Tn5 Transposase And Tagmentation Procedures For Massively Scaled ...

Production of active Tn5 transposase

We cloned a hyperactive Tn5 allele harboring the classical E54K and L372P mutations (but wild-type at M56) into pTBX1 (pTXB1-Tn5) to add a C-terminal intein tag and a chitin-binding domain (CBD) (Fig. 1A; Supplemental Fig. 1). We found that the additional mutations (Y64I, K200R, and S303G) that were introduced in US patent 5,965,443 (Reznikoff and Goryshin 1996) yielded a nearly inactive protein (data not shown). Previously, Tn5 expression from the pTYB4 (NEB) vector was reported (Bhasin et al. 1999), in which the Tn5 CDS is C-terminally fused to the Saccharomyces cerevisiae VMA1 intein with an additional glycine before the catalytic cysteine to achieve efficient cleavage with DTT. Expressing Tn5 in the pTXB1 vector had several advantages: First, the Mycobacterium xenopi GyrA intein is half the size of the S. cerevisiae VMA1 intein, which facilitates higher expression levels of the fusion protein without formation of inclusion bodies; second, the natural C-terminal isoleucine of Tn5 supports efficient intein cleavage in this vector. The T7 expression system was found to suppress production of detectable amounts of Inhibitor (Inh; data not shown). Expression of the M. xenopi GyrA-Tn5 fusion protein was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG), cell DNA in the crude lysate was removed by polyethylenimine (PEI) precipitation, and the cleared lysate was loaded on a chitin column. We observed that oligonucleotides containing the hyperactive ME sequences could be annealed to Tn5 already on the columns (so that excess unannealed oligonucleotides were discarded through the column) or later in solution. Tn5 was released from the column by DTT-induced cleavage of the intein–CBD tag (Fig. 1B). We initially assessed the activity of the assembled Tn5 through its ability to tagment high-molecular-weight (HMW) DNA into 400- to 500-bp fragments, with a representative gel shown in Figure 1C.

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Figure 1.

Production and purification of tagmentation-ready Tn5. (A) Illustration of the Tn5–intein fusion construct. (B) 10% SDS-PAGE run with crude lysate (Lys), supernatant (Sup), flow-through (FT), wash (W), and DTT eluted fractions (DTT) from the chitin column. The expected sizes of the Tn5–intein fusion protein (Mxe-Tn5) and cleaved full-length Tn5 (Tn5) are indicated on the right; marker molecular weights in kDa, to the left. (C) Agarose gel (1% Type LE, in TAE buffer) demonstrating tagmentation of 50 or 100 ng high MW calf thymus DNA with 1 μL of Tn5 prepared by annealing in solution (Free), on chitin columns (Col), or with no Tn5 as a negative control (C). On-column annealing removed excess oligonucleotides present after solution annealing.