title: Recombinant Expression and Characterization of α-Conotoxin LvIA in Escherichia coli.
abstract: 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.
authors.affiliation: Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Laboratory for Marine Drugs of Haikou, Hainan University, Haikou 570228, China. zhuxiaopeng@hainu.edu.cn.|College of Horticulture and Landscapes, Hainan University, Haikou 570228, China. zhuxiaopeng@hainu.edu.cn.|College of Marine Science, Hainan University, Haikou 570228, China. zhuxiaopeng@hainu.edu.cn.
authors.forename: Xiaopeng
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Laboratory for Marine Drugs of Haikou, Hainan University, Haikou 570228, China. jianpeng_bee@yahoo.com.
authors.forename: Jianpeng
authors.initials: J
authors.lastname: Bi
authors.affiliation: Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Laboratory for Marine Drugs of Haikou, Hainan University, Haikou 570228, China. mtying0001@126.com.
authors.forename: Jinpeng
authors.initials: J
authors.lastname: Yu
authors.affiliation: Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Laboratory for Marine Drugs of Haikou, Hainan University, Haikou 570228, China. lixiaodan816@163.com.
authors.forename: Xiaodan
authors.initials: X
authors.lastname: Li
authors.affiliation: Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Laboratory for Marine Drugs of Haikou, Hainan University, Haikou 570228, China. yanin802388@yahoo.com.
authors.forename: Yaning
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Laboratory for Marine Drugs of Haikou, Hainan University, Haikou 570228, China. zhangsundt@163.com.|College of Marine Science, Hainan University, Haikou 570228, China. zhangsundt@163.com.
authors.forename: Dongting
authors.initials: D
authors.lastname: Zhangsun
authors.affiliation: Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Laboratory for Marine Drugs of Haikou, Hainan University, Haikou 570228, China. luosulan2003@163.com.|College of Marine Science, Hainan University, Haikou 570228, China. luosulan2003@163.com.
authors.forename: Sulan
authors.initials: S
authors.lastname: Luo
mesh_terms: D000700:Analgesics / Q000494:pharmacology; D000818:Animals; D020916:Conotoxins / Q000737:chemistry*; D052078:Conus Snail; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D051379:Mice; D010147:Pain Measurement; D051381:Rats; D011978:Receptors, Nicotinic / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry*; D012623:Seawater; D014982:Xenopus laevis
chemical_list: D000700:Analgesics; D020916:Conotoxins; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; C419362:nicotinic receptor alpha3beta2
keywords: electrophysiology; fusion protein; nAChRs; pain assay; recombinant expression; α-conotoxin LvIA