Thesis Defence: "The exploitation of cell-free protein synthesis technology for integrated structural biology"

QUICK INFORMATION
Type
PhD Defense
Start Date
14-06-2019 14:00
End Date
14-06-2019 16:00
Location
ESRF Visitor Centre
Speaker's name
Vinesh Jugnarain
Speaker's institute
ESRF
Contact name
Kimberley ROBERT
Host name
Ed MITCHELL
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Cell-free protein synthesis (CFPS) is an alternative method to
cellular expression for recombinant protein production. Its lack of
cellular boundaries enables direct modification of the CFPS reaction
to allow optimised and rapid production of functional and labelled
proteins. The aim of this industry-funded PhD was to demonstrate the
applicability of the Synthelis CFPS technology for integrated
structural biology by showing that CFPS proteins were functionally
and structurally equivalent to those from in vivo expression
systems.

These objectives were addressed with analytical approaches to the
production and testing of different lysates used in the CFPS
reactions and the use of model proteins for CFPS production and
purification, and biophysical and structural analyses. The model
proteins studied were the GPCR (G-protein coupled receptor) CXCR4
(C-X-C motif chemokine receptor 4) and its soluble ligand SDF1-α
(stromal cell-derived factor alpha -1), and the membrane protein CD4
(cluster of differentiation 4) and its truncated soluble variant
2dCD4. CXCR4 and SDF1-α are essential in the chemotaxis of cells,
and CD4 plays a critical role in immune response. Pathologically,
CXCR4/SDF1-α are involved in cancer progression, and CD4 is the
primary receptor for HIV GP120 (glycoprotein 120) during viral
infection.

For the purposes of integrated structural biology, a primary
objective was to optimise CFPS to generate sufficient amounts of
protein. From a screen of CFPS lysates derived from several E. coli
strains, it was determined that Rosetta lysate enhanced yields of
CXCR4. High-cell density cultures (HCDC) in fermenters were then
established for lysate production. Subsequently, HCDC was used for
the production of deuterated lysates for SANS (small-angle neutron
scattering) studies.

Full-length CXCR4 and CD4 were produced in proteoliposomes or
additionally, for CXCR4, as detergent solubilised protein. However,
the expression and purity levels of solubilised CXCR4 and CD4
remained too low to permit advanced structural work. Nevertheless,
soluble and functional SDF1-α and 2dCD4 were produced using lysate
obtained from the SHuffle E. coli strain, which promotes disulphide
bond pairing. CFPS is therefore advantageous over in vivo
expression, where these occur as insoluble proteins and require an
intensive refolding procedure. Subsequently, CFPS was established as
a method for the rapid production of deuterated SDF1-α and 2dCD4.

The CFPS-generated proteins were assessed using biophysical
techniques. ELISA (enzyme-linked immunosorbent assay) and SPR
(surface plasmon resonance) analyses of CXCR4 in proteoliposomes
using conformational antibodies indicated binding and, therefore, a
proper folding. Additionally, CD4 functionality in proteoliposomes
was confirmed by using GP120 and conformational antibodies in
ELISAs. Mass spectrometry of SDF1-α and its deuterated forms
confirmed disulphide bonding and deuteration levels. The
functionality of SDF1-α variants were confirmed using chemotaxis
assays. With a view to long-term optimisation of CFPS, CXCR4
proteoliposomes were assessed using a NanoSightTM particle tracker,
which provided insights into the changes in size and size
distribution of proteoliposome particles during the CFPS reaction.

Advanced structural studies were performed on SDF1-α and 2dCD4.
Structural determination of CFPS-generated SDF1-α by X-ray
crystallography showed that it was identical to that of the
published E. coli refolded form. The preliminary SANS assessment of
deuterated 2dCD4 in complex with GP120 provided initial confirmation
of the success of CFPS produced protein to provide viable SANS data.
Finally, a SANS feasibility study demonstrated the possibility to
obtain structural information about model proteins despite in the
presence of unfractionated lysates. This novel technique could lead
to the characterisation of CFPS membrane proteins in
proteoliposomes, following a simple partial purification step.5

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