chemistry

peptidaho chemistry

TPG-FLAG

figure inspired by a talk from Professor Jim Wells who has his own solution to the antibody problem: phage display antibodies

Perhaps the single most widely used tool in the molecular biosciences is the antibody. However, there are serious problems with most affordable polyclonal antibodies. (good 2015 Nature News article on the dilemma)These antibodies are typically generated by injecting a host organism (e.g. rabbit, goat, mouse, etc.) with the antigen, provided by the end user, letting the host natural immune system generate an antibody response, and bleeding the animal to collect anti-sera. The anti-sera are tested for a positive ELISA response to the antigen, and responding anti-sera are usually affinity purified (e.g., protein A or protein G), and then sent to the end user. For smaller animals, it is impractical to bleed those animals multiple times, and large animals are too costly for most academics. So, when the end user needs more antibody, the process must begin again. This leads to extreme batch to batch variability in polyclonal antibodies and irreproducible, sometimes contrary results, as every individual organism in a species has its own unique immune signature. Furthermore, the possibility exists that the polyclonal antibodies were responsive to an impurity in the original end user preparation of antigen and not the intended protein. Non-GMP facilities do not have the QA/QC policies to guarantee identical impurities. It is not logical for the end user to spend time and resources preparing indistinguishable antigen the next time they need antibody. This is in sharp contrast to the exhaustively validated mouse M2 monoclonal anti-FLAG antibody manufactured by Sigma.


The TPG group is TriazolylPhenylGlyoxal and was co-invented by Dr. Thompson at The Scripps Research Institute. It modifies the guanidine functionality of arginine (Arg) amino acids in peptides or proteins chemoselectively with whatever payload is attached to the TPG.


The FLAG tag is the antigen, epitope, or binding partner of the anti-FLAG antibody and is a highly soluble, octapeptide sequence: DYKDDDDK. TPG-FLAG will add the FLAG handle to intrinsic Arg in folded proteins, in most cases not interfering with function of the tagged protein.


To overcome the issues with impure polyclonal antibodies stated above, scientists can add the TPG-FLAG handle to their target protein. Then, by using the M2 anti-FLAG antibody described above, perform western blots, ELISA, immuno-precipitation, etc., and not worry about the quality of their antibody.


In addition, therapeutic peptides and proteins must be non-immunogenic in patients (i.e., they should not elicit an immune response). Adding the TPG-FLAG to a biological therapeutic protein will open up an express lane on the biotech superhighway to all the assays available with antibodies. The primary weakness of this method is the reagent is not specific to just your protein, if you have a mixture of proteins TPG-FLAG will ligate to all of them with exposed arginine.

The assays that require an antibody are highly reproducible, even though the antibody itself is not

Many applications

TPG-FLAG labels Beta Endorphin

Beta Endorphin 1-31 Lysine 19-> Arginine is labeled with hydrated TPG-FLAG. Positive ESI-MS result; Positive ELISA


Folded Lysozyme is labeled with TPG-FLAG

Native/Folded lysozyme (11 total Arg) in 100 mM KHCO3 pH 8.5 buffer is labeled with TPG_FLAG, blue ESI-MS spectra of native lysozyme with 2, 3, 4, 5, or 6 TPG-FLAG labels, rp-hplc of native protein reaction after 5x 10 kDa cutoff amicon spin filtration to purify protein away from TPG-FLAG that did not ligate, and black ESI-MS spectra and reconstruct of reduced lysozyme with 2, 3, 4, 5, or 6 TPG-FLAG labels.


Folded RNase A is labeled with TPG-FLAG

Native/Folded RNase A (4 total Arg) in 100 mM KHCO3 pH 8.5 buffer is labeled with TPG-FLAG, blue ESI-MS spectra of native RNase A with primarily 2 labels, A280 rp-hplc of native protein reaction after 2x 10 kDa cutoff amicon spin filtration to purify protein away from TPG-FLAG that did not ligate, black ESI-MS spectra and reconstruct of reduced RNase A with primarily 2 TPG-FLAG labels and reduced unlabeled RNase A (note: dotted line in reconstructed labeled RNase A is where unlabeled protein should appear), and RP-HPLC of reduced labeled and reduced unlabeled RNase A showing nearly identical retention times.


ELISA with TPG-FLAG labeled Biomolecules

Molecules labeled with TPG-FLAG demonstrate positive ELISA results. 2G12 is an antibody to gp120 and is used as a negative control. No signal resulted from plating unreacted molecules. The avidity of the monoclonal antibody (green) is at least an order of magnitude greater than the polyclonal antibody (red). Covalent TPG-FLAGs are binding independently (i.e. no biphasic behavior for RNase A-2FLAG) and are only marginally synergistic (i.e. ~2-fold lower EC50 for RNase A-2FLAG as endorphin-1FLAG).


Western Blot with TPG-FLAG labeled Biomolecules

Western blot and ELISAs performed by Daniel Leaman, Ph.D.

The sensitivity of this approach is illustrated by the "lighting up" of the RNase A mixed disulfide dimer, barely visible in the coomassie stained gel.


OPD-succ NHS ester

Now this little beasty is my first solo invention. It turns amines, lysine or the amino terminus, into thioesters. It does so with chemistry pioneered by Phil Dawson.

Amines are modified with OPD-succ NHS ester


OPD-succ is converted to a thioester


thioester to chemical diversity


POSTERS

Dr. Thompson is also adjunct professor of chemistry at University of Idaho Coeur d'Alene. View some of the posters his students have made.