title: A streamlined method for preparing split intein for NMR study.
abstract: A protein ligase, intein, mediates a protein-splicing reaction. It can be split into two complementary fragments and reconstituted as a whole intein scaffold to perform protein trans-splicing. To understand the association of intein fragments and the splicing mechanism, it is necessary to produce a large quantity of split intein for structural study. Conventionally, two fragments are prepared separately and assembled in solution, but severe aggregation of intein fragments occurs, and precise control of the relative concentration of each fragment is difficult. Here, we present a streamlined method to incorporate a circular permutation concept into the production of split intein. By circular permutation of the native split Nostoc punctiforme DnaE intein (NpuInt), a new backbone opening is relocated to the native split site at residue 102. As the protein splicing activity is preserved, the expressed NpuInt can immediately self-cleave into a two-piece split NpuInt. Because of a tight association between the two complementary fragments, split NpuInt can be purified in one step. The idea is simple and applicable to other split inteins. Employing the new preparation, we use NMR spectra to assign the backbone and side chain resonances for the native split NpuInt.
authors.affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.
authors.forename: Yi-Zong
authors.initials: YZ
authors.lastname: Lee
authors.affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.
authors.forename: Yun-Tzai
authors.initials: YT
authors.lastname: Lee
authors.affiliation: Graduate Institute of Natural Products and Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Yi-Jan
authors.initials: YJ
authors.lastname: Lin
authors.affiliation: Life Science Group, Scientific Research Division, National Synchrotron Radiation Research Center, Hsinchu, Taiwan. Electronic address: chen.yj@nsrrc.org.tw.
authors.forename: Yen-Ju
authors.initials: YJ
authors.lastname: Chen
authors.affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan; Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan. Electronic address: scsue@life.nthu.edu.tw.
authors.forename: Shih-Che
authors.initials: SC
authors.lastname: Sue
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification*; D047668:Inteins / Q000235:genetics*; D046937:Nostoc / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D019154:Protein Splicing
chemical_list: D001426:Bacterial Proteins
keywords: Circular permutation; Intein; NMR; Protein trans-splicing; Split intein