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

Appl Biochem Biotechnol. 2014 Sep;174(2):612-22. doi: 10.1007/s12010-014-1081-1. Epub 2014 Aug 2.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • DNA Primers
  • DnaB Helicases / genetics*
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics*
  • Hydrogen-Ion Concentration
  • Inteins*
  • Polymerase Chain Reaction
  • Soybean Proteins / biosynthesis*
  • Soybean Proteins / chemistry
  • Soybean Proteins / genetics
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • DNA Primers
  • GM2S-1 protein, Glycine max
  • Soybean Proteins
  • dnaB protein, E coli
  • DnaB Helicases