Preparation Of Phi29 DNA Polymerase Free Of Amplifiable ... - PLOS

Cloning, Expression and Purification of Phi29 DNA Polymerase

E. coli BL21 strain was transformed with an expression vector, pGEX-ø29pol, harboring a gene encoding Phi29 DNA polymerase, and expression of GST-ø29pol (approximately 94-kDa) was verified by SDS-PAGE analysis of the whole cell lysate (Fig. 2A lane 1). Following this, approximately 2 g of the overexpressed cells were prepared from large-scale culture using autoinduction systems for further purification.

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Figure 2. Isolation and purification of Phi29 DNA polymerase.

(A) SDS-PAGE analysis of expressed and isolated GST-ø29pol, and purified Phi29 DNA polymerase. Lane; M, molecular mass markers (Bio-Rad); 1, whole cell lysate; 2, supernatant after PEI precipitation; 3, supernatant after dilution; 4, GS4B resin before digestion by PreScission; 5, GS4B resin after digestion by PreScission; 6, purified Phi29 DNA polymerase (before addition of glycerol); 7, purified Phi29 DNA polymerase (after addition of glycerol); 8, commercially available Phi29 DNA polymerase (1 µg, Epicentre, Lot No. RPH-61004). The arrowhead indicates the GST-ø29pol (calculated size, approximately 94 kDa) and the arrow indicates the purified Phi29 DNA polymerase and commercially available polymerase (calculated size, approximately 67 kDa). (B) Agarose gel analysis of the removal of host DNA by PEI precipitation. Lane; M, AccuRuler 1-kb DNA ladder (Maestrogen); 1, whole cell lysate; 2, supernatant after PEI precipitation.

https://doi.org/10.1371/journal.pone.0082624.g002

The collected cells was lysed in BugBuster with benzonase in a buffer containing 100 mM NaCl to gradually degrade the host genomic DNA. Benzonase remained active under this salt condition, and successfully lowered the viscosity of the lysate. Host DNA and cell debris were immediately precipitated with PEI and high salt condition (1 M NaCl). Benzonase got inactive under the high salt condition, so the host DNA was not further fragmented and was easily precipitated with PEI [69]. The resulting soluble GST-ø29pol was recovered in the supernatant with little contaminating host DNA (Fig. 2A lane 1 and 2 and Fig. 2B lane 1 and 2). This supernatant was used for affinity column purification using GS4B resin.

However, soluble GST-ø29pol easily aggregated (data not shown), even when the purification step was performed at 4°C. The aggregated proteins clogged the column and retarded flow, resulting in low yield of purified Phi29 DNA polymerase. Non-ionic detergents to prevent aggregation did not work. Other reagents that prevent or disrupt aggregation, such as arginine [73], [74], the non-detergent sulfobetaine (NDSB) series [75], [76] and trehalose [77], [78], were also tested. The aggregation of GST-ø29pol was suppressed by 0.66 M trehalose or higher concentrations, allowing the isolation of GST-ø29pol using a GS4B affinity column. As a result, GST-ø29pol was collected as the major product on the GS4B resin (Fig. 2A lane 4, arrowhead, approximately 94 kD).

Subsequently, GST-ø29pol bound to the GS4B resin was then treated with EMA, followed by exposure to a white-LED lamp to inactivate the remaining dsDNA. DNA amplification experiment shows that DNA remaining in the sample or binding to the polymerase was not effectively inactivated by the exposure for less than 30 min (Figure S1). This is presumably because that easy precipitation of GS4B resin would inhibit homogeneous exposure to the white light for less than 30 min even if the sample kept being suspended by rotator. Based on this result, irradiation time was designed to be 60 min in our procedure. After washing out the free EMA, the remaining DNA was digested using the 3′-5′ exonuclease activity of GST-ø29pol in 0.8 M trehalose. These buffer conditions retain the activities of GST-ø29pol, including its 3′-5′ exonuclease activity [79].

Finally, Phi29 DNA polymerase was recovered from GS4B using restriction protease (Fig. 2A lane 5, 6 and 7). The purified polymerase was approximately 67 kDa, which was the same as that of a commercial Phi29 DNA polymerase (Fig. 2A lane 8). The average yield of the purified Phi29 DNA polymerase was approximately 1.5 mg per gram of pelleted cells.