Protein production and analysis 3900-BT1-7
Week 1 — Expression, extraction and purification
Preparation of extracts and affinity chromatography. Cell disruption by sonication or high-pressure homogenisation, and the practical consequences of the method chosen for protease release and nucleic acid carryover; clarification; the chemistry of immobilised metal affinity chromatography and the design logic of affinity tags, including tag position, linker composition and protease-cleavable tag removal. Students load the same lysate onto Ni-NTA under three imidazole regimes and compare the outcomes.
Purification and SDS-PAGE (T7 RNA polymerase, Pfu-derived high-fidelity polymerase, Cas9). Second-dimension polishing by ion exchange or heparin affinity; concentration and buffer exchange; construction of the purification table. Laemmli and Tricine gel systems are run side by side, and the class discusses why nucleic-acid-binding proteins frequently migrate anomalously and why a single band is not evidence of homogeneity.
Purification of the VCE–protein complex. Co-expression versus co-lysis strategies for a heterodimeric enzyme; stoichiometry control; the specific problem of separating the fully assembled D1–D12 capping enzyme from free large subunit. This session introduces the idea, developed throughout the course, that a complex has a defined mass and stoichiometry that must be measured rather than assumed.
Week 2 — Functional verification
Activity of 2×T7 RNA polymerase, wild type and variant; VCE activity. In vitro transcription reactions with quantification of yield, run-off fidelity and double-stranded RNA by-product formation; dot-blot or immunoassay detection of dsRNA; capping efficiency assayed by the transfer of a labelled guanylate. The exercise connects directly to the manufacturing constraints of mRNA therapeutics.
In vitro activity of Cas9, catalytically inactive Cas9 and/or Cas13. Ribonucleoprotein assembly and titration; linearised plasmid cleavage analysed by agarose electrophoresis; the catalytically inactive variant as the definitive specificity control; Cas13 collateral cleavage of a quenched reporter as an illustration of how an enzymatic property becomes a diagnostic platform.
Activity of the Pfu-derived high-fidelity DNA polymerase. Comparative amplification against a non-proofreading enzyme; measurement of error rate by a reporter-based or sequencing-based assay; discussion of how fusion of a non-specific DNA-binding domain increases processivity without altering the active site.
Week 3 — Identity, production platforms and first analytical block
Mass spectrometry: introduction, sample preparation, trypsin digestion. Ionisation and the mass analysers in common use; why bottom-up proteomics dominates; reduction, alkylation and digestion chemistry; the trade-offs among filter-aided, bead-based and in-gel preparations; contamination sources that dominate a low-input sample.
Mammalian expression systems; monoclonal antibody purification; SDS-PAGE and/or EM grids. Transient versus stable expression, HEK293 and CHO lineages, glycosylation as a critical quality attribute; Protein A capture followed by polishing chromatography; a negatively stained grid prepared from the purified antibody and examined for aggregation and homogeneity, introducing electron microscopy as a sample quality assay rather than only a structure-determination method.
Analytical ultracentrifugation, DLS and SEC-MALS — demonstration and virtual tours. Instrument architecture, cell assembly, sample requirements and the cost of a poorly conceived experiment. Students load their own Week 1 preparations.
Week 4 — Second analytical block and interpretation
Analytical ultracentrifugation, DLS, SEC-MALS — data analysis. Continuous c(s) distribution analysis of sedimentation velocity data and the interpretation of the resulting peaks; cumulant and distribution analysis of autocorrelation functions, and the reasons a DLS polydispersity index overestimates homogeneity; calculation of absolute molar mass from concurrent light-scattering and concentration signals, and the resolution of the oligomeric state of the capping enzyme complex.
