Biotin Ligase substrate

Dear Colleagues,

I am searching for a target protein tagged with His (or other tags)  and Avi Tag in order to purify via His and then use the protein as substrate for Biotin Liagse activity assay.

Has anyone a construct that can be shared?

Thank you very much in advance

Sabine

Posted on 10-Jan-2014 10:48 CET

Dear Sabine,

luckily, we have generated several constructs for proteins of different size with 5 tags at the C-terminus (His6, HA, Myc, FLAG, Avitag) mainly for using them in our Molecular Weight Marker which could also be used in Western Blots at the same time. We could send you one construct for e.g. SUMO-EGFP with this tag (or the purified protein if you need it quickly). Let me know what you would prefer.

Best regards

Hüseyin

 

Posted on 10-Jan-2014 11:00 CET

Hi,

We have a pET vector with T7promoter [His6-TEV-MBP-avitag].

Very high expression of soluble protein in E. coli. You can purify it on NiNTA or amylose (or both).

Bear in mind that some of it will be naturally biotinylated in normal BL21, even if you don't coexpress BirA or precurse with biotin.

 

Darren

 

Posted on 10-Jan-2014 12:49 CET

Dear Darren and Hüseyin,

thanks so much for your replies! I chose the lazy option, Hüsyeins protein, but will keep in mind Darrens construct.

I am quite surprised about biotinylation capacity of BL21 ????

Best

Sabine

Posted on 10-Jan-2014 15:01 CET
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