Screening optimized protein purification protocols by coupling small-scale expression and mini-size exclusion chromatography.
Testing several different growth and purification conditions by means of a small-scale screening is a valuable approach for identifying optimal protocols for large preparative protein productions. The availability of new mini columns for size exclusion chromatography (SEC) enables the coupling of parallel affinity purification using magnetic beads with the aggregation analysis performed by gel-filtration. Only 35 μL of bead eluate are sufficient to run both experiments and each SEC cycle needs no more than 30 min, washing and equilibration inclusive. We have shown that SEC ideally completes the SDS-PAGE analysis since it allows estimating the amount of soluble aggregates recovered in the elution fraction after affinity purification, although larger aggregates seem to be lost in the pre-column filters. SEC is also useful to identify low molecular mass contaminants that can be underestimated as they migrate together with the front of the acrylamide gel.
Sala
Elisa
E
COGENTECH, IFOM-IEO Campus for Oncogenomics, Milano, Italy.
de Marco
Ario
A
D002850:Chromatography, Gel / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*
D011506:Proteins