title: Efficient production of native lunasin with correct N-terminal processing by using the pH-induced self-cleavable Ssp DnaB mini-intein system in Escherichia coli.
abstract: To develop an efficient and cost-effective approach for the production of small preventive peptide lunasin with correct natural N terminus, a synthetic gene was designed by OPTIMIZER & Gene Designer and cloned into pTWIN1 vector at SapI and PstI sites. Thus, lunasin was N-terminally fused to the pH-induced self-cleavable Ssp DnaB mini-intein linked to a chitin binding domain (CBD) with no extra residues. The resultant fusion protein was highly expressed by lactose induction in Escherichia coli BL21 (DE3) in a 7-l bioreactor and bound to a chitin affinity column. After washing the impurities, the Ssp DnaB intein mediated on-column self-cleavage was easily triggered by shifting pH and temperature to allow the native lunasin released. The final purified lunasin yielded up to 75 mg/l medium. Tricine/SDS-PAGE and matrix-assisted laser desorption time-of-flight (MALDI-TOF)/mass spectrometry (MS) verified the structural authenticity of the product, implying the correct cleavage at the junction between Ssp DnaB intein and lunasin. MTT assay confirmed its potent proliferation inhibitory activity to human cancer cells HCT-116 and MDA-MB-231; however, no cytotoxicity to normal human lens epithelial cell SRA01/04 and hepatoma HepG2. Taken together, we provide a novel strategy to produce recombinant native lunasin with correct N-terminal processing by using the pH-induced self-cleavable Ssp DnaB mini-intein.
authors.affiliation: State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, 211# mailbox, 24 Tongjia Street, Nanjing, 210009, China.
authors.forename: Sarra
authors.initials: S
authors.lastname: Setrerrahmane
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Guangzhi
authors.initials: G
authors.lastname: Dai
authors.forename: Jing
authors.initials: J
authors.lastname: Lv
authors.forename: Shuhua
authors.initials: S
authors.lastname: Tan
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D053488:DnaB Helicases / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D047668:Inteins; D016133:Polymerase Chain Reaction; D030262:Soybean Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D017931:DNA Primers; C120514:GM2S-1 protein, soybean; D030262:Soybean Proteins; C506730:dnaB protein, E coli; D053488:DnaB Helicases