title: [Fusion expression and purification of recombinant ricin A-chain].
abstract: OBJECTIVE
  To prepare recombinant ricin A-chain(RTA) protein with high biological activity.
  
  
  METHODS
  RTA gene containing KDEL sequence at the carboxyl terminal was cloned in pET32a vector, which was fused with thioredoxin. Furthermore, the constructed recombinant plasmid was transformed into the competent cell BL21, and induced with low concentration of IPTG (0.4 mmol/L) under low temperature (20 degrees Celsius). After binding to Co2+ chelating column, the expressed supernatants were eluted by applying imidazole solutions with the concentration from 20 to 100 mmol/L. The purified protein was identified with SDS-PAGE and Western blot analysis and was used to cleave supercoiled dsDNA.
  
  
  RESULTS
  About 60 mg fusion proteins were obtained from 1,000 mL cultures , with purity above 90% and M(r) 45,000. The cleavage of supercoiled dsDNA demonstrated that RTA-Trx fusion proteins could significantly cleave supercoiled dsDNA as native RTA.
  
  
  CONCLUSIONS
  The pET32a vector expression system can be used to produce a mass of soluble RTA-Trx fusion proteins with high biological activity.
authors.affiliation: Institute of Basic Medicinal Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
authors.forename: Shun-tao
authors.initials: ST
authors.lastname: Wang
authors.forename: Mei-ru
authors.initials: MR
authors.lastname: Hu
authors.forename: Jian-wei
authors.initials: JW
authors.lastname: Guo
authors.forename: Jian-nan
authors.initials: JN
authors.lastname: Feng
authors.forename: Bei-feng
authors.initials: BF
authors.lastname: Shen
mesh_terms: D004278:DNA, Superhelical / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012276:Ricin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D004278:DNA, Superhelical; D011993:Recombinant Fusion Proteins; D012276:Ricin