Purification of Bacteria-Derived Recombinant P Domain Proteins of Human Norovirus

Samenvatting

Source: Leuthold, M. M., et al. Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography. J. Vis. Exp. (2016).

This video demonstrates the purification of recombinant human norovirus P domain using size exclusion chromatography, highlighting the separation of the target protein from higher and lower molecular weight impurities based on differential pore accessibility. The process is monitored by UV absorbance and confirmed through SDS-PAGE analysis of eluted fractions.

Protocol

1. 3rd Purification Step

1. Wash pumps and pipes of the HPLC (High-performance liquid chromatography) purification system and pre-equilibrate the SEC (Size exclusion chromatography) column (see Materials List) with GFB (Gel filtration buffer).
2. Inject the P domain into the column at a flow rate of 1 ml/min using a superloop (up to 12 ml) or loop (up to 3 ml), depending on the volume of the concentrated sample. After the injection is finished, increase the flow rate to 2.5 ml/min.
3. As the OD (optical density) increases and the P domain comes off the column, collect fractions of 1.5 ml. Check fractions using SDS-PAGE with a 12% gel (Figure 1A and 1B). Pool only the purest fractions and concentrate to ~ 3 mg/ml and ~ 8 mg/ml.
   Note: After ~110 ml (void volume), most impurities are eluted from a SEC column with 320 ml bed volume. The P domain is usually eluted as a dimer. Elution time/volume of the P domain dimer is dependent on the prep grade (pg) of the SEC column.

Resultaten

Figure 1. Schematic and Representative Results for the 3rd Purification Step.
Chromatogram of P d...

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
P domain DNA	Life Technologies		GeneArt Gene Synthesis
Vivaspin 20	GE Healthcare	various	cutoff of 10 kDa, 30 kDa and 50 kDa used
HiLoad 26/600 Superdex 75 PG	GE Healthcare	28-9893-34	SEC column