Hello Alexander,

The linker between fusion proteins tends to enhance the function but that of course depends of the the protein you are creating. I will refer you to this paper that will give you a good overview of linker types Article Design and characterization of structured protein linkers wi...

It is good that you mentioned that you are creating a GAL4 AD fusion protein as I have seen a paper that created such a protein before (you can take a look and see what it generally looks like):

Article Inducible, tightly regulated and growth condition-independen...

Also here is the addgene link to their fusion protein:

https://www.addgene.org/58438/

Finally addition of the spacer regions from the lambda bacteriophage cI gene enhanced the function of tetracyline transactivator used in Saccharomyces cerevisiae here is the link to the paper (this fusion is to the VP16 activation domain though but you may able to use the same linker they used) :

Article A Set of Vectors with a Tetracycline-Regulatable Promoter Sy...

Also I have attached a map for plasmid pCM183 which contains the linker (I have marked it in red so will be able to find the sequence easily, you can use the freely available snapgene viewer to see the file)

Best of luck,

Hanna

General properties of linkers derived from naturally - occurring multi - domain proteins. In summary, flexible linkers are generally rich in small or polar amino acids such as Gly and Ser to provide good flexibility and solubility. The are suitable choices when movements or interactions ( e.g. scFv ) are required for fusion protein domains. Engineered fusion proteins containing two or more polypeptides joined by a peptide or protein linker are important for many fields of biological research. For more details consult https://www.ncbi.nlm.nih.gov

Dear Alexander

it depends from the nature and dimention of the proteins that you would like to fuse and the use of the proteins. For example in vaccinology generally we try to use short linkers as gsggg ( flexible not structured) or AEAAAKA ( structured alpha elix) GGSLVPRGS (rigid not structured) to minimize their immunogenicity

in my experience in the most cases the first (GSGGG) work well and sometimes if i observed protein degradation was due to the degradation of one of the attached proteins and not of the fragment.

if you fuse big proteins could be better to use more long linker to make the 2 proteins able to indipendetly move and avoiding that one part of a patner cover an important site of the other.

there no solution valid for all cases.. just try..and good luck.

Manuele

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