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Timeline
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- Experiment
- 1. Test synthesis of 40-mer Tyr31 peptide (01/15/2003 - 01/16/2003)
Purpose/Intro: To make the N-terminus of paxillin using an automated peptide synthesizer. The 40-residue polymer can be made to include an unnatural amino acid using the peptide synthesizer.
Results: The major product had a mass that was much lower than what Beth expected. The expected mass was calculated by adding up the weight of each amino acid in the chain. The actual mass of the product can be determined using HPLC (a separation technique that uses a column) and Mass Spectroscopy.
Links:
PDF: See this in the notebook!
Notebook page
- 2. Test cleavage of 40-mer Tyr31 peptide (01/17/2003 - 01/21/2003)
Purpose/Intro: To identify the problematic coupling step that lead to the incomplete synthesis of the 40-mer peptide.
Procedure: Used test cleavages after the Leu24, Lys20, Glu12, and Met1 couplings.
Results: The fragments resulting from cleavage after the Glu12 and Met1 produced complicated spectra (multiple peaks, each with the same large peak, instead of one major peak). Using the mass, Beth determined that the extra peak resulted from an unsuccessful coupling between Fmoc-His(Trt)-OH and Ile18, thereby capping the 19-residue peptide.
Conclusion: The peptide synthesis was proving to be unsuccessful because of a difficult coupling step. Steric interactions between the bulky protecting group on the histidine and the ?-branched isoleucine residue were making the coupling hard.
Links:
PDF: See this in the notebook!
Notebook page
- 3. 2nd attempt at N-terminus of synthesis of 40-mer Tyr31 peptide (01/24/2003 - 02/04/2003)
Purpose/Intro: To manually perform the challenging histidine/isoleucine coupling using two activating agents (PyBOP and DIPEA). Then, to manually add a hexahistine tag and acetyl cap for purification purposes.
Results: The absence of free amines was determined by a TNBS test.
Links:
PDF: See this in the notebook!
Notebook page
- 4. Thioesterification reaction on resynthesized peptide. (02/06/2003)
Purpose/Intro: To convert the carboxylic acid at the C-terminus of the 40-mer peptide into a thioester. The thioester is needed in that position in order to complete the Native Chemical Ligation in later steps of the procedure.
Results: The thioesterification worked well, but it oxidized the Methionine residue. Met can be excluded because the N-terminus Methionine is natively cleaved in the cell.
Conclusion: Synthesis of the N-terminus of Paxillin was complete.
Links:
PDF: See this in the notebook!
Notebook page
- 5. Synthesizing the C-terminal paxillin fragment by cloning. (02/14/2003 - 02/21/2003)
Purpose/Intro: To clone His-IEGRC-Pax(38-557). In order to this, it was necessary to utilize the machinery that bacteria naturally have in order for them to create copies of the paxillin fragment. The steps outlined below basically insert the "original" fragment and ask the bacteria to act as copy machines to produce many more fragments.
Procedure: Design the primer that will be used in the replication of residues 38-557 of Paxillin. Use the primers in a PCR reaction to create copies of the Paxillin fragment. Clone the PCR product into a TOPO vector. Transform bacteria with the TOPO vector and insert. Test the transformation by using a restriction digest, which uses enzymes that recognize a specific sequence of DNA and cuts it at a particular location. Beth used EcoRI, since the DNA template did not have an EcoRI cut site, but the TOPO vector did.
Results: The initial EcoRI digests suggested that none of the bacterial colonies had taken up the TOPO vector. Need to repeat the experiment.
Links:
PDF: See this in the notebook!
Notebook page
- 6. Repeat of transformation of purified PCR product with 3prime-A overhang addition (03/04/2003 - 03/05/2003)
Purpose/Intro: To repeat the transformation and find a successful construct.
Results: The EcoRI digest showed a promising result, and a second digest using BamH1 added credibility to the integrity of the transformation. The purified DNA was sent for DNA sequencing to confirm the transformation.
Conclusion: Ready for sequencing and expression.
Links:
PDF: See this in the notebook!
Notebook page
- 7. Expression and purification attempts (03/06/2003 - 04/10/2003)
Purpose/Intro: Paxillin is a challenging protein to express in bacteria. Beth tried for many days to get paxillin expressed in bacteria, but her attempts were resulted with degraded and truncated products.
