Recombinant GH5_2 Protein Expression
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Corresponding Organization : Max Planck Institute for Chemical Ecology
Other organizations : Canadian Forest Service, Natural Resources Canada
Variable analysis
- Amplification of open reading frames (ORFs) of GH5_2, excluding the stop codon, by polymerase chain reaction (PCR) using RACE-ready cDNA
- Addition of a Kozak sequence at the 5'-end of the PCR product by integrating it into the forward PCR primer
- Cloning of the resulting PCR products into pIB/V5-His TOPO/TA vector in an ORF with a V5-(His)6 epitope at the carboxyl-terminus
- Obtaining codon-optimized synthetic constructs of RBIC5 and RBIC9 cloned into pIB/V5-His TOPO/TA from GenScript
- Expression of recombinant GH5_2 proteins in Sf9 insect cells
- Recovery of recombinant GH5_2 proteins by immunoprecipitation using anti-V5 agarose beads
- Cultured Sf9 insect cells in SF-900 II serum-free medium
- Transfection of Sf9 cells using FUGENE HD as the transfection reagent
- No positive or negative controls were explicitly mentioned in the provided information.
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