Cyclization with Artificially Split Inteins

A truncated Mycobacterium tuberculosis RecA intein (Shingledecker et al. 1998) was used for the in vivo cyclization of the soluble (IIAGLC) and membrane-bound (IICBglc) subunits of the glucose transporter in E. coli. The two subunits sequentially transfer phosphoryl groups from the phosphoryl carrier protein Hpr to glucose. Upon cyclization the activity of the purified soluble (IIAglc) and membrane-bound (IICBglc) proteins was found to have increased 100 and 25%, respectively (Siebold and Erni 2002).

The artificially split Pyrococcus furiosus Pl-Pful intein, which had been previously used for trans-ligation in vitro (Yamazaki et al. 1998; Otomo et al. 1999; Ichiyanagi et al. 2000), served in the in vivo synthesis of cyclic green fluorescent protein (GFP; Crameri et al. 1996). Although the cyclization of GFP using a six amino acid linker had been previously reported (Baird et al. 1999; Topell et al. 1999), this constituted the first report of an artificially split intein functioning in vivo (Iwai et al. 2001). The presence of cyclic protein in the cell was demonstrated by Western blotting, and the pure cyclic form was obtained from E. coli cell extracts with no contamination from the linear protein. This contrasts with czs-splicing using Ssp DnaE, which has been reported to produce linear by-products in vivo (Evans et al. 2000). This is thought to be due to the difference in NIS acyl migration rates between Ssp DnaE and Pl-Pful inteins. The cyclic GFP was shown to have similar structural properties to linear GFP, but it was found to unfold at half the rate of the linear form upon chemical denaturation and be more resilient to unfolding at high temperatures as expected.

The Ssp DnaB split-intein was synthesized de novo from overlapping oligonucleotides with optimal codon usage to ensure good expression in E. coli (Williams et al. 2002). Once suitably rearranged, this synthetic split intein was used for the in vivo cyclization of the NH2 terminal domain of the E. coli rep-licative helicase DnaB, selected because the NMR structural data available indicated the proximity of the N- and C-termini in the folded state. A nine-residue linker was used to join the termini and the cyclization was found to proceed efficiently, with little accumulation of the products of incomplete splicing. The solution structure of cyclic DnaB was not found to differ significantly from that of the linear protein by NMR. As with other cyclized proteins, an increase in thermostability was observed, with the cyclic protein unfolding reversibly -14 °C higher than the linear form. The thermodynamic stabilization of the structure upon cyclization was measured to be about 2 kcal/mol, which was in line with estimates from the reduced conformation entropy in the unfolded form.

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