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Fig. 7 | Mobile DNA

Fig. 7

From: Mobile group I introns at nuclear rDNA position L2066 harbor sense and antisense homing endonuclease genes intervened by spliceosomal introns

Fig. 7

I-DsqI homing endonuclease expression, purification and activity. a 10% SDS-PAGE gel showing expression of MBP-I-DsqI in E. coli CodonPlus cells. Samples were harvested from induced (+) and uninduced (−) cells every thirty minutes. M is the molecular mass standard. Incubation times are shown above lanes. Arrowhead indicates the expressed endonuclease. The protein is theoretically 68 kDa. b After expression, the cells were lysed and centrifuged. The supernatant (cell lysate) and the pellet material (insoluble matter) were run on a 10% SDS-PAGE to clarify if MBP-I-DsqI is soluble or not. Expected size of MBP-I-DsqI is indicated with an arrow. c SDS-PAGE analysis of affinity purified MBP-I-DsqI. Protein purification was executed using amylose resin. Bound MBP-I-DsqI was released from the column using maltose. Nine fractions were sampled. M is a broad range prestained protein marker (New England Biolabs). Purified protein is indicated by an arrow. OD 280 nm measurements of each fraction are shown. Fraction 5 contains the highest concentration of MBP-I-DsqI. d Activity study. The nine fractions from c) were incubated with the linarized target DNA pDan122/747 for 15 min. Two bands at 3.07 kb and 1.53 kb indicate I-DsqI activity. e Temperature range of activity observed after incubation of 0.1 unit affinity purified I-DsqI and linear target DNA for 60 min. Temperatures assessed ranged from 0 °C to 60 °C

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