I read papers about the structure of CAR vector and find inconsistence in the design of FMC63 scFv. VH is placed in front of VL in some papers while VL is placed in front of VH in other papers.
I wonder what's the difference between them and which one should I choose to use?
There also some codon-optimized FMC63 scFv sequence, is the optimization necessary?
Thank you for your answer!
The distance between the C-terminus of the VH domain to the N-terminus of the VL domain frequently is somewhat shorter than the distance between the C-terminus of the VL domain and the N-terminus of the VH domain, therefore you can get away with a slightly shorter linker in the VH-linker-VL orientation. On the other hand, since many Vkappa domain already start with D and others are compatible with replacing the first amino acid by a D, it is possible to attach a very short flag tag of only three amino acids to the free N-terminus of VL in a scFv in VL-linker-VH orientation that is well recognized by the commercially available M1 anti-Flag-tag antibody http://www.bioc.uzh.ch/plueckthun/pdf/APpub0091.pdf
As long as the linker is long enough, either orientation works well for scFv.
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