title: Segmental expression and C-terminal labeling of protein ERp44 through protein trans-splicing.
abstract: Endoplasmic reticulum resident protein 44 (ERp44) is a member of the protein disulfide isomerase family and functions in oxidative protein folding in the endoplasmic reticulum. A structurally flexible C-terminal tail (C-tail) of ERp44 plays critical roles in dynamically regulating ERp44's function in protein folding quality control. The structure-function dynamics of ERp44's C-tail may be studied further using fluorescence and other techniques, if methods are found to label the C-tail site-specifically with a fluorescent group or segmentally with other desired labels. Here we have developed such methods, employing split inteins capable of protein trans-splicing, and identifying atypical S1 split inteins able to function efficiently at a suitable split site in the ERp44 sequence. One method demonstrated segmental expression of ERp44 for segmental labeling of the C-tail, another method efficiently added a commercially available fluorescent group to the C-terminus of ERp44, and both methods may also be generally useful for studying other proteins.
authors.affiliation: College of Textiles, Donghua University, Shanghai, China; Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada.
authors.forename: Xudong
authors.initials: X
authors.lastname: Dai
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada. Electronic address: paul.liu@dal.ca.
authors.forename: Xiang-Qin
authors.initials: XQ
authors.lastname: Liu
authors.affiliation: College of Textiles, Donghua University, Shanghai, China. Electronic address: mengqing@dhu.edu.cn.
authors.forename: Qing
authors.initials: Q
authors.lastname: Meng
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D006801:Humans; D047668:Inteins; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics*; D018832:Molecular Chaperones / Q000032:analysis / Q000235:genetics*; D008969:Molecular Sequence Data; D019154:Protein Splicing; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D020040:Trans-Splicing
chemical_list: C451899:ERP44 protein, human; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins
keywords: C-terminal labeling; ERp44; Intein; Protein trans-splicing; Segmental expression