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Mar Drugs
2016 Jan 05;141:11. doi: 10.3390/md14010011.
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Recombinant Expression and Characterization of α-Conotoxin LvIA in Escherichia coli.
Zhu X
,
Bi J
,
Yu J
,
Li X
,
Zhang Y
,
Zhangsun D
,
Luo S
.
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α-Conotoxin LvIA is derived from Conus lividus, native to Hainan, and is the most selective inhibitor of α3β2 nicotinic acetylcholine receptors (nAChRs) known to date. In this study, an efficient approach for the production of recombinant α-Conotoxin LvIA is described. Tandem repeats of a LvIA gene fragment were constructed and fused with a KSI gene and a His₆ tag in a Escherichia coli (E. coli) expression vector pET-31b(+). The recombinant plasmids were transformed into E. coli and were found to express well. The KSI-(LvIA)n-His₆ fusion protein was purified by metal affinity chromatography and then cleaved with CNBr to release recombinant LvIA (rLvIA). High yields of fusion protein ranging from 100 to 500 mg/L culture were obtained. The pharmacological profile of rLvIA was determined by two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing rat nAChR subtypes. The rLvIA antagonized the α3β2 nAChR subtype selectively with a nano-molar IC50. The rLvIA was analgesic in a mouse hot-plate test model of pain. Overall, this study provides an effective method to synthesize α-conotoxin LvIA in an E. coli recombinant expression system, and this approach could be useful to obtain active conopeptides in large quantity and at low cost.
Figure 1. Strategy for construction, expression, purification and cleavage of fusion protein of recombinant α-conotoxin LvIA (rLvIA). Two primers encoding α-conotoxin LvIA mature peptide were annealed and unidirectionally self-ligated to form an array of tandem repeats. These multimers were then ligated into the AlwNI digested vector pET31b(+) to construct an array of recombinant KSI(rLvIA)nHis6 vectors, where n = 1–11. These gene tandem repeats fused with KSI and His·Tag were expressed in E. coli after the addition of IPTG. The fusion proteins were purified by Ni2+ chromatography and cleaved by CNBr into individual soluble rLvIA, insoluble KSI, and the 6× His·tag tail.
Figure 2. Agarose gel electrophoresis analysis of the LvIA gene tandem repeats. Two phosphorylated oligos were annealed and ligated. These oligos could be ligated to form array of tandem repeats. These multimers were analyzed by 3% agarose gel electrophoresis. Marker, DL 2000 DNA marker. Lane 1, annealed single gene with 51 bp. Lane 2, tandem repeats of rLvIA gene with different size, ranged from 51 to 1000 bp.
Figure 3. SDS-PAGE analysis of recombinant KSI(rLvIA)nHis6 fusion proteins in E. coli. (A) Cell total proteins expressed by IPTG induction. Lane 1–7 were total protein fractions containing KSI(rLvIA)nHis6 fusion constructs, where lane 1–6, n = 0–5. lane 7, n = 11. (B) Recombinant fusion proteins from panel A cell total proteins purified by Ni2+ Chelation Chromatography containing different rLvIA tandem repeats.
Figure 4. RP-HPLC chromatogram (A) and ESI-MS data (B) of rLvIA. (A) The peptide rLvIA was analyzed by RP-HPLC on a Vydac C18 column (5 μm, 4.6 mm × 250 mm), using a linear gradient of 0%–40% Buffer B over 20 min, where A = 0.075% TFA and B = 0.75% TFA, 90% acetonitrile, and the remainder water. Absorbance was monitored at 214 nm. (B) ESI-MS analysis of rLvIA with observed molecular weight of 1810.656 Da, which was consistent with the calculated theoretical molecular weight of 1810.635 Da.
Figure 5. Analgesic effect of rLvIA on a mouse hot-plate test model. (A) Effect of rLvIA (10 nmol), 0.9% saline, and morphine (250 nmol) on 55 °C hot plate test at 30, 60, 90, 120, 150 min after injection as described in Materials and Methods. Latency time (second, s) was used as a measure of thermal hyperalgesia of conopeptide rLvIA (1 nmol/μL). Mice were divided into three groups, each group containing eight mice (n = 8). rLvIA significantly increased the latency time to the thermal stimulus at 30, 90, 120 min after treatment (p < 0.05). The maximum effect of rLvIA was observed at 90 min, which showed a latency of 24.17 s. (B) Corresponding dose response calculated as area under the curve (AUC) for data from each drug in (A) for time points between 0 and 150 min. Asterisks represent significant difference from the saline control group (*p < 0.05, **p < 0.01, ***p < 0.001).
Figure 6. rLvIA selectively blocks α3β2 subtype among the tested nAChRs. nAChR subtypes were expressed as described in Materials and Methods. “C” indicates control responses to ACh. Oocytes were then exposed to 1 μM or 10 μM rLvIA for 5 min, followed by application of a 1 s pulse of ACh. The rLvIA almost blocked α3β2 at 1 μM concentration completely (arrow) (A), and the current resumed quickly within one minute of toxin wash out, For other nAChR subtypes even 10 μM rLvIA had little or no blockage (B–F).
Figure 7. rLvIA concentration-response data for human and rat α3β2 nAChR subtypes. Oocytes expressing human and rat α3β2 nAChR subtypes were voltage clamped and subjected to ACh pulses as described in the Experimental Procedures. Values given are mean ± SEM from 5 to 8 separate oocytes. The IC50 values for rLvIA on hα3β2 and rα3β2 were 46.8 nM and 160.8 nM, respectively.
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