title: A combinatorial approach of N-terminus blocking and codon optimization strategies to enhance the soluble expression of recombinant hIL-7 in E. coli fed-batch culture.
abstract: Human interleukin-7 (hIL-7) is a therapeutically important cytokine involved in lymphocyte development and survival. In previous reports, a uniformly poor expression of hIL-7 has been shown in Escherichia coli host with the problem of inclusion body formation. In this study, the role of codon optimization and N-terminus blocking using various solubility enhancer fusion tags was explored to improve its soluble expression. The use of codon optimization strategy improved its expression to 80 ± 5 mg/L at shake flask level. The utilization of pelB leader sequence resulted in an unprocessed protein in the form of cytoplasmic inclusion bodies with lower expression yields. The N-terminus fusion of small ubiquitin-like modifier (SUMO), thioredoxin (Trx), and NusA tags increased the expression in the range of 90-140 mg/L, where >90 % of the fusion protein was obtained in soluble form. The fed-batch fermentation of SUMO-tagged hIL-7 protein was optimized at bioreactor level, where a high volumetric product concentration of 2.65 g/L was achieved by controlling the plasmid segregation instability using high antibiotic concentration. The specific product yield (YP/X) and volumetric product concentration were 1.38 and 2.55-fold higher compared to batch results, respectively. A preparative scale affinity chromatography resulted in a high recovery yield of 50.6 mg/L with ∼90 % purity. The conformational property of purified recombinant hIL-7 from CD spectroscopy showed a typical helical structure with 31.5 % α-helix and 26.43 % β-sheet. The biological activity of purified protein was tested using IL-7-dependent murine immature B lymphocyte (2E8) cell line by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide salt (MTT) assay, where it showed a similar biological activity as standard control.
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi, 110021, India.
authors.forename: Nirmala
authors.initials: N
authors.lastname: Devi
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi, 110021, India.
authors.lastname: Adivitiya
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi, 110021, India. yogi110@gmail.com.
authors.forename: Yogender Pal
authors.initials: YP
authors.lastname: Khasa
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects / Q000502:physiology; D061249:Batch Cell Culture Techniques; D019149:Bioreactors / Q000382:microbiology; D049109:Cell Proliferation; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D015851:Interleukin-7 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D013057:Spectrum Analysis
chemical_list: C508605:IL7 protein, human; D015851:Interleukin-7; D011993:Recombinant Fusion Proteins
keywords: Codon optimization; Fed-batch; MTT assay; Plasmid stability; SUMO tag; hIL-7