Publication protocol
All DNA manipulations were performed in Escherichia coli strain DΗ5α (15). All DNA PCR primers were obtained from Integrated DNA Technologies. All mutant Tnp constructs contain hyperactive EK54 and LP372 mutations (7,16) and the MA56 mutation (17) which prevents translation of the Inh protein. C-terminal point mutations and truncations were first cloned into pET-21d(+) (Novagen) for in vivo analysis. C-terminal truncations were constructed by amplifying the bases corresponding to aa 1 to the truncation point from pRZ10300 by polymerase chain reaction (PCR) using Pfu polymerase (Stratagene), a primer which amplified the N-terminal XbaI site of pRZ10300 and an EcoRI tailed primer. The PCR products were digested with XbaI (Promega) and EcoRI (Promega) and cloned into the large XbaI-EcoRI fragment of pET-21d(+). pRZ10300 was originally constructed by L. Mahnke as follows. The XbaI-SalI fragment from pRZ7075 (18) was cloned into the large XbaI-SalI fragment from pBAD18-Cm (19) to create pRZ10250. The hyperactive EK54 and LP372 mutations were then introduced by substituting the XbaI-BglII Tnp fragment from pRZPET2 (6) into the large XbaI-BglII fragment of pRZ10250 to construct pRZ10300. Point mutants GD462 and SA458 were constructed by overlap PCR (20). Bases corresponding to the C-terminal region of Tnp (aa 301-476) were amplified from pRZPET2 by PCR using Pfu polymerase and internal mismatched primers containing the point mutation. The external primers included the NheI site in Tnp and the DraIII site C-terminal of the Tnp gene. PCR products were digested with NheI (NEB) and DraIII (NEB) and ligated to the large NheI-DraIII fragment of pRZPET2. The point mutation AD466 was originally cloned into pRZ7075 by L. Mahnke as follows. Overlap PCR using mismatched internal primers was used to amplify bases corresponding to the Cterminal region of Tnp. External primers included the N-terminal NheI site of Tnp and the C-terminal BclI site of Tnp. The PCR product was digested with NheI and BclI (NEB) and cloned into the large NheI-BclI fragment of pRZ7075 to create pRZ10350. For this study, EK54 and LP372 mutations were incorporated by digesting both pGRPET2 and pRZ10350 with PvuI (Pharmacia Biotech) and AlwNI (NEB). The PvuIAlwNI fragment from pRZ10350 containing the AD466 mutation was then ligated to a 847 bp PvuI-AlwNI 7 fragment from pRZPET2 and a 4556 bp AlwNI-AlwNI fragment from pRZPET2 containing the EK54 and LP372 mutations. All C-terminal point mutations and truncations were also substituted into pGRTYB35 (9) for protein purification. The C-terminal region of each mutant was amplified by PCR using a primer which included the NheI site of Tnp and a KpnI tailed primer. Each PCR product was digested with NheI and KpnI (NEB) and ligated to the large NheI-KpnI fragment of pGRTYB35. All mutants were purified as described previously (9). It should be noted that proteins expressed from this vector each contain one extra C-terminal glycine residue. For purposes of this manuscript, C-terminal truncations will be denoted by Tnp∆ followed by the number of amino acids remaining in the transposase, the C-terminal point mutations will be denoted as Tnp followed by the amino acid change and the amino acid number, and the full length Tnp containing just EK54, LP372 and MA56 mutations will be referred to as Tnp-FL (See Table 1).
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