[Fusion expression and purification of recombinant ricin A-chain].
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.
Wang
Shun-tao
ST
Institute of Basic Medicinal Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
Hu
Mei-ru
MR
Guo
Jian-wei
JW
Feng
Jian-nan
JN
Shen
Bei-feng
BF
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
D004278:DNA, Superhelical; D011993:Recombinant Fusion Proteins; D012276:Ricin