3 replies to this topic

[Dr.Kurva]

Hello everybody,
could anybody help me with my problem? I want to express recombinant protein (antibody fragment) in HEK293-E cells and I have a signal sequence (for kappa chain of Antibody) for secretion into medium. As a control I have other recombinant protein (different recombinant antibody fragment) under the same signal sequence in the same vector. On western blot I see for control protein nice expression into medium but for "my" protein I don't see any signal in medium but quite strong signal in the cells. Does anybody of you know any tips and tricks how to push the protein from cells into medium or what can be wrong with "my" protein.
Thanks a lot,
Dr. K.

[pDNA]

do you express heavy and light chain from two promoters or just one?

Regards,
p

[bob1]

Are you sure that the plasmid sequence is correct - does it need a secretion marker?

[Dr.Kurva]

bob1, on 02 December 2011 - 02:12 PM, said:

Are you sure that the plasmid sequence is correct - does it need a secretion marker?

pDNA, on 02 December 2011 - 08:23 AM, said:

do you express heavy and light chain from two promoters or just one?

Regards,
p

Sequence of the plasmid is correct (I did sequencing) also the secretion signal is ok.  I do have variable domains of light and heavy chains joined via peptide linker (it's normal scFv) so there is just one promoter and  because I do have as N-terminal VL domain I used secretion signal for light chain.
Thanks for suggestions and if you can think of something else I would appreciate it.
Dr. K