Procedure: (1) Transformation of construct: Purified DNA was transformed into E. coli cells. The bacteria were incubated overnight in LB media, which is a nutrient rich broth that bacteria love to grow in. (2) Split the culture into two groups. Added IPTG to one of the bacterial batches - IPTG is a chemical very similar to lactose that is used to induce the expression of cloned genes. (3) Visualized the protein using gel electrophoresis, which separates proteins based on size, to determine purity of the expressed protein.
Links:
PDF: See this in the notebook!
Notebook page
- 8. Repeat expression and purification using codon-enhanced cells (04/10/2003)
Purpose/Intro: To understand the problems of expression and purification of paxillin.
Results: A Post-doctoral researcher in the lab suggested that Beth should search for rare codons in paxillin. After examining the DNA sequence, she realized that not only were there were 27 rare ccc (proline) codons, but many were close together and at the beginning of the sequence. This is a result of the fact that seven codons in humans appear very rarely in E. coli, which can become a problem if the E. coli have to produce a lot of them. As the protein is being transcribed, the E. coli does not have the tRNAs available, so the protein becomes truncated.
Conclusion: Repeat the transfection using codon enhanced cells, where the E. coli have an extra plasmid that allows them to make more of the rare tRNAs.
Links:
PDF: See this in the notebook!
Notebook page
- 9. Transformation into Rosetta (codon-enhanced) cells (04/10/2003 - 05/01/2003)
Purpose/Intro: To repeat the transfection with Rosetta cells, which are genetically engineered E. coli that are equipped with the machinery to make all the rare tRNAs.
Results: The expression was still not great. Since the Rosetta cells encode all rare codons, Beth had problems because the vector put in them was so big that it would fall out.
Links:
PDF: See this in the notebook!
Notebook page
- 10. Transformation into BL(21) Codon Plus (RP) cells (05/01/2003 - 06/10/2003)
Purpose/Intro: To repeat the transfection with BL(21) Codon Plus (RP) cells, which only code for the AGG (Arg), AGA (Arg) and CCC (Pro) tRNAs.
Results: Expression of the paxillin construct was significantly improved. However, there were still major truncation products.
Links:
PDF: See this in the notebook!
Notebook page
- 11. Redesign construct to increase expression (06/10/2003)
Purpose/Intro: To design a new construct, GST-IEGRC-Pax(38-557)-FLAG to increase expression and have a C-terminal tag for purification (to isolate full-length product from truncation).
Procedure: (1) Design new primers that included a GST-tag, which E. coli really likes to express. By placing GST before the paxillin construct, the bacteria are essentially “tricked” into expressing a construct that would normally be difficult. (2) The FLAG tag, an 8 amino acid sequence, is added to the C-terminus. Since proteins are expressed from the N to C terminus, the existence of the FLAG tag at the end of the protein means that the entire protein has been expressed. Purifying the protein for FLAG eliminates all of the truncated products.
Links:
PDF: See this in the notebook!
Notebook page
- 12. Correct constructs found- ready for sequencing/expression (07/14/2003)
Results: The minipreps and digests showed that most of the colonies had the correct vector/insert.
Links:
PDF: See this in the notebook!
Notebook page
- 13. Transformed into RP enhanced cells (07/15/2003 - 09/01/2003)
Purpose/Intro: To transform the plasmid into the BL21-CodonPlus-RP Competent Cells. The cells are engineered to express codons for arginine and proline.
Results: Transformation worked. Resulting protein was purified using the GST-tag and FLAG-tag.
Links:
PDF: See this in the notebook!
Notebook page
- 14. Paxillin cleavage using Factor Xa (09/01/2003)
Purpose/Intro: To reveal this N-terminal cysteine, a protein-cleaving enzyme, called a protease, was used. The original construct for the C-terminal paxillin fragment had a sequence that could be recognized and cleaved using a Factor Xa protease. Factor Xa was selected because it was previously used widely with NCL.
Results: “The Factor Xa seems to be completely chewing up my previous protein!”
Notes: In order to perform Native Chemical ligation, the C-terminal peptide must have an N-terminal cysteine.
Links:
PDF: See this in the notebook!
Notebook page
- 15. Changing the conditions of Factor Xa cleave to try to eliminate non-specific cleavage (09/01/2003)
Purpose/Intro: To eliminate the unselective proteolysis of paxillin.
