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The paths below with colored circles as bullet points correspond to experiments in the timeline of that same color. Paths with black squares as bullet points have no associated experiments.

• Synthesis of the three 41-residue N-terminal thioesters (representing residues 2-36 of paxillin)

Peptide synthesizers are commonly used to make peptides up to 50 residues in length. Vogel used solid phase peptide synthesis (SPPS) to create three 40 residue peptides, one of which had an unnatural amino acid of caged phosphotyrosine that they developed in the lab. The carboxylic acid was then converted to a thioester to enable it to be used in the NCL reaction. Finally, the product was purified using reversed phase high performance liquid chromatography (HPLC) and stored in powder form.

• Synthesis of the C-terminal fragment of paxillin (representing residues 37-557)
• Cloning of the C-terminal fragment of paxillin

This was the first cloning that Vogel had ever done. The DNA encoding for the paxillin fragment and a protease recognition site was transformed into E. coli cells.

• Expression of the C-terminal fragment of paxillin in E. coli

Paxillin is a notoriously challenging protein to express in bacteria, since it is a human protein and has a significant number of rare codons and proline-rich regions. She solved the problem by using codon-enhanced cells, which are E. coli with an extra plasmid in them that allow them to make more of the rare tRNAs.

• Design of a new construct to increase expression and allow for better purification

To solve problems with poor yields, truncation and degradation, a new construct was designed. It incorporated a GST-fusion protein at the N-terminal, to essentially trick the bacteria into expressing the protein, and a purification tag at the C-terminal, to remove the truncation and degradation products.

• Cleavage of the fragment from cells

The construct was designed for use with a Factor Xa protease. However, this protease was found to completely degrade the protein through unselective proteolysis. Eventually, TEV, a different protease, was found to work effectively. A new construct was designed to have a TEV protease cleavage site.

• Testing of Native Chemical Ligation (NCL) technique using a small, simple thioester

In the interest of saving precious quantities of 41 residue N-terminal thioester, a test NCL was performed to join a short thioester with the C-terminal paxillin fragment. The NCL went smoothly and a proper tag for visualization was determined.

• NCL with actual test thioesters and paxillin fragments

Once the practice ligation was successfully completed, it was time to perform the real ligation using the 41 residue N-terminal thioester. Unfortunately, the ligations consistently failed, regardless of alterations in the concentrations of the proteins and pH and variations of the thioester. The ligation only worked under very low pH conditions. After emailing Tom Muir, one of the developers of NCL, it turned out that the glycerol that was being used to store the paxillin fragment in the freezer was capping the cysteine residue and thereby preventing the chemical reaction. The first glycerol-free attempt at NCL worked perfectly. Next, the procedure was optimized.

• In vitro characterization of semisynthetic paxillin

Several assays were performed to prove the function of the reconstituted paxillin. The assays showed that the semisynthetic paxillin was able to bind to paxillin-binding partners and that it was recognized by natural kinases.