The effects of induction conditions on production of a soluble antitumor sFv in Escherichia coli

CC49 is a second generation monoclonal antibody(mAb) with highaffinity to a pancarcinoma antigen, TAG-72. A single-chain Fv(sFv)ofCC49 may have a role in managing human carcinomas. Most reportedsFvs have been expressed as insoluble products that must besolubilized and renatured. Soluble sFv expression is advantageousas activity can be assayed directly from the periplasmic fraction.Also, gene-level immunoconjugates may not be amenable to refoldingprotocols. Using a vector that carries the tac promoter andomp A signal, we have examined the effects of four variableson the expression and accumulation of soluble CC49 sFv: (i)linker sequence joining VL and VH, (ii) isopropylthio-ß-D-galactosideconcentration for induction, (iii) temprature, and (iv) theaddition of nonmetabolizable sugars to the medium. We have beenable to demonstrate, using rapidly prepared periplasmic extracts,that the yield of soluble sFv improves by the addition of 0.4Msucrose to the medium and by inducing expression with a verylow concentration of IPTG (0.02–0.03 mM). Under theseinduction conditions periplasmic extracts demonstrate increasedexpression of the sFv, as shown by the larger amount of a 27kDa band on SDS-polyacrylamide gel, and an increased abilityto inhibit binding of the mAb CC49 to immobilized tumor extracts.