title: Straightforward approach to produce recombinant scorpion toxins-Pore blockers of potassium channels.
abstract: Scorpion venom peptide blockers (KTx) of potassium channels are a valuable tool for structure-functional studies and prospective candidates for medical applications. Low yields of recombinant KTx hamper their wide application. We developed convenient and efficient bioengineering approach to a large-scale KTx production that meets increasing demands for such peptides. Maltose-binding protein was used as a carrier for cytoplasmic expression of folded disulfide-rich KTx in E. coli. TEV protease was applied for in vitro cleavage of the target peptide from the carrier. To produce KTx with retained native N-terminal sequence, the last residue of TEV protease cleavage site (CSTEV) was occupied by the native N-terminal residue of a target peptide. It was shown that decreased efficiency of hydrolysis of fusion proteins with non-canonical CSTEVcan be overcome without by-product formation. Disulfide formation and folding of a target peptide occurred in cytoplasm eliminating the need for renaturation procedure in vitro. Advantages of this approach were demonstrated by producing six peptides with three disulfide bonds related to four KTx sub-families and achieving peptide yields of 12-22mg per liter of culture. The developed approach can be of general use for low-cost production of various KTx, as well as other disulfide-rich peptides and proteins.
authors.affiliation: Biological Faculty, Lomonosov Moscow State University, Leninskie Gory 1, Moscow, 119992, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia.
authors.forename: Oksana
authors.initials: O
authors.lastname: Nekrasova
authors.affiliation: Biological Faculty, Lomonosov Moscow State University, Leninskie Gory 1, Moscow, 119992, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia.
authors.forename: Ksenia
authors.initials: K
authors.lastname: Kudryashova
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia.
authors.forename: Arkadiy
authors.initials: A
authors.lastname: Fradkov
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia.
authors.forename: Sergey
authors.initials: S
authors.lastname: Yakimov
authors.affiliation: Biological Faculty, Lomonosov Moscow State University, Leninskie Gory 1, Moscow, 119992, Russia.
authors.forename: Maria
authors.initials: M
authors.lastname: Savelieva
authors.affiliation: Biological Faculty, Lomonosov Moscow State University, Leninskie Gory 1, Moscow, 119992, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia.
authors.forename: Mikhail
authors.initials: M
authors.lastname: Kirpichnikov
authors.affiliation: Biological Faculty, Lomonosov Moscow State University, Leninskie Gory 1, Moscow, 119992, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia. Electronic address: avfeofanov@yandex.ru.
authors.forename: Alexey
authors.initials: A
authors.lastname: Feofanov
mesh_terms: D010450:Endopeptidases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D058619:Maltose-Binding Proteins / Q000235:genetics; D026902:Potassium Channel Blockers / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D012604:Scorpion Venoms / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D013104:Spheroplasts / Q000187:drug effects
chemical_list: D058619:Maltose-Binding Proteins; D026902:Potassium Channel Blockers; D011993:Recombinant Fusion Proteins; D012604:Scorpion Venoms; D010450:Endopeptidases; C099488:TEV protease
keywords: Disulfide rich peptide; Maltose-binding protein; Potassium channel blocker; Recombinant peptide; Scorpion toxins; TEV protease cleavage site