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Pichia pastoris cells (X33 strain) were transformed with recombinant plasmids by electroporation. X33 transformant selection was performed using a medium containing Zeocin, and successful integration of the CLP gene into the P. pastoris genome was confirmed by PCR. |
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| Protocol tips |
| Pichia pastoris cells (X33 strain) were transformed with recombinant plasmids by electroporation. X33 transformant selection was performed using a medium containing Zeocin, and successful integration of the CLP gene into the P. pastoris genome was confirmed by PCR. |
Publication protocol
Pichia pastoris cells (X33 strain) were transformed with recombinant plasmids by electroporation. X33 transformant selection was performed using a medium containing Zeocin, and successful integration of the CLP gene into the P. pastoris genome was confirmed by PCR. The recombinant TbCLP with C-terminus His-tag was expressed by induction with 0.5% methanol for 24–72 h in 200 ml of Buffered Methanol-complex Medium (BMMY), and then purified by immobilized metal ion affinity chromatography using Protino Ni-NTA agarose (Macherey-Nagel). The protein samples were analyzed qualitatively by SDS PAGE and Western blotting, and their concentration was measured using Pierce BCA Protein Assay Kit (Thermo Scientific).
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