title: Bioprocess development for extracellular production of recombinant human interleukin-3 (hIL-3) in Pichia pastoris.
abstract: Human interleukin-3 (hIL-3) is a therapeutically important cytokine involved in the maturation and differentiation of various cells of the immune system. The codon-optimized hIL-3 gene was cloned in fusion with the N-terminus α-mating factor signal peptide of Saccharomyces cerevisiae under an inducible alcohol oxidase 1 (AOX1) and constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. A Zeocin concentration up to 2000 mg/L was used to select hyper-producers. The shake flask cultivation studies in the Pichia pastoris GS115 host resulted a maximum recombinant hIL-3 expression level of 145 mg/L in the extracellular medium under the control of AOX1 promoter. The batch fermentation strategy allowed us to attain a fairly pure glycosylated hIL-3 protein in the culture supernatant at a final concentration of 475 mg/L with a high volumetric productivity of 4.39 mg/L/h. The volumetric product concentration achieved at bioreactor level was 3.28 folds greater than the shake flask results. The 6x His-tagged protein was purified using Ni-NTA affinity chromatography and confirmed further by western blot analysis using anti-6x His tag antibody. The glycosylation of recombinant hIL-3 protein was confirmed in a PNGase F deglycosylation reaction where it showed a molecular weight band pattern similar to E. coli produced non-glycosylated hIL-3 protein. The structural properties of recombinant hIL-3 protein were confirmed by CD and fluorescence spectroscopy where protein showed 40 % α-helix, 12 % β-sheets with an emission maxima at 343 nm. MALDI-TOF-TOF analysis was used to establish the protein identity. The biological activity of purified protein was confirmed by the human erythroleukemia TF-1 cell proliferation assay.
authors: Department of Microbiology, University of Delhi South Campus, New Delhi, 110 021, India.
authors: Vikas Kumar
authors: VK
authors: Dagar
authors: Department of Microbiology, University of Delhi South Campus, New Delhi, 110 021, India.
authors: Adivitiya
authors: Department of Microbiology, University of Delhi South Campus, New Delhi, 110 021, India.
authors: Nirmala
authors: N
authors: Devi
authors: Department of Microbiology, University of Delhi South Campus, New Delhi, 110 021, India. yogi110@gmail.com.
authors: Yogender Pal
authors: YP
authors: Khasa
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics; D019149:Bioreactors; D003062:Codon; D005285:Fermentation; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D007377:Interleukin-3 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D003062:Codon; C508596:IL3 protein, human; D007377:Interleukin-3; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
keywords: Bioprocess optimization; Circular dichroism (CD); Fed-batch fermentation; Human interleukin-3 (hIL-3); Pichia pastoris; TF-1 cell