I must also thank Jost Vielmetter and his team at the Protein Expression Center (PEC) for being so incredibly helpful throughout. Thank you for being one of the most caring and considerate people I have ever met.
- Protein-Catalyzed Click (PCC) Peptide Capture Agents for Biomarker Detection and
- Epitope Targeting Strategies
- In Situ Click Screening Using Azide-Containing Phage Display Libraries
- References
The physical size of the library limits the number of total sequences that can be screened. These phage libraries can be screened in place of the OBOC peptide libraries described in previous chapters for faster development of PCC agents.
Introduction
- Iterative In Situ Click Chemistry for Protein-Catalyzed Capture (PCC) Agent
- Prostate Specific Antigen (PSA)
This chapter describes the transformation of OBOC's iterative in situ click technology into an efficient and robust technique. In the presence of the protein and an OBOC library with the opposite click handle added, the anchor can click on the library to form a biligand.
Materials and Methods
- Standard Materials
- Peptide Library Construction
- Bulk Peptide Synthesis
- Typical Screening Protocol for Fluorescent Dye-labeled Protein Target Detection
- Typical Screening Protocol for Antibody Signal Amplification Target Only Screens
- Typical Screening Protocol for an Anti-Screen
- Typical Target Screening Procedure During a Multi-Step Screen (Figure 2-3)
- Typical Screening Protocol for a Preclear
- Typical Screening Protocol for a Click Product Screen
- Peptide Sequencing Strategies
The beads were then washed three times with high salt TBS and then incubated on the rocker arm with the high salt buffer for one hour. The beads were then washed three times with BCIP and developed as for the anti-screen.
Results and Discussion
- Screening via Fluorescent Dye-labeled Protein Target Detection
- Screening via Antibody Signal Amplification Target Only Screens
- Introduction of an Anti-screen
- Introduction of a Click Product Screen
- Introduction of a Preclear
- Use of Alkyne Versus Azide Libraries
- Typical Flow of Screening
The product screen is an elegant step in the screening process that allows for a very specific narrowing of the sequence space. The secondary ligands themselves also did not show any binding to the PSA protein, independent of the anchor ligand.
Conclusions
This methodology has an incredibly high success rate that is only getting better as the process continues to grow and evolve.
Acknowledgements
Introduction
- The E17K Mutation in the Pleckstrin Homology Domain of Akt1
- A General Strategy for Targeting Single Amino Acid Point Mutations in Proteins 53
- Akt1 PH Domain Expressions
- Design and Synthesis of Epitope-Targeting Anchor/Target Peptide
- CD Spectroscopy of 33-mer Target Peptide Epitope
- Screen for Initial Anchor Ligand Peptide
- Hit Library Bead Sequence Analysis
- Streptavidin-Agarose Immunoprecipitation (Pull-down) Assays for Binding Affinity
- HPLC-Detected Immunoprecipitation (Pull-down) Assays (Epitope Targeting
- Ligand-Directed Tosyl Labeling Experiments
- Details of the MALDI-TOF Analysis of Tryptic Peptide Fragments
- Images of Anchor Ligand in HEK-293T Cells Expressing PH Domains
Beads remaining from the preclear were washed three times with 1x TBS, then incubated with 5 ml of a 100 nM dilution of the 33-mer epitope target in 0.5% milk for either 5 h or 12 h to allow for ' an in situ click response to occur. The beads were then washed three times with 1x TBS, and incubated for one hour with a 7M Guanadine-HCl buffer (pH = 2.0) to remove all the 33-mer epitope target that was not covalently attached to the beads. These beads were then washed ten times with 1x TBS, blocked for two hours in 5% milk, then incubated for one hour with a 1:10,000 dilution of streptavidin-alkaline phosphatase conjugate in 0.5% milk in TBS to track for the presence of the 33-mer epitope target clicked on a bead.
Assays were performed using 50 μL streptavidin-agarose slurry (25 μL resin) in Spin-X tubes (Sigma) to allow easy removal of the solutions. The resin was then incubated with 200 μL of 7M guanidine-HCl (pH = 2.0) buffer used to remove beads on the sieve. The peak observed on HPLC indicated how much of the 33-mer epitope bound to either the yleaf anchor or the empty beads.
