Dr Manish S Kushwah

Next-generation Purification matrix development for accelerating drug and therapeutic discovery

Secondary Supervisor(s): Dr Sam Parkinson

University of Registration: Aston University

BBSRC Research Themes:

• Integrated Understanding of Health (Pharmaceuticals)

Project Outline

Background:

Protein purification is central to academic & industrial biotechnology and is critical for therapeutic development. Biologics, antibodies, and vaccines all rely highly purified on efficient purification, where even modest gains in yield, reproducibility, and selectivity can translate into lower costs, faster timelines, and broader patient access. Current purification matrixes, however, remain ineffective against non-specific binding. Distinguishing specific from non-specific interactions is particularly challenging when target proteins are present at low abundance in complex mixtures such as lysates, serum, or plasma. Passivation strategies, including PEG coatings or blocking layers (BSA, casein), reduce some background binding but remain inconsistent across a broad variety of proteins.

Inspired by lipid bilayers, which combine inertness with selective recognition, we aim to engineer synthetic polymer matrices that mimic bilayer passivation while providing mechanical stability. Using innovative polymer synthesis approaches, Reversible Addition Fragmentation Chain Transfer (RAFT) polymerisation, we will generate block copolymers, with hydrophobic & hydrophilic blocks, for passivation, and binding motifs (e.g., Ni-NTA) for specific capture. Mass photometry (MP), a label-free single-molecule counting method, allows us to directly quantify non-specific protein binding and optimise polymer composition. This quantitative approach will enable rational design of next-generation purification matrices with superior yield, selectivity, and reusability.

Aims:

Develop a library of RAFT-derived block copolymers with tunable hydrophobic & hydrophilic blocks.

Use mass photometry to quantitatively assess non-specific binding and binding-site density to identify optimal candidates.

Benchmark the new matrices against commercial standards (e.g., Sepharose) using His-tagged proteins as proof-of-concept, with future expansion to ion exchange and size exclusion format.

Understanding Principles of ESCRT-III Membrane Remodelling by Single-Molecule Imaging and Functional Reconstitution

Secondary Supervisor(s): Professor Roslyn M. Bill

University of Registration: Aston University

BBSRC Research Themes:

• Understanding the Rules of Life (Immunology,Microbiology,Neuroscience and Behaviour,Stem Cells)

Project Outline

Vesicular transport maintains lipid and protein homeostasis across cellular compartments and is essential for cell survival. This requires reshaping membranes into nanometre-scale tubes or vesicles (remodelling) and separating them from the parent membrane (fission). ESCRT-III (E-III) is a protein complex that catalyses membrane remodelling and fission in range of functions such as lysosome formation, wound repair, and cell division. Cancer cells and viruses hijack E-III function for their survival. Current models of ESCRT-III function focus on polymer architecture but lack a quantitative link between polymerisation and the resulting modulation of membrane affinity. This parameter is central to understanding polymer-driven membrane remodelling and to explain the diversity of ESCRT-III functions, the effects of partner proteins, and its hijacking in disease.

To address this gap, we will combine single-molecule imaging with fluorescence based functional reconstitution of ESCRT-III membrane remodelling on surface-stabilised membrane tubes. Specifically, we will apply Dynamic Mass Photometry (DMP), a label-free method that simultaneously quantifies the mass of membrane-bound proteins and their diffusion, the latter serving as a direct measure of membrane affinity. This will allow us to correlate ESCRT-III polymer growth with changes in membrane affinity. These measurements will form the basis of a quantitative model of membrane remodelling. We will then integrate this framework with ESCRT-III reconstitution on membrane tubes, linking polymer-membrane affinity to polymerisation rates and polymer-driven membrane remodelling.

Aims:

Use DMP to quantify how E-III (Snf7/CHMP4) polymer growth modulates membrane affinity, with and without partner proteins

Reconstitute remodelling/fission on membrane tubes to link polymer kinetics to curvature and scission

Test viral adaptors (e.g., HIV-1 Gag p6) for their effects on ESCRT-III nucleation, polymerisation, and affinity