Soluble scFv production and purification

Inoculate 10 ml of 2TYGA in a SO ml Falcon tube with an individual colony of the clone of interest and grow overnight at 30 C with shaking (200 r.p.m.). 

Split the culture between two 50 ml Falcon tubes and centrifuge at 4000 r.p.m, for 15 min at 4 C in a pre-chilled Sorvall SS 34 rotor. 

Pour off the supernatant and resuspend the cell pellet in 50 ml of 2TY containing 100 ng/ml ampicillin and 1 mM isopropyl p-S-thiogalactopjrranoside (IPTG) prewarmed to 30 C. 

Return the cells to a 250 ml flask and grow for 4 h at 30 C with shaking. 

Pellet the cells as before and resuspend the cell pellet in 1 ml of 50 mM Tris-HCl pH 8.0 containing 2056 (w/v) sucrose and 1 mM EDTA. Transfer the susptension into a 1.5 ml Eppendorf tube and leave on ice for 15 min. 

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