Hi,

does anyone have experience with cloning of heavy and light chain domains for generating scFv's and expression in E. coli? We would need an appropriate protocol and vector(s) for cloning a couple of hundred constrcuts in 96-well format and expression in E. coli. Thanks for any suggestions in advance.

Best regards,

Hüseyin

Hi Huseyin,

Have you seen Jo's paper in PEP 2008 using pOPINVL and pOPINVH vectors? Expression is in HEK but might contain  some useful hints for the cloning. I think this is along the right lines.

Nick

Hi Huseyin,

Following up Nick's comment, we published the exact protocol recently in Methods in Molecular Biology: Nettleship JE, Flanagan A, Rahman-Huq N, Hamer R, Owens RJ. Converting monoclonal antibodies into Fab fragments for transient expression in mammalian cells. Methods Mol Biol. 2012;801:137-59.

This paper describes the vectors and degenerate primer design in detail.

The pOPINVH and pOPINVL vectors are available on Addgene (along with maps), or just ask me.  They are in the TriEx background, but contain RTPTu signal sequence which you wouldn't want.  pOPINVH puts a C-term His tag on the heavy chain, and then the light chain isn't tagged, but is co-purified after co-transfection.  The vectors contain the constant region for human Fabs so that you can clone in the variable region.

Thanks and see you all soon in Porto

Jo