Title
High-level expression and efficient refolding of therapeutically important recombinant human Interleukin-3 (hIL-3) in E. coli
Author
Vikas Kumar Dagar, Adivitiya, Yogender Pal Khasa
Year
2017
Journal
Protein Expression and Purification
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.
Instrument
J-815
Keywords
Circular dichroism, Secondary structure, Biochemistry