[Studies on the optimal expression condition, purification and its characterization of ScFv-2F3].
The expression vectors of the gene encoding ScFv-2F3 were transformed into E. coli BL21(DE3). Clones of higher expression were first selected, then were grown in the presence of IPTG at 37 degrees C to induce its expression. The culture conditions were carefully optimized. It was found that optimal conditions were as follows: the induction was started as OD590 reached to 1.0-1.8; the concentration of IPTG was 0.3-0.5 mmol/L and induction time is 7 h. The yield of ScFv-2F3 expressed in the selected clones is about 20% of the total proteins. The optimal culture conditions were successfully applied to fermenter of 50 L. The conditions of washing the inclusion bodies were also optimized. A two-step method was used to renature the inclusion body. The expression product of interest and its biological activities were characterized with Western blotting and ELISA. A novel selenium-containing single-chain abzyme with GPX activity was prepared.
Luo
Yuan-Ming
YM
Key Laboratory of Molecular Enzymology and Engineering of Educational Ministry, Jilin University, Changchun 130023, China.
Mu
Ying
Y
Wei
Jing-Yan
JY
Yan
Gang-Lin
GL
Luo
Gui-Min
GM
D017151:Antibodies, Catalytic / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019149:Bioreactors / Q000382:microbiology; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002479:Inclusion Bodies / Q000378:metabolism; D017510:Protein Folding; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012643:Selenium / Q000378:metabolism
D017151:Antibodies, Catalytic; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D012643:Selenium