Cloning of Human Adiponectin Gene by PCR-driven Overlap?Extension and Expression in Pichia pastoris

Abstract:

Gene of human adiponectin (ADPN) was cloned by PCR-driven overlap?extension. The ADPN gene was linked into pGEM-T vector. After the sequence was determined, the ADPN gene was subcloned into expression vector pPIC3.5K to yield the recombinant expression vector pPIC3.5K-ADPN. The recombinant plasmid was transformed into Pichia pastoris GS115 by electroporation, then the recombinant strain was identified by PCR and Southern blotting. After induction by methanol, ADPN was expressed in GS115, then the protein was identified by Western blotting. The results showed that the ADPN was expressed successfully. The optimum conditions of expression were 30°C and 1% methanol inducing 48 h.

Key words:human adiponectin, PCR-driven overlap?extension, P. pastoris, Southern blotting, Western blotting