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.
Protein
Starting material (g)
Fraction
Total protein (mg)
Total volume (mL)
Total activity (unitsa)
Specific activity (U/mg)
α
400
Fr I (cell lysate)
30420
1600
Fr II (35% AS)b
850
200
1.1 × 108
0.15 × 106
Fr III (butyl pool)
71
300
1.1 × 108
1.6 × 106
Fr IV (S-200 pool)c
10
7.5
2.2 × 107
2.3 × 106
FR IV (reassay)d
10
7.5
8.0 × 106
0.8 × 106
β
200
Fr I (cell lysate)
8780
760
Fr II (40% AS)
1000
200
9.4 × 107
0.1 × 106
Fr II (heat shock)
150
380
8.1 × 107
0.5 × 106
Fr III (Q pool)
50
60
3.2 × 107
0.67 × 106
Fr IV (butyl pool)
23
20
1.7 × 107
0.75 × 106
DnaX
300
Fr I (cell lysate)
24500
1140
Fr II (35% AS)
1258
250
5.0 × 108
0.4 × 106
Fr II (heat shock)
523
600
5.0 × 108
0.9 × 106
Fr III (SP pool)
200
440
2.8 × 108
1.4 × 106
300
Fr I (cell lysate)
15300
930
Fr II (45% AS)
1850
2400
3.4 × 109
1.8 × 106
Fr III (Q pool)
115
2471
3.6 × 109
32.0 × 106
Fr IV (methyl pool)
14
100
1.7 × 109
123.0 × 106
Fr V (GF pool)
5
24
5.8 × 109
122 × 106
200
Fr I (cell lysate)
19000
1400
Fr II (45% AS)
4000
540
1.5 × 1010
3.5 × 106
Fr III (butyl pool)
90
970
1.0 × 1010
15.5 × 106
Fr IV (octyl pool)
15
200
1.5 × 109
90 × 106
Fr V (GF pool)
4.1
54
8.2 × 108
200 × 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.