title: Expression and purification of toxic anti-breast cancer p28-NRC chimeric protein.
abstract: BACKGROUND
  Chimeric proteins consisting of a targeting moiety and a cytotoxic moiety are now under intense research focus for targeted therapy of cancer. Here, we report cloning, expression, and purification of such a targeted chimeric protein made up of p28 peptide as both targeting and anticancer moiety fused to NRC peptide as a cytotoxic moiety. However, since the antimicrobial activity of the NRC peptide would intervene expression of the chimeric protein in Escherichia coli, we evaluated the effects of two fusion tags, that is, thioredoxin (Trx) and 6x-His tags, and various expression conditions, on the expression of p28-NRC chimeric protein.
  
  
  METHODS
  In order to express the chimeric protein with only 6x-His tag, pET28 expression plasmid was used. Cloning in pET32 expression plasmid was performed to add both Trx and 6x-His tags to the chimeric protein. Expression of the chimeric protein with both plasmids was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis following optimization of expression conditions and host strains.
  
  
  RESULTS
  Expression of the chimeric protein in pET28a was performed. However, expression yield of the chimeric protein was low. Optimization of culture conditions and host strains led to reasonable expression yield of the toxic chimeric protein in pET32a vector. In cases of both plasmids, approximately 10 kDa deviation of the apparent molecular weight from the theoretical one was seen in SDS-PAGE of purified chimeric proteins.
  
  
  CONCLUSIONS
  The study leads to proper expression and purification yield of p28-NRC chimeric protein with Trx tag following optimizing culture conditions and host strains.
authors.affiliation: Department of Pharmaceutical Biotechnology, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Meysam
authors.initials: M
authors.lastname: Soleimani
authors.affiliation: Department of Pharmaceutical Biotechnology, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Hamid
authors.initials: H
authors.lastname: Mirmohammad-Sadeghi
authors.affiliation: Department of Medicinal Chemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Hojjat
authors.initials: H
authors.lastname: Sadeghi-Aliabadi
authors.affiliation: Department of Pharmaceutical Biotechnology, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Ali
authors.initials: A
authors.lastname: Jahanian-Najafabadi
keywords: Anticancer peptide; NRC; apparent molecular weight; chimeric protein; p28