Procedure: Beth tried changing the conditions of the cleavage by lowering the concentration of Factor Xa (4-41), lowering the temperature from room temperature to 4 degrees C (4-43), adding zinc to "improve folding and decrease secondary site cleavage by organizing the four LIM domains (double Zn fingers)."
Results: The protease was either chopping the protein into pieces or not cutting at all. The unwanted proteolysis seemed to be occurring after glycine-arginine pairs.
Links:
PDF: See this in the notebook!
Notebook page
- 16. Test cleavage with enterokinase, an alternate protease (09/01/2003)
Purpose/Intro: To test for non-specific cleavage with enterokinase, which cleaves C-terminal to a DDDDK recognition sequence.
Results: There was significant proteolysis of the paxillin fragment in the presence of enterokinase.
Notes: Since paxillin was being non-specifically chopped up by Factor Xa, a different protease had to be tried.
Links:
PDF: See this in the notebook!
Notebook page
- 17. Synthesis of phosphoTyr31 and caged phosphoTyr31 thioesters. (09/15/2003 - 11/01/2003)
Purpose/Intro: To synthesize phosphoTyr31 and caged phosphoTyr31 thioesters using the automated peptide synthesizer.
Results: HPLC traces shown at various stages.
Links:
PDF: See this in the notebook!
Notebook page
- 18. Test cleavage with Furin, an alternate protease (10/07/2003)
Purpose/Intro: To test for non-specific cleavage with a different protease, furin.
Results: The paxillin was degraded, though not as much as with enterokinase or Factor Xa.
Links:
PDF: See this in the notebook!
Notebook page
- 19. Test cleavage with Tobacco Etch Virus (TEV), an alternate protease (10/08/2003)
Purpose/Intro: To test for non-specific cleavage with a different protease, tobacco etch virus (TEV).
Results: No undesired cleavage with GST_IEGR-Cys-Pax(38-557)-FLAG!
Links:
PDF: See this in the notebook!
Notebook page
- 20. Design a new construct to incorporate a TEV cleavage site (11/04/2003)
Purpose/Intro: To design a new construct to incorporate a TEV cleavage site. Since the TEV protease did not cause undesirable cleavage, it was now necessary to change the construct for TEV to cleave N-terminal to the paxillin insert. The new construct is: GST-ENLYFQC-Pax(38-557)-FLAG.
Links:
PDF: See this in the notebook!
Notebook page
- 21. Test TEV cleavage of paxillin construct (11/17/2003)
Purpose/Intro: To test the cleavage of the paxillin construct (GST-ENLYFQC-Pax(38-557)-FLAG with TEV.
Results: TEV cleaved the construct perfectly on the first try to give Cys-Pax(38-557)-FLAG. The top band on the gel is the band for the full paxillin construct with GST. The trop is about 27-30 kDa. After treatment with TEV, there was no full-length protein left. Finally, the cysteine piece was ready to use for NCL.
Links:
PDF: See this in the notebook!
Notebook page
- 22. Synthesize a small construct for to test Native Chemical Ligation (01/13/2004)
Purpose/Intro: To test the native chemical ligation with a mock construct. The real N-terminal paxillin construct, which is the thioester piece, has to be made in the peptide synthesizer and is therefore available in very limited quantities. Beth wanted to test the ligation, purification, and visualization using a trial thioester that she designed to be easy to examine.
Procedure: Synthesized Ac-HHHHHHGKWG-COSBn for test NCL. The rationalization behind the construct design is as follows. The histidine tag (H6) was used for visualization using a dot blot (as seen on the lower left corner of 4-111). The lysine (K) residue was inserted because it has a positive charge that allows it to go very quickly through mass spectroscopy, making it easy to determine the mass of the peptide. The tryptophan (W) residue makes the peptide easier to visualize in HPLC.
Links:
PDF: See this in the notebook!
Notebook page
- 23. Compare tetra vs. hexahistidine tag for visualization of Native Chemical Ligation (01/14/2004)
Purpose/Intro: To compare the visualization using a tetra- (H4) versus hexa- (H6) histidine tag. (Remember that this peptide AcHHHH(HH)GKWG-CosBn was synthesized using the automated peptide synthesizer. Surprisingly, it is much easier to synthesize a 39-mer peptide than a 41-mer.)
