystals, respectively, using a synchrotron source.展开更多
The crystal structure of trichosanthin (TCS) in space group C2 hasbeen refined by PROLSQ and XPLOR to an R-factor of 0. 204 for 33, 531 reflectionswith F。≥2-σ(F。) between 6 to 1. 9 A resolution. The root mean squa...The crystal structure of trichosanthin (TCS) in space group C2 hasbeen refined by PROLSQ and XPLOR to an R-factor of 0. 204 for 33, 531 reflectionswith F。≥2-σ(F。) between 6 to 1. 9 A resolution. The root mean square(r. m. s. ) devi-ations of bond lengths and bond angles from the standard values are 0. 015 A and 2.77°, respectively. The overall fold of the molecule of TCS is, in general, similar to oth-er RIPs, but there are some differences in secondary structure and in the active sitecleft. An overlay of the two molecules (shown by Mol. A and Mol. B), which are notrelated by symmetry in an asymmetric unit, results in an r. m. s. deviation of 0. 850Afor the main chain atoms. The backbones of the C-terminus of the two molecules arequite different. The structures of the water in two molecules are not completely analo-gous. In the active site cleft of Mol. B, the bonding sites of three water molecules toprotein are similar to those of N atoms of formycin ring in the structure of pokeweedantiviral protein complexed with formycin 5-monophosphate, and the side chains of twoTyr70 in the two crystalline forms of TCS show a similar orientation, which is differentfrom that of the corresponding residue in ricin A-chain.展开更多
文摘Trichosanthin is a valuable protein in medical applications. An NMR analytical of the trichosanthin solution conformation is made to study the structural-functional relationship of this protein. For preparing a sample labled with 15 N, the tcs gene was cloned into the expression vector pQE 30, and produced a high level expression of trichosanthin in the E coli strain M15. The amount of fusion TCS synthesized in E coli was about 6 4% of the total cellular protein. The fusion protein was purified by using affinity chromatography with Ni NTA resin.
基金support of National Key Research and Development Program of China(2021YFE0103100,China)National Natural Science Foundation of China of China(81925035,81673382,and 81521005,China)+3 种基金National Special Project for Significant New Drugs Development(2018ZX09711002-010-002,China)Shanghai SciTech Innovation Initiative(19431903100,18430740800,China)Shanghai Collaborative Innovation Group of Early Diagnosis and Precise Treatment of Hemangiomas and Vascular Malformations(SSMU-ZDCX20180701,China)Chinese Pharmaceutical Association-Yiling Pharm Joint Grants(CPAYLJ201901,China)for the support。
文摘An essential step for cancer vaccination is to break the immunosuppression and elicit a tumor-specific immunity.A major hurdle against cancer therapeutic vaccination is the insufficient immune stimulation of the cancer vaccines and lack of a safe and efficient adjuvant for human use.We discovered a novel cancer immunostimulant,trichosanthin(TCS),that is a clinically used protein drug in China,and developed a well-adaptable protein-engineering method for making recombinant protein vaccines by fusion of an antigenic peptide,TCS,and a cell-penetrating peptide(CPP),termed an"allin-one"vaccine,for transcutaneous cancer immunization.The TCS adjuvant effect on antigen presentation was investigated and the antitumor immunity of the vaccines was investigated using the different tumor models.The vaccines were prepared via a facile recombinant method.The vaccines induced the maturation of DCs that subsequently primed CD8^(+)T cells.The TCS-based immunostimulation was associated with the STING pathway.The general applicability of this genetic engineering strategy was demonstrated with various tumor antigens(i.e.,legumain and TRP2 antigenic peptides)and tumor models(i.e.,colon tumor and melanoma).These findings represent a useful protocol for developing cancer vaccines at low cost and time-saving,and demonstrates the adjuvant application of TCSdan old drug for a new application.
基金the NNSF of China (No.39970872,No.30772587)the Natural Science Foundation of Fujian Province (C97052,C0510012)+1 种基金Special Fund of Fujian Development and Reform Commissionthe National Science Foundation of USA
文摘β-Trichosanthin, a type 1 ribosome-inactivating protein （RIP） isolated from the root tuber of Trichosanthe kirilowii Maxim, is an isoform of trichosanthin. Here we report its crystallization in two crystal forms using the hanging-drop vapor-diffusion method. The form A and form B crystals belong to the orthorhombic space group P212121 and monoclinic space group P21, respectively. X-ray data have been collected to 1.6 and 1.2 A resolution for form A and form B crystals, respectively, using a synchrotron source.
文摘The crystal structure of trichosanthin (TCS) in space group C2 hasbeen refined by PROLSQ and XPLOR to an R-factor of 0. 204 for 33, 531 reflectionswith F。≥2-σ(F。) between 6 to 1. 9 A resolution. The root mean square(r. m. s. ) devi-ations of bond lengths and bond angles from the standard values are 0. 015 A and 2.77°, respectively. The overall fold of the molecule of TCS is, in general, similar to oth-er RIPs, but there are some differences in secondary structure and in the active sitecleft. An overlay of the two molecules (shown by Mol. A and Mol. B), which are notrelated by symmetry in an asymmetric unit, results in an r. m. s. deviation of 0. 850Afor the main chain atoms. The backbones of the C-terminus of the two molecules arequite different. The structures of the water in two molecules are not completely analo-gous. In the active site cleft of Mol. B, the bonding sites of three water molecules toprotein are similar to those of N atoms of formycin ring in the structure of pokeweedantiviral protein complexed with formycin 5-monophosphate, and the side chains of twoTyr70 in the two crystalline forms of TCS show a similar orientation, which is differentfrom that of the corresponding residue in ricin A-chain.
