2. We routinely use a Spectra/Por
R 7 dialysis membrane which is
chemically treated to remove any trace levels of heavy metals
and sulfide compounds.
3. PSY reaction buffer needs to be prepared always fresh. Stock
solutions of all compounds can be made and stored at room
temperature (Tris, Tween 80, MgCl 2 , NaCl). MnCl 2 and
TCEP stocks needs to be stored in small aliquots at À20
C
to avoid oxidation during multiple freezing–defreezing cycles.
4. It is recommended to use plastic bottles with high capacity
(e.g., 500 ml) to reduce centrifugation time. Bacteria pellets
in different centrifugation bottles can be pooled after resuspension in 20 ml LB-A/C and recentrifugation.
5. Washed inclusion body pellets can be stored at À20
C for
several months.
6. A constant drip rate can be achieved by using a separatory
funnel equipped with a stopcock, mounted on a tripod above
the beaker with the stirring refolding buffer. Ideally, place the
whole apparatus in the cold room to ensure cooling of resuspended inclusion bodies. Check flow from time to time as
inclusion body aggregates might lead to clogging.
7. Highest binding efficiency and yields were achieved with Co
2+ -
coupled Fractogel EMD Chelate if compared with equivalent
metal affinity resins from other suppliers. This may be due to
the tentacle technology used consisting of long, linear polymer
chains which might reduce aggregation of bound 6ÂHis-PSY
proteins in close proximity. We observed a yield drop if regular
matrices were used.
8. Eluate can be transferred into a 2 ml reaction tube which
contains a hole in the lid. Cover the filled tube with one layer
of dialysis membrane, carefully close the lid and confirm tightness by reverting the closed tube. Place tube upside down in a
floating rack swimming in PSY dialysis buffer.
9. This step is to remove remaining Fractogel and PSY aggregates
which might have formed during refolding/elution/dialysis
procedure and which compromises efficiency of enzyme activity assays. Refolded PSY can be stored at À80
C without
significant loss in enzyme activity for at least 6 months.
10. Protein concentrations of purified GGPS and PSY should be
determined photometrically (OD 280 , Bradford, etc.) and confirmed on an SDS-PAGE gel with BSA (between 0.5 and 5 μg)
and staining with Coomassie. Equimolar amounts of the
enzyme pair AtGGPS11 and AtPSY, each truncated by their
transit peptide, for one assay of 200 μl volume are 1 μg
AtGGPS11 and 1.22 μg AtPSY.
50
Maurizio Camagna and Ralf Welsch
R 7 dialysis membrane which is
chemically treated to remove any trace levels of heavy metals
and sulfide compounds.
3. PSY reaction buffer needs to be prepared always fresh. Stock
solutions of all compounds can be made and stored at room
temperature (Tris, Tween 80, MgCl 2 , NaCl). MnCl 2 and
TCEP stocks needs to be stored in small aliquots at À20
C
to avoid oxidation during multiple freezing–defreezing cycles.
4. It is recommended to use plastic bottles with high capacity
(e.g., 500 ml) to reduce centrifugation time. Bacteria pellets
in different centrifugation bottles can be pooled after resuspension in 20 ml LB-A/C and recentrifugation.
5. Washed inclusion body pellets can be stored at À20
C for
several months.
6. A constant drip rate can be achieved by using a separatory
funnel equipped with a stopcock, mounted on a tripod above
the beaker with the stirring refolding buffer. Ideally, place the
whole apparatus in the cold room to ensure cooling of resuspended inclusion bodies. Check flow from time to time as
inclusion body aggregates might lead to clogging.
7. Highest binding efficiency and yields were achieved with Co
2+ -
coupled Fractogel EMD Chelate if compared with equivalent
metal affinity resins from other suppliers. This may be due to
the tentacle technology used consisting of long, linear polymer
chains which might reduce aggregation of bound 6ÂHis-PSY
proteins in close proximity. We observed a yield drop if regular
matrices were used.
8. Eluate can be transferred into a 2 ml reaction tube which
contains a hole in the lid. Cover the filled tube with one layer
of dialysis membrane, carefully close the lid and confirm tightness by reverting the closed tube. Place tube upside down in a
floating rack swimming in PSY dialysis buffer.
9. This step is to remove remaining Fractogel and PSY aggregates
which might have formed during refolding/elution/dialysis
procedure and which compromises efficiency of enzyme activity assays. Refolded PSY can be stored at À80
C without
significant loss in enzyme activity for at least 6 months.
10. Protein concentrations of purified GGPS and PSY should be
determined photometrically (OD 280 , Bradford, etc.) and confirmed on an SDS-PAGE gel with BSA (between 0.5 and 5 μg)
and staining with Coomassie. Equimolar amounts of the
enzyme pair AtGGPS11 and AtPSY, each truncated by their
transit peptide, for one assay of 200 μl volume are 1 μg
AtGGPS11 and 1.22 μg AtPSY.
50
Maurizio Camagna and Ralf Welsch