[Optimizing expression and purification of recombinant Salvia miltiorrhiza copalyl diphosphate synthase protein in E. coli and preparation of rabbit antiserum against SmCPS].
The expression plasmid pET32CPS harboring SmCPS gene was transformed into E. coli BL21 trxB (DE3) resulting in recombinant strain E. coli [pET32CPS]. The induction of E. coli [pET32CPS] in different temperatures, induction time, IPTG concentrations and A600 values of E. coli were performed. The optimal expression conditions of SmCPS were characterized according to the orthogonal analysis, and the ratio of the interest protein to total proteins reached to 35.6%. The recombinant SmCPS protein purified by Ni2+ affinity chromatography column was identified by SDS-PAGE and Western blotting, and then used for rabbit immunization. The titer of the rabbit antiserum against SmCPS was about 1:24 300 after the third immunization, and could specifically recognize the antigen of SmCPS protein by Western blotting analysis. The successful preparation of polyclonal antibody against SmCPS laid a foundation for further correlative study between expression of SmCPS and the production of tanshinones in protein level.
Gao
Wei
W
Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Science, Beijing 100700, China.
Cui
Guang-hong
GH
Kong
Jian-qiang
JQ
Cheng
Ke-di
KD
Wang
Wei
W
Yuan
Yuan
Y
Huang
Lu-qi
LQ
D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D000917:Antibody Formation; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D007106:Immune Sera / Q000096:biosynthesis* / Q000276:immunology; D007544:Isopropyl Thiogalactoside / Q000737:chemistry; D008297:Male; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018517:Plant Roots / Q000737:chemistry; D010946:Plants, Medicinal / Q000737:chemistry; D010957:Plasmids; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D027561:Salvia miltiorrhiza / Q000737:chemistry; D013696:Temperature; D013997:Time Factors; D014170:Transformation, Genetic
D007106:Immune Sera; D010940:Plant Proteins; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D019883:Alkyl and Aryl Transferases; C090963:ent-kaurene synthetase A