A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide

• Published in: Journal of Venomous Animals and Toxins including Tropical Diseases
• Main Authors: Herlinda Clement, Vianey Flores, Elia Diego-Garcia, Ligia Corrales-Garcia, Elba Villegas, Gerardo Corzo
• Format: Article in Journal/Newspaper
• Language: English
• Published: SciELO 2015
• Subjects: Insecticidal peptides; Protein expression; Bacteria; Theraphosid spider; Chemical synthesis; Cysteine-rich peptides; Arctic medicine. Tropical medicine; RC955-962; Toxicology. Poisons; RA1190-1270; Zoology; QL1-991; Arctic
• Online Access: https://doi.org/10.1186/s40409-015-0018-7; https://doaj.org/article/6c94e2b6eec5436586dab9ab1b07453d

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 ...