title: Expression analysis and ATR-FTIR characterization of the secondary structure of recombinant human TNF-α from Escherichia coli SHuffle®T7 Express and BL21 (DE3) cells.
abstract: TNF-α, a prototype member of the TNF family of ligands, has both pro-inflammatory and immune-regulatory functions, which make it as an appropriate therapeutic target for selective blockade in antibody therapy of many diseases like in rheumatoid arthritis. Using two models of SHuffle®T7 Express and BL21 (DE3) cells, we have expressed this protein and recognized it by SDS-PAGE analysis. FTIR biospectroscopy of the resulted purified proteins has been performed and mathematical calculations has been done for further identification of the structural and conformational differences between the two products. Our results showed some differences in disulfide bond formation and β-sheet turns between these two recombinant proteins. To the best of our knowledge, this is the first study that compare secondary structure of recombinant proteins in both conventional and next generation Escherichia coli based expression systems using reliable, simple, rapid and economic ATR-FTIR analysis. Whether these differences might have significant effects on TNF-α inflammatory and immune-regulatory function in biological systems might be very much important and need further investigations.
authors.affiliation: Pharmaceutical Sciences Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Hossein
authors.initials: H
authors.lastname: Safarpour
authors.affiliation: Pharmaceutical Sciences Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Sahar Barzegari
authors.initials: SB
authors.lastname: Banadkoki
authors.affiliation: Department of Toxicology, School of Pharmacy, Shahid Beheshti University of Medical Science, Tehran, Iran.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Keshavarzi
authors.affiliation: Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: Mohammad Hossein
authors.initials: MH
authors.lastname: Morowvat
authors.affiliation: Biochemistry Department, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran.
authors.forename: Mahdieh
authors.initials: M
authors.lastname: Soleimanpour
authors.affiliation: Biochemistry Department, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran.
authors.forename: Shokoufeh
authors.initials: S
authors.lastname: Pourmolaei
authors.affiliation: Pharmaceutical Sciences Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran; Department of Toxicology, School of Pharmacy, Shahid Beheshti University of Medical Science, Tehran, Iran. Electronic address: F.shirazi@sbmu.ac.ir.
authors.forename: Farshad H
authors.initials: FH
authors.lastname: Shirazi
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D017550:Spectroscopy, Fourier Transform Infrared; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha
keywords: BL21 (DE3); E. coli expression; FTIR structural analysis; SHuffle(®) T7 Express; TNF-α