We have to use E. coli only to produce Trispecific scfv of size 100kda
Problem is that E. coli is not good with the expression of large proteins.
Is it possible that we use N terminal signal sequences and allow the bacteria to secret the protein.
There are a total of 6 disulfide bonds in full protein. Will disulfide bonds will form in broth? Will folding occur in broth?
Will slowing down the expression allow the protein to fold in periplasm if expressed there?
Individual scFv vary widely in their ability to fold and their stability - upon periplasmic expression in E.coli, these range from 100% aggregation in inclusion bodies to 100% soluble, natively folded product under the same expression conditions. First test each scFv for its expressibility, and if this is less than optimal, do whatever you can to optimise the scFv sequence (see https://plueckthun.bioc.uzh.ch/publications/?topic=ab for various publications on the topic). Only if each scFv by itself is well expressed, you are ready to go to the trivalent constructs. Another potential problem is the potential mispairing of VL and VH domains, you need to design linker lengths and domain orders such as to discourage this, and may even need to do some interface engineering.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes