title: High-level expression and efficient refolding of therapeutically important recombinant human Interleukin-3 (hIL-3) in E. coli.
abstract: Human interleukin-3 (hIL-3) is a pleiotropic cytokine that stimulates the differentiation and proliferation of multipotent hematopoietic cells thus making it a therapeutically important molecule. In this study, its poor expression yield was improved by addressing various upstream bottlenecks in E. coli heterologous system. The codon-optimized hIL-3 gene was cloned under various signal sequences and solubility enhancer fusion tags for its hyper-expression under a strong T7 promoter. The optimization of shake flask expression studies resulted in a hIL-3 protein concentration of 225 mg/L in the form of inclusion bodies (IBs). Lowering of inducer concentration and cultivation temperature did not improve its solubility. The hIL-3 protein was refolded from IBs and resulted a protein recovery yield of 53% after optimization of refolding conditions. The refolded protein was subsequently purified using Ni-NTA affinity chromatography and gave ∼95% pure protein. The conformational properties of the refolded hIL-3 protein were studied by CD and fluorescence spectrometry where protein showed 40% α-helix and 12% β-sheets with a fluorescence emission maxima at 344 nm. The molecular identity was further confirmed by MALDI-TOF/TOF and western blot analysis. The biological activity of refolded protein was confirmed via cell proliferation assay on human erythroleukemia TF-1 cells where commercial hIL-3 was taken as a standard control.
authors.affiliation: Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110 021, India.
authors.forename: Vikas Kumar
authors.initials: VK
authors.lastname: Dagar
authors.affiliation: Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110 021, India.
authors.lastname: Adivitiya
authors.affiliation: Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110 021, India. Electronic address: yogi110@gmail.com.
authors.forename: Yogender Pal
authors.initials: YP
authors.lastname: Khasa
mesh_terms: D002460:Cell Line; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007377:Interleukin-3 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: C508596:IL3 protein, human; D007377:Interleukin-3; D011994:Recombinant Proteins
keywords: Circular dichroism; Codon-optimization; Escherichia coli; Human interleukin-3 (hIL-3); Refolding; TF-1 cells