I am looking to clone a 120kDa protein with a C-terminal SBP tag (38 aa) into a lentiviral vector. Is it ok to add a linker between the gene and the SBP tag (followed by the stop codon)? If so, how do i choose the length of the linker? Does the length also depend on the size of the tag? Please help!
Thanks!
Hi Radhika, it depends what you are planning to do with your tagged protein. If you intend to purify it or use it for immunoprecipitation, adding a linker might help to improve accessibility of the tag und thus can improve product purity during affinity purification. Also, it is known that tag removal by proteases is more efficient if about 10-15 amino acids of a flexible, disordered linker containing e.g. glycine or alanine residues is included.In previous work (see https://www.researchgate.net/publication/235732987_Immunisation_with_foamy_virus_Bet_fusion_proteins_as_novel_strategy_for_HIV-1_epitope_delivery) we used the linker SGSAGAGAGGG to separate antigen epitopes of 2.5-9.7 kDa from the carrier protein.
Best regards, Michael
Article Immunisation with foamy virus Bet fusion proteins as novel s...
Hello Micheal
Thank you for your answer. I am looking to immunoprecipitate the protein eventually. However, I have decided to shift the tag to the N-terminus and add a GSGSG linker. Hopefully this should help improve the yield. I will also look into your paper for further information.
Warm regards, Radhika
Personally, I am not a big fan of N-terminal tags for IP, since one will detect all the truncated forms of protein, which usually results in quite messy Western blots. Particularly for a protein of 120 kDa, it might be useful to reconsider this decisions.
Best regards, Michael
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