The human interferon alpha 2b (IFN α2b) belongs to the interferon family of cytokines that exerts many biological functions like inhibition of virus multiplication, repression of tumour growth and other immunological functions. Herein, a synthetic gene coding for human IFN α2b was cloned and integrated into a methylotropic yeast—Pichiapastoris. The recombinant human IFN α2b protein (∼19 kDa) could be successfully expressed in Pichiapastoris to a level of nearly 300 mg/L with nearly 93% recovery on purification using a single anion exchange chromatography step. A novel media component dimethyl sulphoxide (DMSO) was found to aid in expression of rightly processed IFN α2... More
The human interferon alpha 2b (IFN α2b) belongs to the interferon family of cytokines that exerts many biological functions like inhibition of virus multiplication, repression of tumour growth and other immunological functions. Herein, a synthetic gene coding for human IFN α2b was cloned and integrated into a methylotropic yeast—Pichiapastoris. The recombinant human IFN α2b protein (∼19 kDa) could be successfully expressed in Pichiapastoris to a level of nearly 300 mg/L with nearly 93% recovery on purification using a single anion exchange chromatography step. A novel media component dimethyl sulphoxide (DMSO) was found to aid in expression of rightly processed IFN α2b form with dramatic reduction in the expression of a 20 kDa IFN isoform contaminant frequently observed by other workers. The identity of the 20 kDa isoform was confirmed by N terminal sequencing which showed extra eleven amino acids at the N terminal portion of the IFN molecule obtained due to incorrect processing by the host KEX2 protease. The purified IFN α2b (19 kDa) preparation was confirmed by N terminal sequencing, and characterized by MALDI-TOF and Agilent 2100 Bioanalyzer. The bioassay of the recombinant protein gave a specific activity of >2 × 108 IU/mg.
