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. Author manuscript; available in PMC: 2023 Feb 15.
Published in final edited form as: Biochemistry. 2022 Jan 24;61(4):294–302. doi: 10.1021/acs.biochem.1c00788

Figure 3. In vitro protein splicing activity of H. walsbyi inteins as separate fusion proteins.

Figure 3.

SDS-PAGE analysis of in vitro protein splicing of MIH, stained with Coomassie blue. Splicing of precursor MIH (68 kDa) results in MH (46 kDa) and I (22 kDa). In panels A and C, purified precursor fusion protein MIH-A or MIH-D (0.2 mg/mL) was incubated for 2 h or 6 h at 20°C at the total NaCl concentrations indicated above the lanes in 20 mM HEPES buffer, pH 7.5, with 2 mM TCEP and 5 mM EDTA, as described in the Materials and Methods. The percentage of splicing was estimated by densitometry using ImageJ and represents the average of two independent trials.47 In panels B and D, the gels were probed with InVision stain, which detects proteins with a His-tag. Hence, we would expect to see bands corresponding to MIH, MH, or IH, but not MI or I. UR is unreacted protein, nd is not determined.