Efficient production of soluble recombinant single chain Fv fragments by a XXXX cell factory.

@entity1 @entity2355 @entity15250 @entity26731 @entity10390 @entity2624 @entity1851
BACKGROUND: Recombinant antibody fragments have a wide range of applications in research, diagnostics and therapy. For many of these, small fragments like single chain fragment variables ( @entity10390 ) function well and can be produced inexpensively in bacterial expression systems. Although @entity2624 production systems are convenient, yields of different fragments, even those produced from codon-optimized expression systems, vary significantly. Where yields are inadequate, alternative production systems are needed. @entity26731 is a versatile biosafety strain known for good expression of heterologous genes, so we have explored its utility as a cell factory for production of scFvs. RESULTS: We have generated new broad host range @entity10390 expression constructs and assessed their production in the @entity26731 host. Two scFvs bind either to @entity1 @entity1851 or to @entity2355 , proteins of significant medical diagnostic and therapeutic interest, whereas a third is a model anti-lysozyme @entity10390 . The @entity26731 antibody expression systems produce scFvs targeted to the periplasmic space that were processed precisely and were easily recovered and purified by single-step or tandem affinity chromatography. The influence of promoter system, codon optimization for @entity15250 , and medium on @entity10390 yield was examined. Yields of up to 3.5 mg/l of pure, soluble, active @entity10390 fragments were obtained from shake flask cultures of constructs based on the original codon usage and expressed from the Ptac expression system, yields that were 2.5-4 times higher than those from equivalent cultures of an @entity2624 expression host. CONCLUSIONS: @entity26731 is a good cell factory for the production of scFvs, and the broad host range constructs we have produced allow yield assessment in a number of different expression hosts when yields in one initially selected are insufficient. High cell density cultivation and further optimization and refinement of the @entity26731 cell factory will achieve additional increases in the yields of scFvs.
