Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To increase the selection efficiency and productivity of stable clones expressing recombinant antibody-IL-2 fusion protein, a dicistronic expression vector containing the anti-erbB2 scFv-Fc-IL-2 fusion gene followed by a weakened neo gene, was constructed to allow for the concurrent translation of the recombinant protein and mutated neomycin phosphotransferase from a single mRNA. The presence of the mutant enzyme in the transfectomas resulted in a decreased resistance of cells in the presence of an elevated level of G418 and retarded cell growth. The transfectomas containing the mutant enzyme expressed considerably higher levels of the fusion protein than those containing the normal enzyme. Furthermore, these positive clones had an almost identical level of recombinant gene expression, which was very stable even when the concentration of G418 was significantly increased. Thus, the selection efficiency of strongly positive producers was remarkably increased. Our results demonstrate that the dicistronic expression vector is useful for the selection of highly expressing clones. Combined with an amplification system, this vector may have potential usage for the expression of recombinant antibody and the yield may be further improved by this expression system.
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PMID:Highly efficient selection of the stable clones expressing antibody-IL-2 fusion protein by a dicistronic expression vector containing a mutant neo gene. 1562 10

The administration of antibodies against the cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is a promising approach in the upregulation of immune responses in many cancers and infectious diseases. The single-chain variable fragment of antibody against CTLA4 is also useful in developing immunotoxins that might be used in the treatment of cancer, transplant rejection, and autoimmune diseases. Here, we report the production of a soluble and functional scFv antibody against CTLA4 by using Pichia pastoris as the expression system. The gene encoding scFv hS83 with an additional 6His-tag at the 5'-end was inserted into the expression vector pPIC9K. Then, the transformants were double-screened on plates containing 0.25 mg/mL and 1.5 mg/mL of neomycin G418 and many clones with different levels of G418-resistance were selected for further studies on expression. After induction by the addition of methanol, various levels of hS83 were detected in the supernatant of P. pastoris containing pPIC9K-hS83. Clones with low G418-resistance produced more hS83 than those with higher G418-resistance. Under the optimized conditions (initial inoculum, 40 A(600nm) AU/mL; pH 6.0; methanol concentration, 3.0%; induction time, 72 h), approximately 16-20 mg protein could be recovered from 1 L of the culture. The purified hS83 had a stronger binding ability towards CTLA4-positive Raji cells than CTLA4-negative ECV304 cells. This finding indicates that the antibody produced by P. pastoris is functional and may be used in immunotherapy for cancer, infection, transplant rejection, and autoimmune diseases.
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PMID:High-level expression of a functional humanized anti-CTLA4 single-chain variable fragment antibody in Pichia pastoris. 1893 37