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    Progress

    For a complete look at the research I have conducted for my GCS experience, please see my thesis on the GCS thesis page.

    Updates

    April 2020

    Micelle-Forming Diblock Polypeptides Project

    Due to Duke’s transition to online learning as a result of the Covid-19 pandemic, further research on this project has been stalled. My future plans for the remainder of my time at Duke were as follows. More details about these planned experiments can be found in my thesis.

    • Assess the antigen presentation of Ova-RLP1-40-ELP1-80-K12 by performing flow cytometry. Verify that the Ova peptide is being properly cross-presented.
    • Test the activity of CpG in the Ova-RLP1-40-ELP1-80-K12:CpG complex using a HEK-Blue TLR9 Assay
    • Confirm CpG uptake using confocal imaging.
    • Further characterize the self-assembly of Ova-RLP1-40-ELP1-80-K12 with CpG using cyroTEM.
    • Finish cloning remaining constructs in Ova-RLP-ELP-K12 library.
    • Express and purify remaining Ova-RLP-ELP-K12 constructs.
    • Characterize the remaining Ova-RLP-ELP-K12 constructs (with and without CpG) using UV-Vis spectroscopy, dynamic light scattering, and cryoTEM. Perform additional assays on viable vaccine candidates.
    • Determine ideal candidate for nanoparticle-based vaccine platform.
    • Perform initial animal study to assess therapeutic potential.

    March 2020

    Micelle-Forming Diblock Polypeptides Project

    In the beginning of the Spring 2020 semester I was able to visualize the structure of the Ova-RLP1-40-ELP1-80-K12 protein complex with CpG using cryoTEM, with the help of a lab member, Dr. Mike Dzuricky. In the absence of CpG, Ova-RLP1-40-ELP1-80-K12 forms both spherical and wormlike micelles. In the presence of CpG, Ova-RLP1-40-ELP1-80-K12 forms spherical and wormlike micelles in addition to larger, zipper-like structures. These zipper-like structures range in size, and are on the scale of 100s of nm.

    Additional work has been completed to clone, express and purify other Ova-RLP-ELP-K12 constructs. Ova-RLP1-80-ELP1-160-K12 was also successfully expressed and purified.The status of these other proteins can be seen below.

    RLP-ELP Diblock K12 cloned? Ova cloned? Expressed? Solublility Successfully Purified?
    RLP140-ELP180 Yes Yes Yes Soluble Yes
    RLP1100-ELP180 Yes Yes Yes Moderately soluble No
    RLP180-ELP1160 Yes Yes Yes Moderately soluble Yes
    VA5-RLP140-ELP180 No No No N/A N/A
    VA4-RLP180-ELP180 No No No N/A N/A
    RLP180-ELP2160 Yes Yes No N/A N/A
    RLP180-ELPS160 Yes Yes Yes Moderately soluble No
    RLP280-ELP2160 Yes No No N/A N/A
    RLP280-ELPS160 Yes Yes Yes Moderately soluble No

    Please see my thesis for a more comprehensive overview of my research, including data and figures.

    December 2019

    The choice to switch projects

    After much consideration, I have decided to switch the focus of my GCS research to another project that I have been working on in the Chilkoti laboratory, the development of micelle-forming diblock polypeptides for a cancer immunotherapy. I had struggled with my previous project to produce and purify the Flt3L cytokine. After struggling with issues such as protein production and purification, and obtaining unideal results for an in vitro activity assay, I have decided to shift focus to another project with which I have already had tremendous success and is another biomaterial-based cancer immunotherapy. This new project is a nanoparticle vaccine that targets dendritic cells to prime a cancer specific immune response. Nanoparticles have demonstrated tremendous potential for the enhancement of immunotherapies by promoting increased antigen uptake, programming accumulation in the lymph nodes, and decreasing clearance. Here we describe the use of a diblock consisting of reslin-like polypetides (RLPs), hydrophobic repetitive proteins, and elastin-like polypeptides (ELPs), hydrophilic repetitive protein biopolymers for a cancer vaccine consisting of antigen and negatively charged adjuvants, such as CpG. RLP-ELP diblocks, a novel class of amphiphilic diblock polypeptides, are a strong choice for such as they provide a robust platform for creating micelles of a given size and morphology.

    Thus far, I have been able to express and purify Ova-RLP40 -ELP-80 -K12. I have characterized the temperature dependent phase of the protein cytokine complex using UV-Vis spectroscropy, and determine the hydrodynamic radius using dynamic light scattering. I have shown that the protein’s successful micelle formation and its binding to CpG. Finally, I have also completed an in vitro activity which suggests that micelle-formation enhances the activity of the adjuvant, perhaps by increasing cellular uptake.

    May 2019

    Flt3L Cancer Immunotherapy Project

    Cytokine development

    After much research and careful consideration, I have selected a cytokine, murine Flt3-L (mFlt3-L), suitable for the project. A codon optimized DNA sequence was designed to encode the protein. Cloning was performed to fuse mFlt3-L to an elastin-like polypetide (ELP). Histidine (His) tags were added to explore alternative purification methods. Soluble expression of the fusion protein was achieved, and was increased by changing the host. The fusion protein was successfully purified.

    I am currently working expressing, purifying and characterizing new constructs, with different ELP sequences. The construct I was working with previously did not exhibit the thermoresponsive behavior that is characteristic of ELPs, and I am hoping that by increasing the length of the ELP this issue will be resolved. I am also exploring whether this issue is a function of the melting temperature of mFlt3L, itself.

