Tagging recombinant proteins with a Sel-tag for purification, labeling with electrophilic compounds or radiolabeling with 11C
Selenocysteine (Sec; U in one-letter code) is the twenty-first naturally occurring amino acid, with a selenium atom that gives this cysteine (Cys) homolog unique biochemical properties, including a high nucleophilicity and significant reactivity with electrophilic agents. This can be used in biotechnological Sec-dependent applications. Here, we describe how Sec can be introduced into a carboxy-terminal tetrapeptide motif (-Gly-Cys-Sec-Gly-COOH, known as a Sel-tag) for recombinant proteins by tailoring the encoding gene to become compatible with the Escherichia coli selenoprotein synthesis machinery. We also describe how the Sel-tag can be used as a basis for efficient one-step protein purification, rapid Sec-targeting protein labeling with electrophilic compounds, or radiolabeling with the positron emitter 11C.
Comments
PAPAO is one of the precursor compounds for PAO sepharose synthesis. We used to purchase this product from the Canadian based company Toronto Research Chemicals (cat. no. A622500) until we experienced an error in shipment of the product, whereby the company sent the wrong chemical labeled under the correct product name. PAPAO should be a yellowish powder in appearance and it dissolves easily in DMSO and further addition of water should not change the solubility, whereas the chemical sent at that occassion was a yellowish compound which dissolved in DMSO but immediately precipitated when water was added. After several email communications with the company, they admitted their mistake and stated in another email that ?It is possible that there was a problem with multiple customers?. The company has also claimed that, at present, they have the correct product in stock, but since the occassion we have synthesized this particular compound on our own as indicated by reference nine in the protocol. We feel sorry for any researcher having experienced problems in the synthesis or performance of the PAO sepharose as a result from the mistake made by this vendor of PAPAO.
Qing Cheng
Biochemistry, MBB, Karolinska Institutet
Posted by: Qing Cheng | April 10, 2007 04:43 PM