[An easy way to purify the inclusion body protein with high purity from prokaryotic expression cells].
To clone partial ORF of SjBMP and to construct the recombinant SjBMP-pET-28a(+) plasmids, and then to transform them into the competent cells E. coli BL21 (DE3), finally a positive clone was used to be induced by IPTG. The bacterial aggregates with target protein expressed as inclusion bodies were purified by the methods of Ni(2+)-NTA affinity purification under denaturation condition and SDS-PAGE gel extraction. The purified protein was used to immune rabbits and make antiserum against the SjBMP, and the antiserum were then used to identify the rSjBMP by Western blotting. The target protein obtained by Ni(2+)-NTA Agarose affinity purification was not pure with unspecific proteins, but the protein further purified by SDS-PAGE gel extraction and the dialysis bag horizontal electrophoresis was quite pure, and the recovery rate was more than 11.0%. Meanwhile, Western blotting was used to identify the recombinant SjBMP protein by antiserum, only a specific single strip appeared, which suggested the protein purified by this method kept its antigenicity, and could be used for common immunological studies. Therefore, the SDS-PAGE gel extraction combining with electroosmosis and dialysis recycling are good and easy to purify the inclusion body proteins.
Rong
R
Liu
Qin-Ping
QP
Zhong
Ming-Sen
MS
Jiang
Hui-Fen
HF
Dong
D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D015801:Helminth Proteins / Q000302:isolation & purification*; D007106:Immune Sera; D002479:Inclusion Bodies / Q000276:immunology*; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification*; D012549:Schistosoma japonicum / Q000235:genetics*
D015801:Helminth Proteins; D007106:Immune Sera; D011994:Recombinant Proteins