By Krawetz S.A., Womble D.D. (eds.)
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8), who created two circularly permuted variants of phosphoribosylanthranilate isomerase, with new termini located in surfaceexposed loops. Both variants were similar to the wild-type in their three-dimensional structure and biologically active in vivo. The influence of circular permutations on the α-spectrin SH3 domain has been studied in great detail. All circular permutations of the protein with new termini in loop regions folded correctly, as probed by circular dichroism and nuclear magnetic resonance spectroscopy.
For random circular permutation, the circular gene is partially digested with DNase I (c) and a library of relinearized genes is isolated. The library is treated with T4 DNA ligase and T4 DNA polymerase (d), yielding blunt-ended, repaired fragments that can be cloned into an appropriate expression plasmid. The circular gene can also be used as template for PCR amplification to generate rationally designed, circularly permuted variants (e,f). degraded by E. coli proteases. In difficult cases, efficient development of the chromophore may take several days of incubation at 4°C.
1999) Genetic engineering of E. coli for the production of precorrin-3 in vivo and in vitro. Bioorg. Med. Chem. 7, 2215–2219. 6. Spencer, J. , Stolowich, N. , Roessner, C. , and Scott, A. I. (1993a) The Escherichia coli cysG gene encodes the multifunctional protein, siroheme synthase. FEBS Lett. 335, 57–60. 7. , Singh, D. , Smith, A. , Woodcock, S. , Awan, S. , and Warren, M. J. (1997) Siroheme biosynthesis in higher plants: Analysis of an S-adenosyl-L-methionine-dependent uroporpyrinogen III methyltransferase from Arabidopsis thaliana.