Hello Jean-Francois, probably the best option is to use your construct with the 1-Step Human Coupled IVT Kit as you say (in order to use the rabbit reticulocyte translation system you would have to first do an in vitro transcription by yourself).

The GST is fused without a cutting sequence, but you could think o putting in a prescission recognition sequence, although on the C-Term of your protein it will leave 6 aa overhang... still better than the whole GST, anyway (you can easily insert the short sequence with the Agilent quick mutagenesis kit... but you can also simply buy the Pfu and the Dpn1 separately, it is much cheaper; I attach the protocol for your information. Otherwise you can clone the little sequence using a classical RE approach or a Gibson Assembly or InFusion system in the C-Term restriction site of your gene).

If you need to purify, you can use the GST tag on a Glutathione-Sepharose 4B beads column, the prescission is also GST-fused, so it will cut and remain in the column, and you can elute the cut purified protein with any mild buffer.

http://www.gelifesciences.com/webapp/wcs/stores/servlet/ProductDisplay?categoryId=11741&catalogId=10101&productId=16822&storeId=11762&langId=-1

http://www.chem.agilent.com/library/usermanuals/Public/200523.pdf

Dear Pietro / Liebe Pietro,

Thank you VERY much for this answer and the attached links. This is perfect!

I just hope now to be successful and to have the opportunity to help you back.

All the best,

jfb

Salut Jean-Francois, you are very welcome and I definitely wish you to succeed!

All the best,

Pietro