title: Protein trans-splicing of multiple atypical split inteins engineered from natural inteins.
abstract: Protein trans-splicing by split inteins has many uses in protein production and research. Splicing proteins with synthetic peptides, which employs atypical split inteins, is particularly useful for site-specific protein modifications and labeling, because the synthetic peptide can be made to contain a variety of unnatural amino acids and chemical modifications. For this purpose, atypical split inteins need to be engineered to have a small N-intein or C-intein fragment that can be more easily included in a synthetic peptide that also contains a small extein to be trans-spliced onto target proteins. Here we have successfully engineered multiple atypical split inteins capable of protein trans-splicing, by modifying and testing more than a dozen natural inteins. These included both S1 split inteins having a very small (11-12 aa) N-intein fragment and S11 split inteins having a very small (6 aa) C-intein fragment. Four of the new S1 and S11 split inteins showed high efficiencies (85-100%) of protein trans-splicing both in E. coli cells and in vitro. Under in vitro conditions, they exhibited reaction rate constants ranging from ~1.7 × 10(-4) s(-1) to ~3.8 × 10(-4) s(-1), which are comparable to or higher than those of previously reported atypical split inteins. These findings should facilitate a more general use of trans-splicing between proteins and synthetic peptides, by expanding the availability of different atypical split inteins. They also have implications on understanding the structure-function relationship of atypical split inteins, particularly in terms of intein fragment complementation.
authors.affiliation: Institute of Biological Sciences and Biotechnology, Donghua University, Shanghai, PR China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Lin
authors.forename: Mengmeng
authors.initials: M
authors.lastname: Li
authors.forename: Huiling
authors.initials: H
authors.lastname: Song
authors.forename: Lingling
authors.initials: L
authors.lastname: Xu
authors.forename: Qing
authors.initials: Q
authors.lastname: Meng
authors.forename: Xiang-Qin
authors.initials: XQ
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics; D047668:Inteins / Q000235:genetics*; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D020040:Trans-Splicing
chemical_list: D011506:Proteins