This study aimed to investigate the synergism of the TAT PTD （ protein transduction domain in HIV-1 transactivator of transcription protein） to antibacte- rial peptide tachyplesin from Tachp/eus tr/dentatus. Treated...This study aimed to investigate the synergism of the TAT PTD （ protein transduction domain in HIV-1 transactivator of transcription protein） to antibacte- rial peptide tachyplesin from Tachp/eus tr/dentatus. Treated with Pichia pastoris preferred codon optimization, using the eDNA sequence of tuchyplesin mature pep- tide （54 aa） harboring TAT FFD sequence （11 aa） as reference template, six single-stranded oligonueleotides were designed, the sequences of restriction sites EcoR I and Xba I were introduced to the 5＇ end of primers P1 and P6, respectively. TAT PTD ＋ Tachyplesin fusion gene with a full length of 219 bp was artificially synthesized by overlap extension PCR, which laid the preliminary foundation for subsequent functional and synergism studies.展开更多
CD59 may inhibit the cytolytic activity of complement by binding to C8/C9 and protect host cell membranes against homologous membrane attack complex （MAC）. However, CD59 is widely overexpressed on tumor cells, which...CD59 may inhibit the cytolytic activity of complement by binding to C8/C9 and protect host cell membranes against homologous membrane attack complex （MAC）. However, CD59 is widely overexpressed on tumor cells, which has been implicated in tumorigenesis. The active site of CD59 relative to MAC is still confused. As reported the MAC binding site is located in the vicinity of a hydrophobic groove on the membrane distal face of the protein centered around residue W40. Here two site-directed mutagenesis were performed by overlapping extension PCR to delete residue W40 site （Mutant 1, M1） or to change C39W40K41 to W39W40W41 （Mutant 2, M2）. Then we constructed mutant CD59 eukaryotic expression system and investigated their biological function on CI-IO cells compared with wild-type CD59. Stable populations of CHO cells expressing recombinant proteins were screened by immunotechnique. After 30 passages culturing, proteins could be tested. Dye release assays suggest that M1CD59 loses the activity against complement, while M2CD59 increases the anti-complement activity slightly. Results indicate that W40 of human CD59 is important to its activity, and prohibition of this site may be a potential way to increase complement activity and to treat tumors. Cellular ＆ Molecular Immunology.展开更多
基金Supported by the National High Technology Research and Development Program of China(863Program)(2006AA03Z451)Shanghai Basic Research Key Program(10JC1400300)+1 种基金National Natural Science Foundation of China(31070698)the Fundamental Research Funds for the Central Universities(12D10520)~~
基金Supported by Natural Science Foundation of Guangdong Province(06025389)Post-doctoral Project of Fujian Province
文摘This study aimed to investigate the synergism of the TAT PTD （ protein transduction domain in HIV-1 transactivator of transcription protein） to antibacte- rial peptide tachyplesin from Tachp/eus tr/dentatus. Treated with Pichia pastoris preferred codon optimization, using the eDNA sequence of tuchyplesin mature pep- tide （54 aa） harboring TAT FFD sequence （11 aa） as reference template, six single-stranded oligonueleotides were designed, the sequences of restriction sites EcoR I and Xba I were introduced to the 5＇ end of primers P1 and P6, respectively. TAT PTD ＋ Tachyplesin fusion gene with a full length of 219 bp was artificially synthesized by overlap extension PCR, which laid the preliminary foundation for subsequent functional and synergism studies.
基金National Natural Science Foundation of China （No.30671936）.
文摘CD59 may inhibit the cytolytic activity of complement by binding to C8/C9 and protect host cell membranes against homologous membrane attack complex （MAC）. However, CD59 is widely overexpressed on tumor cells, which has been implicated in tumorigenesis. The active site of CD59 relative to MAC is still confused. As reported the MAC binding site is located in the vicinity of a hydrophobic groove on the membrane distal face of the protein centered around residue W40. Here two site-directed mutagenesis were performed by overlapping extension PCR to delete residue W40 site （Mutant 1, M1） or to change C39W40K41 to W39W40W41 （Mutant 2, M2）. Then we constructed mutant CD59 eukaryotic expression system and investigated their biological function on CI-IO cells compared with wild-type CD59. Stable populations of CHO cells expressing recombinant proteins were screened by immunotechnique. After 30 passages culturing, proteins could be tested. Dye release assays suggest that M1CD59 loses the activity against complement, while M2CD59 increases the anti-complement activity slightly. Results indicate that W40 of human CD59 is important to its activity, and prohibition of this site may be a potential way to increase complement activity and to treat tumors. Cellular ＆ Molecular Immunology.