Protein blank cells were also incubated with 50 nM of leaf anchor to ensure that binding was due to the presence of the E17K mutant protein.
Results and Discussion
- In situ Click Epitope-Targeted Screening Strategy for E17K PH Domain-Specific
- Verification of the Epitope Targeting Strategy
- Ligand-Directed Labeling Experiment to Confirm Epitope Targeting and Ligand
- In Cell Imaging
Two of the four other candidates also favored the E17K mutant protein. Therefore, peptide binding to the E17K fragment should have a surface similar to that of the full-length PH domain protein. The selectivity of the yleaf peptide for the E17K 33-mer epitope was also tested in an ELISA assay format.
The approach yields information relative to the binding site of the ligand on the protein target. For this method, a payload is attached to the N-terminus of the target yl ligand via an electrophilic tosylate linker. Live cell-based assays may provide a demanding environment to demonstrate the selectivity of the yllleaf PCC agent to the E17K Akt1.
Green fluorescence indicates equivalent expression of WT and Mut proteins in cells.
Conclusions
These measurements demonstrate the selectivity of the E17K capture agent to its target in the demanding environment of living cells.
Acknowledgements
Jost Vielmetter, Angela Ho, and Sravya Keremane of the Protein Expression Center were indispensable in the expression of these proteins. Felicia Rusnak and Jie Zhou performed trypsin digestion and LC/MS for protein labeling experiments. Mona Shahghola's advice on MS techniques and experimental setup was essential to the success of the labeling experiments.
- Introduction
- Materials and Methods
- Screen for Biligand Peptide
- Streptavidin-Agarose Immunoprecipitation (Pull-down) Assays to Test Biligand
- Screen for Triligand Peptide
- Full ELISA Curves for Ligands
- Point ELISA Assays for Triligand Binding to Akt1 and Akt2 Wildtype and E17K
- PIP3 Agarose Inhibition Assays
- Results and Discussion
- Biligand Development
- Triligand Development
- Inhibition Assays
- Conclusions
- Acknowledgements
- References
The beads were incubated with a 7.15μM solution of the anchor peptide - Biot-PEG5-yleaf-Pra for one hour, then washed three times with 1xTBS. The presence of the his-tagged PH domain is detected via an anti-His antibody. These assays were performed to test the binding of the triligand to the off-target Akt2 wild-type and mutant proteins.
To test the inhibition of each of the ligands, anchor biligand and triligand, 20 μl of resin slurry was added to each of the four tubes and washed three times with 1x TBS. The iryrn triligand shown in red shows a significant increase in affinity, but loses much of the selectivity for the E17K mutant PH domain (triangles). The ivdae retains the selectivity and affinity of the anchor ligand and was continued as the triligand.
This assay shows significant selective inhibition (by about 103) of the E17K mutant over the WT.
Introduction
- Azide-Containing Phage Display Libraries
- Mirror-Image Phage Display
- G6PD Capture for Malaria Eradication
To screen each sequence once in a 7-mer sized library would require 447 g of beads – a virtually impossible task using our current methods. In the phage display screen, however, it is trivial to fully screen this 7-mer library 100 times in each screen – a major advantage of the biodisplay technique. In this technique, an L-amino acid phage library is screened against a target synthesized from D-amino acids in order to find a D peptide that binds to the L-target4, where the D target forms an exact mirror image of the L-target.
Therefore, screening against a mirror image of the target and reversing the stereochemistry of the hit peptide linker produces a D ligand that binds to the original L target. With the epitope targeting strategy currently used for PCC drug discovery in the laboratory, where a chemically synthesized portion of the protein is used as a target for screening, it is trivial to prepare a D-amino acid epitope for use in a mirror phage screen. This is due to the higher than normal incidence of young red blood cells, which express a higher G6PD copy number than mature red blood cells, compensating for the reduced activity of the enzyme caused by the mutation6.
The goal of this project is to develop a capture agent that universally detects G6PD in all possible mutant forms in order to capture the protein on a chip where G6PD concentration and activity in blood can be measured simultaneously.