Results: Simply by reducing the length of the peptide by 2 residues, Beth was able to synthesize much more of the product. However, as seen in the dot blots on 4-108, there was a strong signal when using the mouse anti-hexahistidine antibody but nearly no signal for the mouse anti-tetrahistidine antibody. Thus, Beth found out that it was worth the extra effort to use a hexahistidine tag.
Links:
PDF: See this in the notebook!
Notebook page
- 24. Testing Native Chemical Ligation with the hexahistadine-tagged thioester (01/20/2004)
Purpose/Intro: To use the hexhistidine-tagged thioester to test native chemical ligation.
Procedure: 1) Thawed, isolated, purified, and cleaved the C-terminal paxillin fragment from E. coli, as described previously. 2) Performed a NCL with the test peptide to get Ac-HHHHHHGKWG-Cys-Pax(38-557)-FLAG. 3) Visualized the ligation product by running the products on gels and treating with a mouse anti-FLAG antibody and a mouse anti-hexahistidine antibody. Found that lanes 6 and 7 of the anti-hexahistidine blot contained the ligation product.
Results: Successful test NCL.
Links:
PDF: See this in the notebook!
Notebook page
- 25. Synthesis of complete hexahistidine-tagged Tyr31, pTyr31, and caged-pTyr31 thioesters (02/01/2004 - 05/01/2004)
Purpose/Intro: To synthesize complete hexahistidine-tagged Tyr31, pTyr31, and caged-pTyr31 thioesters. (Now that Beth knew that the Native Chemical Ligation would work, it was time to synthesize multimilligram quantities of the thioesters to perform NCL on later.)
Procedure: 1) Make the carboxylic acid derivatives using the automated peptide synthesizer. 2) Treat convert the carboxylic acid into a thioester. 3) Purify the thioester using columns. 4) Repeat for the different types of thioester.
Results: Successful synthesis.
Links:
PDF: See this in the notebook!
Notebook page
- 26. Native Chemical Ligation with paxillin thioesters (05/01/2004 - 10/31/2004)
Purpose/Intro: To perform NCL on the paxillin thioesters and cysteine constructs.
Results: Beth tried nearly every combination of protein and peptide concentration, pH, thioester, etc. to try to get the real ligation to work. She tried different thioesters since they catalyze the reaction. The only time she could get the NCL to work was using a really low pH of 4, even though the reaction should have worked at physiological conditions. This was a very confusing, upsetting time period since the science indicated that the reaction should work, but it was not! In fact, the results were counter-intuitive because the reaction should work better at a higher pH, since the thioester loses a proton to become a negatively charged sulfur instead of a -SH.
Links:
PDF: See this in the notebook!
Notebook page
- 27. Optimization of concentrations, pH, thiols (10/01/2004 - 11/30/2004)
Purpose/Intro: To optimize the concentrations of the thioesters, cysteines, and pH.
Results: Using 5-73 as an example, found the optimal concentrations. Beth didn't worry about achieving perfectly clean gels, since after ligation is it possible to just pick out the desired band on the gel (as shown in the notebook.)
Links:
PDF: See this in the notebook!
Notebook page
- 28. Advice from Professor Tom Muir (10/18/2004)
Purpose/Intro: To ask one of the developers of NCL, Tom Muir of the Rockefeller Institute, for insight into why the reaction was not working.
Results: Dr. Muir basically responded with the comment that it was strange that the reaction was not working, and that the only reason he could think of was if the thioester was exposed to glycerol at any point. Beth realized that she had been adding glycerol in order to store her proteins in the -20 degree freezer, since glycerol allows safe storage of proteins by preventing them from completely freezing. Glycerol inherently should not cause problems, but it also contains small amounts of glyceraldehde. A cysteine and aldehyde react, causing the cysteine to become capped. Once capped, the cysteine cannot participate in the NCL reaction. This also rationalized Beth's previous finding that the NCL worked under low pH conditions, since the capping of the cysteine was reversible in acid. Suddenly, everything worked! Beth described it as, "This was the best day ever. All of a sudden everything made sense. Now I know I have all my pieces and know that it's going to work."
Conclusion: Beth now knows that all of the reagents of her reaction must be prepared fresh (to eliminate the necessity to freeze using glyercol.)
Links:
PDF: See this in the notebook!
