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The Magnet Platform in Action: VHL-ElonginB-ElonginC (VCB)

Part 1 — Optimizing VCB Expression for Crystallography — Yield gain of 330%

Producing multi-protein complexes like VCB is a bit of a balancing act. It requires expressing three subunits in sync, ensuring they fold and assemble correctly, to pull out enough material for crystallography or screening. Let’s dive in.

In early trials, when simply reproducing literature conditions, VCB expression in E. coli yielded just 0.5 mg of purified complex per liter of TB medium – a yield too low for meaningful biophysical or structural work.

So our team went back and ran a systematic expression optimization, testing:
• Different media (LB, TB, autoinduction),
• IPTG concentrations (0.5 vs. 1 mM),
• Expression temperature (adjusted to 25 °C).

The real breakthrough came with the autoinduction medium, which gave not only the highest cell density (7.3 g/L), but also a clear increase in expression levels – confirmed by SDS-PAGE and Western blot.

Following this, expression was scaled up to 6 liters and the complex was purified using a multi-step protocol:
1. Affinity chromatography,
2. Tag cleavage,
3. Ion exchange chromatography,
4. Size exclusion chromatography.

The final outcome? 10.5 mg of highly pure VCB complex from a 6 L expression, representing a 330% yield increase per gram of biomass compared to the original condition. Clear bands for all three components (VHL, EloB, EloC) were observed on SDS-PAGE, confirming proper ratio.

This case illustrates the importance of:
• Testing different media early on,
• Exploring a range of IPTG and temperature combinations,
• Following through with optimization for a low-yielding construct until conditions are fully optimized.

Part 2 will focus on crystallography and SmartSoak application on VCB crystals.

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