N-terminus tagging and signalP prediction - (Dec/10/2017 )

Hi all.

I am planning to add a tag at the N-terminal of a multipass transmembrane protein.

I wonder if I should add it immediately after the start codon, or after the signal peptide. 

From signalP prediction, it seems like it predicted the protein with possible signal peptide btwn amino acid 1-19, but the D value also predicted it as non-signal peptide (results as pasted below).

Does that mean there's likely no signal peptide for the protein sequence? 

Anyone used signalP before would really love to hear your input. Thanks

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# Measure Position Value Cutoff signal peptide?

max. C 60 0.291

max. Y 20 0.270

max. S 6 0.596

mean S 1-19 0.434

D 1-19 0.336 0.500 NO

Name=Sequence SP='NO' D=0.336 D-cutoff=0.500 Networks=SignalP-TM

Sems a bit ambiguous, so hard to say for sure.

Are residues ~1-19 predicted to be transmembrane? Those residues may not be true a signal sequence in terms of export to periplasm or extracellular space but just a signal for membrane embedding. At the end of the day, I would try both options and create two constructs-- one with with tag at residue 1 and one with tag starting at residue 19. Test both side by side for expression/function and you'll have your answer.

But as a word of caution, typically when working with membrane proteins we stick tags at the C-terminus for this very reason. Nterminal segments are too often crucial for proper localization to be messing with, so sticking them at the Cterminus is more likely to work. Just depends on your application if this is an option.

It's a multispanning membrane protein with the first transmembrane helix starting after aa 37. 

I don't have any other information beside amino acid sequence. Everthing else are based on prediction tools.

Another prediction tool (Phobius) also predicts signal peptide from 1-22. So means I have to try 3 options. One after start codon, one after aa19, and another after aa22.

My supervisor wants me to try random insertion mutation of a luminesent tag in order to scan the possible topology/functional domains of the transmembrane protein so we can have some information for future works. So beside C-terminal, I wish to try on other hydrophilic domains as well. :)