Signal peptide capable of efficiently directing many protein secretion in mammalian cells is one of the key elements in recombinant protein production,gene therapy and the development of DNA vaccines.In order to explo...Signal peptide capable of efficiently directing many protein secretion in mammalian cells is one of the key elements in recombinant protein production,gene therapy and the development of DNA vaccines.In order to explore the possibility of rat growth hormone signal peptide as such an element,a new vector based on the mammalian expression vector pcDNA3 was constructed by employing rat growth hormone(rGH) signal peptide as leading sequence,followed by multiple cloning sites,the myc epitope-tag and 6×his purification tag in the expression cassette.The vector was validated by successfully expressing and secretion of chick MMP-2 C-terminal PEX domain,a potential angiogenesis inhibitor,and tandem peptide repeats of myc epitope-tag in COS-7 cells.These results suggest that rat growth hormone signal peptide is effective in the mediation of recombinant protein expression and secretion,and this vector provides a new tool for universal cloning and secretion of exogenous proteins in mammalian cells.展开更多
The wild-type human Fas-associated death domain （FADD） protein was expressed as a His-tag fusion protein in Escherichia coll. Recombinant FADD proteins were purified under the denatured condition. After denatured pr...The wild-type human Fas-associated death domain （FADD） protein was expressed as a His-tag fusion protein in Escherichia coll. Recombinant FADD proteins were purified under the denatured condition. After denatured protein purification, it was refolded and obtained at a yield of about 23 mg/L. Purified FADD exhibited as a homogenous band corresponding to the molecular weight of 31 kDa. Immunization of rabbits against the refolded FADD protein was allowed the production of high titre polyclonal antiserum. This new polyelonal antibody could recognize recombinant FADD protein in Western blot. Immunoreactivity was also observed in immunofiuorescence assay. The low cost polyclonal antiserum was applicable to extensive detection of FADD in various immunoassays. Cellular ＆ Molecular Immunology. 2008;5（6）：471-474.展开更多
文摘Signal peptide capable of efficiently directing many protein secretion in mammalian cells is one of the key elements in recombinant protein production,gene therapy and the development of DNA vaccines.In order to explore the possibility of rat growth hormone signal peptide as such an element,a new vector based on the mammalian expression vector pcDNA3 was constructed by employing rat growth hormone(rGH) signal peptide as leading sequence,followed by multiple cloning sites,the myc epitope-tag and 6×his purification tag in the expression cassette.The vector was validated by successfully expressing and secretion of chick MMP-2 C-terminal PEX domain,a potential angiogenesis inhibitor,and tandem peptide repeats of myc epitope-tag in COS-7 cells.These results suggest that rat growth hormone signal peptide is effective in the mediation of recombinant protein expression and secretion,and this vector provides a new tool for universal cloning and secretion of exogenous proteins in mammalian cells.
基金financially supported by the following funds to Zi-Chun Hua: the National Nature Science Foundation of China (No. 30425009, 30600320, 30730030, 30330530, 30270291)Jiangsu Provincial Nature Sciences Foundation (No. BK2007715)Jiangsu Provincial Department of Health (No. H200524, H200742)
文摘The wild-type human Fas-associated death domain （FADD） protein was expressed as a His-tag fusion protein in Escherichia coll. Recombinant FADD proteins were purified under the denatured condition. After denatured protein purification, it was refolded and obtained at a yield of about 23 mg/L. Purified FADD exhibited as a homogenous band corresponding to the molecular weight of 31 kDa. Immunization of rabbits against the refolded FADD protein was allowed the production of high titre polyclonal antiserum. This new polyelonal antibody could recognize recombinant FADD protein in Western blot. Immunoreactivity was also observed in immunofiuorescence assay. The low cost polyclonal antiserum was applicable to extensive detection of FADD in various immunoassays. Cellular ＆ Molecular Immunology. 2008;5（6）：471-474.
