title: A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide.
abstract: BACKGROUND
  The choice between heterologous expression versus chemical synthesis for synthesizing short cysteine-rich insecticidal peptides from arthropods may impact the obtainment of yields and well-folded bioactive molecules for scientific research. Therefore, two recombinant expression systems were compared to that of chemical synthesis for producing Ba1, a cysteine-rich spider neurotoxin.
  
  
  METHODS
  The transcription of the insecticidal neurotoxin Ba1 was obtained from a cDNA library of venom glands of the spider Brachypelma albiceps. It was cloned into the pCR®2.1-TOPO® cloning vector and then introduced in two different expression vectors, pQE40 and pET28a(+). Each vector was transfected into E. coli M15 and BL21 cells, respectively, and expressed under induction with isopropyl thiogalactoside (IPTG). The chemical synthesis of Ba1 was performed in an Applied Biosystems 433A peptide synthesizer.
  
  
  RESULTS
  Both expression systems pQE40 and pET28a(+) expressed the His-tagged recombinant protein products, HisrDFHRBa1 and HisrBa1, respectively, as inclusion bodies. The recombinant proteins HisrDFHRBa1 and HisrBa1 presented respective molecular masses of 28,289 and 8274.6 Da, and were not biologically active. These results suggested that both HisrDFHRBa1 and HisrBa1 were oxidized after cell extraction, and that their insecticidal activities were affected by their N-terminal pro-peptides and different disulfide bridge arrangements. The respective protein expression yields for HisrDFHRBa1 and HisrBa1 were 100 μg/L and 900 μg/L of culture medium. HisrBa1 was reduced and folded under in vitro conditions. The in vitro folding of HisrBa1 produced several isoforms, one of which, after removing its N-terminal pro-peptide by enzymatic cleavage, presented elevated insecticidal activities compared to the native Ba1. Furthermore, the His-tagged protein HisrDFHRBa1 underwent enzymatic cleavage to obtain recombinant Ba1 (rBa1). As expected, the molecular mass of rBa1 was 4406.4 Da. On the other hand, Ba1 was chemically synthesized (sBa1) with a yield of 11 mg per 0.1 mmol of amino acid assembly.
  
  
  CONCLUSIONS
  The two recombinant insecticidal peptides and the one synthesized chemically were as active as the native Ba1; however, toxin yields differed drastically.
authors.affiliation: Department of Molecular Medicine and Bioprocesses, Institute of Biotechnology, National Autonomous University of Mexico (UNAM), apartado postal 510-3, Cuernavaca, Morelos 61500 Mexico.
authors.forename: Herlinda
authors.initials: H
authors.lastname: Clement
authors.affiliation: Department of Molecular Medicine and Bioprocesses, Institute of Biotechnology, National Autonomous University of Mexico (UNAM), apartado postal 510-3, Cuernavaca, Morelos 61500 Mexico.
authors.forename: Vianey
authors.initials: V
authors.lastname: Flores
authors.affiliation: Department of Molecular Medicine and Bioprocesses, Institute of Biotechnology, National Autonomous University of Mexico (UNAM), apartado postal 510-3, Cuernavaca, Morelos 61500 Mexico.
authors.forename: Elia
authors.initials: E
authors.lastname: Diego-Garcia
authors.affiliation: Department of Food, School of Pharmaceutical and Food Sciences, University of Antioquia (UdeA), Medellín, Colombia.
authors.forename: Ligia
authors.initials: L
authors.lastname: Corrales-Garcia
authors.affiliation: Laboratory of Function-Structure and Protein Engineering, Center for Research on Biotechnology, Autonomous University of Morelos State (UAEM), Cuernavaca, Morelos Mexico.
authors.forename: Elba
authors.initials: E
authors.lastname: Villegas
authors.affiliation: Department of Molecular Medicine and Bioprocesses, Institute of Biotechnology, National Autonomous University of Mexico (UNAM), apartado postal 510-3, Cuernavaca, Morelos 61500 Mexico.
authors.forename: Gerardo
authors.initials: G
authors.lastname: Corzo
keywords: Bacteria; Chemical synthesis; Cysteine-rich peptides; Insecticidal peptides; Protein expression; Theraphosid spider