This research experience was at The Scripps Research Institute (TSRI) in La Jolla, California.
The Scripps Research Institute (TSRI) — one of the world’s largest, private, non-profit research organizations — stands at the forefront of basic biomedical science, a vital segment of medical research that seeks to comprehend the most fundamental processes of life.(www.scripps.edu)
In here I had the experience of working in the International AIDS Vaccine Initiative (IAVI) Neutralizing Antibody Center under the expertise of Dr. Richard Wyatt and Dr. Javier Guenaga in HIV-1 gp140 trimer design for protein-based vaccine approach.
Abstract
Recent studies show that ~20-30% of HIV-1 infected individuals develop broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) and, it is likely that a native-like and stable Env mimic will be required for an effective HIV-1 vaccine. We designed and characterized two soluble Env mimics derived from Donor 45 Env sequences, from whom VRC01, VRC02, VRC03 and other bNAbs targeting the CD4-binding site (CD4bs) were isolated. In this study, we used the Native Flexible Linker (NFL) platform for the design and development of two native like, soluble, cleavage independent and stable Env trimers by de novo synthesis. This platform replaces the furin cleavage site in gp140 with a flexible “Glycine-Serine” peptide linker (2xG4S) and has a series of stabilizing substitutions to achieve highly stable and homogeneous covalently linked gp120-gp41 trimers in native-like association and structure. Size-exclusion chromatography profiles of the lectin-purified trimers showed a low degree of aggregation and low levels of dissociation into monomeric forms of Env. Differential scanning calorimetry (DSC) analysis revealed thermal transition midpoints (Tm) for both NFL trimers of over 73°C. Biomolecular binding assays using Bio-layer light interferometry showed reactivity with trimer preferring bNAbs while excluding binding by non-neutralizing antibodies. Weak but detectable recognition was observed with the germline-reverted precursors of the 3BNC60, VRC13 and VRC16 bNAbs targeting the CD4bs. We conclude that these trimers may have utility to trigger neutralizing antibody responses in vivo.
In this research experience I gained deep knowledge of laboratory techniques including and not limited to :
- Bacteria transformation using heat shock and competent cells
- Bio-layer light interferometry
- Differential Scanning Calorimetry
- Size-Exclusion Chromatography
- Cell Transfection for protein expression
- Site-directed mutagenesis
- Liquid bacteria culture inoculation
- Plasmid DNA purification (Maxi-Prep and Mini-prep)
- Engineering of mutation inducing primers
Also encountering troubleshooting in the following areas:
- DNA from bacteria cell transformation without desired mutation due to Dpn1 treatment not being sufficient
- Low or no protein production from transfection due to insufficient use of transfection agent
- Genomic contamination after Plasmid DNA purification due to excess of Lysing time
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