Research Article

Long-Range PCR Amplification of DNA by DNA Polymerase III Holoenzyme from Thermus thermophilus

Table 2

Purification summary for the Tth pol III holoenzyme subunits.

ProteinStarting material
(g)
FractionTotal protein
(mg)
Total volume
(mL)
Total activity
(unitsa)
Specific activity
(U/mg)

α400Fr I (cell lysate)304201600
Fr II (35% AS)b8502001.1 × 1080.15 × 106
Fr III (butyl pool)713001.1 × 1081.6 × 106
Fr IV (S-200 pool)c107.52.2 × 1072.3 × 106
FR IV (reassay)d107.58.0 × 1060.8 × 106

β200Fr I (cell lysate)8780760
Fr II (40% AS)10002009.4 × 1070.1 × 106
Fr II (heat shock)1503808.1 × 1070.5 × 106
Fr III (Q pool)50603.2 × 1070.67 × 106
Fr IV (butyl pool)23201.7 × 1070.75 × 106

DnaX300Fr I (cell lysate)245001140
Fr II (35% AS)12582505.0 × 1080.4 × 106
Fr II (heat shock)5236005.0 × 1080.9 × 106
Fr III (SP pool)2004402.8 × 1081.4 × 106

300Fr I (cell lysate)15300930
Fr II (45% AS)185024003.4 × 1091.8 × 106
Fr III (Q pool)11524713.6 × 10932.0 × 106
Fr IV (methyl pool)141001.7 × 109123.0 × 106
Fr V (GF pool)5245.8 × 109122 × 106

200Fr I (cell lysate)190001400
Fr II (45% AS)40005401.5 × 10103.5 × 106
Fr III (butyl pool)909701.0 × 101015.5 × 106
Fr IV (octyl pool)152001.5 × 10990 × 106
Fr V (GF pool)4.1548.2 × 108200 × 106

One unit of activity is 1 pmol of total deoxyribonucleotide incorporated per min.
bThe initial assays in purification used a gap-filling assay to monitor nonprocessive polymerase activity [31].
cOne-fourth of Fr III was used to prepare Fr IV.
  α Fr IV was reassayed in the reconstitution assay described in Experimental Procedures.