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

Abstract

BackgroundThe 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.MethodsThe 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.ResultsBoth 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.ConclusionsThe two recombinant insecticidal peptides and the one synthesized chemically were as active as the native Ba1; however, toxin yields differed drastically.

Synthetic hepcidin from fish: Uptake and protection against Vibrio anguillarum in sea bass (Dicentrarchus labrax)

“…Thus, the development of techniques in peptide chemical synthesis has been a great advance for large-scale use of bioactive peptides [44]. Synthesis of cysteine rich peptides like hepcidin are more biologically active molecules than the peptides produced by the recombinant system [45]. In addition, oxidative folding is a key step for the four disulfide bonds of hepcidin to generate a tightly folded peptide [46].…”

“…In chemical synthesis these disulfide bonds are usually performed in a one-step oxidative procedure that is sufficient for the correct peptide folding. Consequently, solid-phase peptide synthesis is more attractive for producing this molecule and is less expensive and time-consuming compared to heterologous expression [45].…”

Successful refolding and NMR structure of rMagi3: A disulfide‐rich insecticidal spider toxin

“…Since uncontrolled disulfide‐bond oxidative scramble of cysteine‐rich arachnid toxins could occur during bacterial expression, the trx‐6His‐rMagi3 fusion protein was submitted to a refolding process to improve the yield of a well‐structured rMagi3. After that, the trx‐6His‐rMagi3 was enzymatically cleaved with thrombin, and the trx‐His6‐LVPR residue was satisfactorily removed from the reaction mixture by a second IMAC separation [Fig.…”

Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli

“…In addition, gene expression in bacteria is regulated by strong promoters, leading to the accumulation of recombinant proteins as insoluble aggregates or inclusion bodies. Different technologies have been developed to promote the correct oxidation of cysteine residues in recombinant proteins expressed in bacteria [10]. Exporting the proteins to the E. coli oxidative periplasm is a well-established strategy although levels of recombinant protein can be limited by protein export [11].…”