A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide
Periodo de realización: 1900/01/01 al 2015/01/01
Tipo: Artículo científico
Lugar(es) de estudio: Av. Universidad 2001, Chamilpa, 62210 Cuernavaca, Morelos, México, 2431 Veerle, Bélgica
Resumen: "Background: The choice between heterologous expression versus chemical synthesis for synthesizing shortcysteine-rich insecticidal peptides from arthropods may impact the obtainment of yields and well-folded bioactivemolecules for scientific research. Therefore, two recombinant expression systems were compared to that of chemicalsynthesis 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 ofthe spider Brachypelma albiceps. It was cloned into the pCR®2.1-TOPO® cloning vector and then introduced in twodifferent 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 Ba1was 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 HisrBa1presented respective molecular masses of 28,289 and 8274.6 Da, and were not biologically active. These resultssuggested that both HisrDFHRBa1 and HisrBa1 were oxidized after cell extraction, and that their insecticidal activitieswere affected by their N-terminal pro-peptides and different disulfide bridge arrangements. The respective proteinexpression yields for HisrDFHRBa1 and HisrBa1 were 100 µg/L and 900 µg/L of culture medium. HisrBa1 was reducedand folded under in vitro conditions. The in vitro folding of HisrBa1 produced several isoforms, one of which, afterremoving its N-terminal pro-peptide by enzymatic cleavage, presented elevated insecticidal activities compared to thenative Ba1. Furthermore, the His-tagged protein HisrDFHRBa1 underwent enzymatic cleavage to obtain recombinantBa1 (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 thenative Ba1"