Differential scanning calorimetry and microcalorimetry (virtual tour); circular dichroism; FTIR; mass photometry. Transition midpoint and calorimetric enthalpy, reversibility and the validity of two-state fitting; the high-throughput fluorimetric surrogate and where it disagrees with DSC; secondary structure estimation from far-UV CD spectra and the systematic error introduced by inaccurate protein concentration; amide I band decomposition and the particular utility of FTIR for aggregated and membrane samples; mass photometry as a single-molecule mass distribution measurement on a few microlitres of dilute sample.
Analysis of MS results. Database searching, precursor mass tolerance and the effect of search space size; target–decoy false discovery rate estimation at the peptide-spectrum-match and protein level; label-free quantification; identification of the contaminating proteins that were carried through the Week 1 purification, and reconciliation of that finding with the Coomassie gel.
Course coordinators
Type of course
Mode
Prerequisites (description)
Learning outcomes
Knowledge
1. Explains the basis of affinity, ion-exchange and size-exclusion separation, and predicts how buffer conditions affect IMAC selectivity and capacity.
2. Describes SDS-PAGE separation, anomalous migration, and the limits of a stained gel as a purity criterion.
3. Accounts for the enzymology of the model systems: T7 RNA polymerase promoter recognition and processivity, Cas9 HNH and RuvC nuclease chemistry and its inactivation, Cas13 RNA targeting and collateral cleavage, family-B polymerase proofreading, and the three capping-enzyme reactions.
4. Compares bacterial and mammalian expression platforms by yield, modification, cost and regulatory position, and outlines an industrial monoclonal antibody process.
5. States the observable measured by each method (sedimentation coefficient, hydrodynamic radius, molar mass, transition enthalpy and midpoint, ellipticity, amide I band shape, interferometric contrast) and the assumptions behind its interpretation.
6. Explains bottom-up proteomics from proteolytic specificity to target–decoy FDR control.
Skills
1. Prepares a clarified extract and performs a two-step purification, documenting yield and specific activity in a purification table.
2. Runs and interprets an SDS-PAGE gel, estimating purity and stoichiometry by densitometry.
3. Designs and performs a quantitative in vitro activity assay with appropriate negative controls.
4. Prepares a sample for LC-MS/MS and critically evaluates the search output.
5. Acquires and analyses AUC, DLS, SEC-MALS, DSC, CD and FTIR data on one sample set, reconciling them into one self-consistent description.
6. Reports work as a reproducible methods section and figure set.
Social competences
1. Collaborates at the bench and defends a data interpretation before peers.
2. Recognises the limits of a single-method conclusion and seeks orthogonal confirmation.
Assessment criteria
Preparation and presentation of a project.
Examination form: preparation and presentation of a project.
Each team carries one protein preparation through the entire course, from the lysate produced in Week 1 to the final biophysical description in Week 4, reporting it as a single, integrated project. The project is not a summary of four separate exercises. The project focuses on one preparation, which is described using all the applied methods. The central requirement is that the final picture is self-consistent: the inferred oligomeric state from SEC-MALS must agree with the sedimentation coefficient distribution and mass photometry histogram; the claimed purity from gel analysis must be reconcilable with the mass spectrometry search output; and the specific activity must be reported against the purification table rather than in isolation.
Where methods disagree, the project must acknowledge this and explain which measurement to trust and why. A project that reports only concordant data is incomplete because, in a four-week purification and characterisation campaign, the measurements do not all agree in fact.
Deliverables: A written project dossier comprising:
- a reproducible methods section
- the purification table with yield and specific activity at each step
- the annotated figure set (gels, activity assay with its controls and other results collected)
- a reconciliation section providing one integrated description of the preparation
- a statement of individual contributions signed by both team members must be submitted one week before the final session. Raw data and analysis files must be submitted with the dossier.
Presentation. A 20-minute team seminar in the final session followed by 10 minutes of questions, delivered to the class and instructors. Both members present. Each team additionally acts as assigned discussant for one other team, submitting two substantive questions in advance.
A maximum of 2 absences is allowed. For multi-stage experiments conducted in pairs, at least one member of the team must be present to continue the work.
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