pET28a+-BL1

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Tuner(DE3) is a BL21(DE3)-derived strain lacking the gene encoding β-galactosidase, which is the protein that is recognized by the model recombinant protein, the scFv BL1, used in this study.	Tuner(DE3) (Novagen) transformed with pLemo was used (23). The gene encoding DsbA BL1 was expressed from a pET28a+-derived vector as described before (23). As a control, an empty vector was used as described before (23).

Upstream tips
Tuner(DE3) is a BL21(DE3)-derived strain lacking the gene encoding β-galactosidase, which is the protein that is recognized by the model recombinant protein, the scFv BL1, used in this study.
Protocol tips
Tuner(DE3) (Novagen) transformed with pLemo was used (23). The gene encoding DsbA BL1 was expressed from a pET28a+-derived vector as described before (23). As a control, an empty vector was used as described before (23).

Publication protocol

The Lemo setup was used in this study for the production of the scFv BL1 (25, 59). However, rather than BL21(DE3) transformed with pLemo, Tuner(DE3) (Novagen) transformed with pLemo was used (23). Tuner(DE3) is a BL21(DE3)-derived strain lacking the gene encoding β-galactosidase, which is the protein that is recognized by the model recombinant protein, the scFv BL1, used in this study. The genetic information encoding the scFv BL1 was fused at the 5′ end to the genetic information encoding the DsbA signal sequence and at the 3′ end to the genetic information encoding a His6 tag. The DsbA signal sequence directs BL1 to the Sec translocon, and the His6 tag enables the detection of both the precursor form and the processed form of the scFv BL1 by means of immunoblotting (23). The gene encoding DsbA BL1 was expressed from a pET28a+-derived vector as described before (23). As a control, an empty vector was used as described before (23).

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