Materials and Methods
- Preparation of Plasmid for Incorporation of Azidophenylalanine and Amp
- Test of Azidophenylalanine Incorporation into a Protein in E.coli
- Test of Azidophenylalanine Incorporation into M13KE Phage
- Synthesis of M13KE Azidophenylalanine-Terminated 7-mer Random Library
- Design and Synthesis of G6PD Target and Scrambled Target for Screening
- Optimized Phage Library Target Screening Conditions
- Testing Phage Plaques for Library Inserts
- Incorporation of Azidophenylalanine into Phage Libraries
- Optimized Phage Library Click Screening Conditions
This leads to a greater prevalence of the amino acid glycine (G) throughout the distribution (Figure 5-5). Recall that the third position is limited to T and G due to the NNK library format. The high prevalence of the G base pair at the first site resulted in a much higher prevalence of amino acid G than expected.
In the crystal structure (PDB ID: 1QKI) in Figure 5-8, the region encoded by exons 2 and 3 is unstructured in red at the bottom right of the protein. Therefore, the capture agent must be targeted to the amino acids shown in black to ensure that it does not bind to a highly mutated portion of the protein. This mutant G6PD in complex with NADP+ indicates that the unstructured regions encoded by exons 2 and 3 (lower right in red, not completely present in structure) are far from the active site of the protein.
A stock solution of azidophenylalanine was freshly prepared by dissolving 8 mg of amino acid in 250 μL of DMSO and 250 μL of acidic water (pH = 2.0). The entire volume of the stock solution of azidophenylalanine was added to the culture to achieve the final concentration of 2 mM.
Results and Discussion
- Test of Azidophenylalanine Incorporation into a Protein in E.coli
- Test of Azidophenylalanine Incorporation into M13KE Phage
- Phage Library Screening Conditions and Results
- Focused Library Screening
This result indicates that the mutated MjTyrRS for azidophenylalanine is functional and incorporates the azidophenylalanine amino acid into the amber stop codon of the protein. Following the successful incorporation of the azidophenylalanine amino acid into a protein in E. coli, incorporation was tested using a small number of M13KE phage clones containing representative display peptide sequences similar to the library designed for the subsequent in situ click screen . This allowed fluorescence visualization of the dye-labeled pIII protein resolved by SDS-PAGE.
The high prevalence of naked phage in the library complicates subsequent incorporation of azidophenylalanine, as naked phages that infect rapidly and do not require repression of the amber codon have a significant growth advantage. For this reason, traditional phage display screening methods—“target” screens that only require binding of the library component to the target of interest—were used to minimize the number of naked phages present in the incorporation step. The first band above corresponds to ~65 kDa, the known mass of pIII protein.
Therefore, from this target screen, 11 of the amplified stocks containing inserts (Table 5-4) were pooled to create a "focused" library of phage known to bind to the target.
Conclusions
Acknowledgements
Regular Use
- Loading a Bead
- Solvents
- Ordering
- Contacting AB
- Idle machine
- Settings
Most of the solvents used are purchased from somewhere other than Applied Biosystems and mixed in-house. To loosen or tighten this bottle, you must first press hard on the ratchet to hold it in place while you screw or unscrew the bottle. The machine should never be left completely stationary, otherwise the column may dry out and must be replaced.
If the machine is not running, press the "Manual" button on the front of the pumps. It should turn on the pumps at 5uL/min at 50%B and the bottom of this window will change to "idle". This low solvent flow keeps the solvents moving and the column wet. These are our normal settings for the machine in case something gets lost or goes to rest and needs to be reprogrammed.
These are also the settings used for all the amino acid sequencing in this thesis.
Troubleshooting
- Computer Errors, Freezing, or Not Saving Spectra
- Machine Bottle Runs Dry
- HPLC Bottle Runs Dry
- Baseline Errors – Dip at front of Spectra
- Baseline Errors – Stretched Out Amino Acid Standards
- Pressure Errors – Change Bottle Seals
- Pressure Errors – Clogged Lines
The pump must then be bled manually using the buttons on the front of the pumps. When cleaning is complete, press "Manual" on the front of the pump to idle the pumps. Allow the pumps to run freely until bubbles stop coming from the end of the column (as seen in the waste tube).
To do this, you need to unscrew the gray metal screw that goes into the pump cylinder, then unscrew the black ring around the top of the cylinder. Remove the cylinder using the pump opener function to bleed the pump you are working on and PUSH the cylinder out. One of the pump seals that needs to be replaced is the one at the end of the rod.
If one of the metal alignment pieces cave in, it will be VERY difficult to remove.
