Abstract: T4 DNA polymerase (T4 DP) is an indispensable tool in genetic engineering and other related fields. To obtain a large quantity of high purity and bioactive soluble T4 DP, the recombinant expression vector pET-22b(+)-T4 dp was constructed by cloning the T4 dp gene from the T4 bacteriophage genome into pET-22b(+) plasmid, which was then transformed into Escherichia coli Transetta (DE3) competent cells after the positive clones were confirmed by sequencing. The expression of soluble T4 DP was efficiently and accurately detected by magnetic beads. The temperature and time of auto-induction were optimized to be 30 ℃ and 10 h, respectively. The auto-inducing recombinant bacteria were disrupted by ultrasonic treatment, ultrasonic treatment after preincubation with lysozyme and chemical cleavage, and chemical cleavage was determined as the optimal disruption method. The soluble T4 DP in the supernatant after disintegrating recombinant bacteria was purified by Ni SepharoseTM 6 Fast Flow affinity column or MagNi magnetic beads. High purity T4 DP was efficiently purified to a concentration of 3 073.960 μg/mL by MagNi magnetic beads. The high purity soluble T4 DNA polymerase was successfully applied in the construction of low background cloning vector demonstrating its terminal blunting activity.

Key words: T4 DNA polymerase, auto-induction, magnetic bead method, low background cloning vector, terminal blunting