2 replies to this topic

[Kb Homes]

Hello,

I have a question about peptide expression in e. coli. I am planning on expressing a 16-aa peptide with a 6xHis tag and a Met site in between to cleave the His-tag after purification. So the gene would be: Met-HHHHHH-Met-16aa-STOP downstream of a T7 promoter. I realize most people express small peptides as a fusion with other proteins or larger tags. I've been looking and I can't really find the reason why a small peptide like mine cannot be expressed as is. The peptide itself should be very soluble and the 6xhis tag should help with the solubility as well.
So, the question is, what is the reason for people to use larger fusion partner (eg GST, SUMO)? Is it just due to the small size of the peptide and you want something tangible for gels and columns? or is there any other properties that I'm unaware of such as effects on expression level?

Thank you for your help and input.

[snowchild]

I think so... For detection and increasing solubility and stability.

In my case, I express a peptide which is 10 amino acids. So I fused it with a C terminal GST and thrombin cleavage site in between. It expressed in E.coli well.

Sometimes a N-terminal tag like GST or MBP will help the translation initiation and thus expression levels.

Edited by snowchild, 07 February 2013 - 11:14 AM.

[andy_lord]

I have had some experience expressing a 14 aa peptide with both N-terminal His-tag and GST tag separately and I'd have to say it's much better expressing your peptide with a GST tag rather than a His-tag. My experience has taught me that GST being a highly soluble protein has better chances to "pull" your peptide to solubility than a normal 6X His-tag with a cleavage site.

But before you go for cloning and expressing a peptide this short I'd advice you to use a few bioinfo tools like DisEMBL or RONN to predict the disorder of your expressed peptide (the whole gene in case of the His-tag).

I'd agree with snowchild regarding detection and increased solubility and stability too. The shift in the gel for GST alone and GST tagged 16 aa peptide might be a little difficult to differentiate. Don't forget to use protease inhibitors. A peptide this small would be vulnerable to protease attacks.

Good luck!

Edited by andy_lord, 20 February 2013 - 02:37 AM.