A novel expression vector for the improved solubility of recombinant scorpion venom in Escherichia coli

Investigation of Binding Modes and Functional Surface of Scorpion Toxins ANEP to Sodium Channels 1.7

“…Plasmid pSYPU-1b-Tag (his) -ANEP [ 17 ], E. coli strains DH5α and BL21 (λDE3) were stored in our laboratory. Restriction endonucleases, Taq DNA polymerase and T4 DNA ligase were purchased from TaKaRa (Dalian, China.…”

“…The first PCR products were generated by the general sense primer ANEP-F and the antisense primer of each specific mutant, while the second step were introduced by the mutant sense primer and ANEP-R. After being purified by DNA Gel Extraction Kit, the first and second PCR products were amplified by the pair of general primers, ANEP-F and ANEP-R, using an overlapping extension method. All of the target genes were inserted into plasmid pSYPU-1b, as described to obtain E. coli BL21 (λDE3) cells harboring various recombinant pSYPU-1b plasmids [ 17 ]. All clones were screened and selected by sequencing the entire PCR fragments.…”

“…The expression and purification procedure of rANEP and its mutants were performed according to the previous report [ 17 ]. The purity of the sample was checked on 15% SDS-PAGE and RP-HPLC.…”

“…In addition to the classical four disulfide bridges in the structure of β-toxin, ANEP is a compact of a α-helix and three antiparallel β-strands, and binds to receptor site 4 of sodium channels. The recombinant ANEP that is expressed in our Escherichia coli expression system exhibited similar analgesic activity to the native peptide in a mouse-twisting model and the hot plate assay [ 17 ]. In order to investigate the binding modes and functional surfaces of ANEP to its target sodium channels, here we generated structural models of the IIS1–S4 domain of mice Na v 1.7 resting and activated state by using molecular dynamics (MD) simulation.…”

Study of Anti-Inflammatory and Analgesic Activity of Scorpion Toxins DKK-SP1/2 from Scorpion Buthus martensii Karsch (BmK)

“…Since scorpion toxins are rich in disulfide bonds, there would be some hurdles to leap to achieve soluble expression in the prokaryotic system. Our previous efforts had been made to establish a series of vectors with high solubility recombinant protein expressions, the major improvements could be attributed to the co-expression of solubility-enhancing genes, such as N-Utilization substance (NusA), thioredoxin A (TrxA) and intein [ 30 ].…”