: Paxillin Ligation Difficulties
Notebook page
- 29. Cloning and expression of GST-tagged paxillin binding regions of FAK and GIT (12/14/2004 - 02/16/2005)
Purpose/Intro: To examine the functionality of the reconstituted paxillin by testing its ability to bind with known paxillin binding partners, FAK and GIT1.
Paxillin is a molecular adaptor protein but not an enzyme. Therefore, to prove its functionality, it is not possible to simply observe the catalysis of a reaction. Rather, paxillin is known to bind with proteins like focal adhesion kinase (FAK), GRK interactor 1 (GIT1), and PTP-PEST. By proving that paxillin can still be recognized by these proteins, Beth proved that the structure of paxillin was intact.
Procedure: First, Beth needed to clone and express GST-tagged paxillin binding regions of FAK and GIT. The GST tag is necessary for purifications using a GST-affinity column. In order to do this, she inserted the paxillin-interacting regions of FAK and GIT into GST-fusion vectors using the appropriate primers. The proteins were then expressed by the bacteria and isolated from their cell lysates.
Results: Paxillin binding was "detected strongly with the FAK and GIT constructs, negligibly with GST alone."
Links:
PDF: See this in the notebook!
Notebook page
- 30. First GST pull down attempt between paxillin and FAK and GIT (03/03/2005)
Purpose/Intro: To try to "get a positive readout as a starting point" between GST-FAK and paxillin.
Results: The pull down attempt was successful.
Links:
PDF: See this in the notebook!
Notebook page
- 31. Expression of GST-PTP-PEST (05/03/2005)
Purpose/Intro: To show binding between PTP-PEST and paxillin.
PTP-PEST binds at the C-terminal zinc-binding LIM domains of paxillin.
Results: The binding was weak. Beth "evaluated whether improper folding of these domains due to insufficient Zn(II) in the purified construct contributed to the poor PTP-PEST binding characteristics. The addition of ZnCl2 to the paxillin solution significantly increased PTP-PEST binding."
Links:
PDF: See this in the notebook!
Notebook page
- 32. Phosphorylation of full length paxillin with upstream kinases (06/28/2005)
Purpose/Intro: To show that the full-length paxillin was recognized and phosphorylated by these kinases.
In the body, paxillin is recognized by several upstream kinases, including Src, ERK, and JNK. These kinases cause the phosphorylation of paxillin at different residues: Src phosphorylates Tyr31 and Tyr118, ERK phosphorylates Ser126, JNK phosphorylates Ser178.
Procedure: 1. Treat paxillin with Src, ERK, and JNK.
2. Visualize the products by Western blots using a general anti-paxillin antibody (to show the presence of un-phosphorylated paxillin) and 4 phosphorylation-specific anti-paxillin antibodies.
Explanation of blots:
Lane 1: blank.
Lane 2: Paxillin without a kinase.
Lane 3: Paxillin with Src.
Lane 4: Paxillin with ERKII.
Lane 5: Paxillin with JNK.
Results: Paxillin was recognized by upstream kinases.
Links:
PDF: See this in the notebook!
Notebook page
- 33. Phosphorylation and chemiluminescent trials with ligated vs. truncated paxillin (07/27/2005)
Purpose/Intro: To repeat the assays using the semisynthetic construct Y31Pax.
Results: "Considering the extremely high sensitivity of chemiluminescent detection, it was encouraging that visualization of ligated paxillin (Y31Pax) with the anti-paxillin pan antibody resulted in a clean gel containing one major band for the ligated paxillin, no detectable band for uncleaved protein (GST-ENLYFQ-Cys-Pax(38-557)-FLAG), and only a light band for unligated material (Cys-Pax(38-557)." "The identical results for the kinase assays with expressed and semisynthetic paxillin, as well as the agreement with in vivo and in vitro reports of paxillin phorphorylation, further confirmed that the semisynthetic proteins behave comparably to native paxillin."
Links:
PDF: See this in the notebook!
Notebook page
- 34. Example of uncaging detection by chemiluminescence (10/18/2005)
Purpose/Intro: To quantify the uncaging of NPE-caged paxillin. Needed to show that uncaging worked on the whole protein and not just the peptide.
Procedure: Used the technique of quantitative Western Blots.
Results: Achieved about 87% uncaging after 90 seconds.
Links:
PDF: See this in the notebook!
Notebook page
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