    Antigen development

    A modified antigen sequence (a peptide of the Trp2 antigen) was successfully designed, cloned and ligated to an ELP. The antigen has been expressed and purified. Current work entails decreasing endotoxin levels so that the protein is ready for our upcoming animal study.

     

    Relevant Coursework

    For the GCS Program

    BME 493: Recombinant Synthesis of Micelle-Forming Diblock Polypeptides Cancer Immunotherapy

    Supervisor: Dr. Ashutosh Chilkoti

    Dates: January 2019-May 2020

    Hours: 35 hours/week

    Continuation of research conducted in the Fall of 2019. Characterized structure of Ova-RLP40-ELP80-K12: CpG with cryo-TEM with the help of lab member Dr. Mike Dzuricky. Cloned two additional Ova-ELP-RLP-K12 constructs, which were successfully expressed, though largely insoluble. Cloned two new Ova-Ova-ELP-RLP-K12 constructs, which successfully expressed and purified.

    BME 494: Recombinant Synthesis of Depot-Forming–Peptide Polymer for Flt3-L Cancer Immunotherapy

    Supervisor: Dr. Ashutosh Chilkoti

    Dates: August 2019-December 2019

    Hours: 25 hours/week

    Continuation of research conducted in the Summer of 2019. Worked to purify the ELP-mFlt3L fusion. Switched construct design to use human version of the cytokine (much more prevalent in literature; possible due to cross-reactivity). Cloned new constructs of ELP-hFlt3L-His and His-hFlt3L-ELP. Characterized of the isolated proteins via SDS-page, Cary UV-Vis Scans, and DLS.

    Began to work on RLP-ELP project. Cloned five different Ova-ELP-RLP-K12 constructs. Expressed and purified Ova-RLP40-ELP80-K12. Verified protein binding to CpG. Characterized the temperature dependent phase behavior of the protein and protein CpG complex with UV-Vis spectroscopy. Characterized the hydrodynamic radius with DLS. Completed an in vitro assay which suggested that micelle formation ameliorated CpG activity, perhaps due to enhanced uptake.

    Pratt Fellows Summer Research Program (same project as GCS)

    Supervisor: Dr. Ashutosh Chilkoti

    Dates: May 2019-August 2019

    Hours: 40 hours/week

    Continuation of research conducted in the Fall of 2018.

    Continued to to produce and purify the ELP-mFlt3-L fusion by exploring a different expression host, and additional purification protocols. Continued to optimize his-tag purification, by optimizing buffers, resin, and columns. Expressed and purified additional antigen protein for the depot, and the ELP-K12 protein (to bind the protein electrostatically). Characterized of the isolated proteins via SDS-page, Cary UV-Vis Scans, and DLS. Completed bioactivity assays for the other components of the depot.

    BME 493: Recombinant Synthesis of Depot-Forming–Peptide Polymer for Flt3-L Cancer Immunotherapy

    Supervisor: Dr. Ashutosh Chilkoti

    Dates: January 2019-May 2019

    Hours: 20 hours/week

    Continuation of research conducted in the Fall of 2018.

    Worked to produce and purify the ELP-mFlt3-L fusion by exploring a different expression host, and additional purification protocols. Explored ion exchange for purification. Worked to optimize his-tag purification, by optimizing buffers, resin, and columns. Expressed antigen protein for the depot, and the ELP-K12 protein (to bind the protein electrostatically). Cloned and purified soluble controls for parallel animal study with different cytokine. Characterized of the isolated proteins via SDS-page, Cary UV-Vis Scans, and DLS.

    BME 493: Recombinant Synthesis of Depot-Forming–Peptide Polymer for Flt3-L Cancer Immunotherapy

    Supervisor: Dr. Ashutosh Chilkoti

    Dates: August 2018-December 2018

    Hours: 16 hours/week

    The cytokine for the project was selected after careful consideration. A DNA gBlock was designed to encode the mFlt3-L protein. This was ligated into a DNA vector via Gibson Assembly. Two different constructs were formed via ligation: one with the mFlt3-L protein located at the N terminus of the protein (mFlt3-L-ELP), and one with the mFlt3-L protein located at the C terminus of the protein (ELP-mFlt3-L). His tags were also added to explore alternative purification methods, generating two additional proteins: His-mFlt3-L-ELP and ELP-mFlt3-L-His. His tag purification and inverse transition cycling (ITC) were tested. Cloning for antigen component, Trp2-long, was completed.

     

    Prior to GCS

    EGR 393: Tandem PTM of Polypeptides 

    Supervisor: Dr. Ashutosh Chilkoti

    Dates: January 2018-May 2018

    Hours: 12 hours/week

    Continuation of research conducted in the Fall of 2017. Additional protein was expressed and purified. The protein was analyzed using several different methods including SDS-page, Reverse Phase (RP) HPLC, dynamic light scattering (DLS) and MALDI.

    EGR 393: Tandem PTM of Polypeptides

    Supervisor: Dr. Ashutosh Chilkoti

    Dates: August 2017-December 2017

    Hours: 12 hours/week

    Worked in the Chilkoti Laboratory to control the self-assembly of a peptide-polymer ELP, via dual post-translational lipidation. A DNA vector containing the relevant genetic material was cloned. The vector was transformed into competent E. Coli and expressed. The expressed protein was purified via ITC. The purified protein underwent a reaction with cholesterol.