Strategies to maximize expression of rightly processed human interferon alpha2b in Pichia pastoris.
The human interferon alpha 2b (IFN alpha2b) 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 alpha2b was cloned and integrated into a methylotropic yeast-Pichiapastoris. The recombinant human IFN alpha2b protein (approximately 19kDa) could be successfully expressed in Pichiapastoris to a level of nearly 300mg/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 alpha2b form with dramatic reduction in the expression of a 20kDa IFN isoform contaminant frequently observed by other workers. The identity of the 20kDa 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 alpha2b (19kDa) 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 >2x10(8)IU/mg.
Salunkhe
Shardul
S
Lupin Limited, Biotechnology R & D, Gat #. 1156, Village Ghotawade, Taluka Mulshi, Pune, India.
Soorapaneni
Sudheerbabu
S
Prasad
Ketaki Sabnis
KS
Raiker
Veena A
VA
Padmanabhan
Sriram
S
D001681:Biological Assay; D002845:Chromatography; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D007372:Interferons / Q000235:genetics / Q000378:metabolism*; D008297:Male; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D016207:Cytokines; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D007372:Interferons