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Protein Expr Purif. 2015 Aug;112:29-36. doi: 10.1016/j.pep.2015.04.005. Epub 2015 Apr 20.

Segmental expression and C-terminal labeling of protein ERp44 through protein trans-splicing.

Author information

1
College of Textiles, Donghua University, Shanghai, China; Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada.
2
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada. Electronic address: paul.liu@dal.ca.
3
College of Textiles, Donghua University, Shanghai, China. Electronic address: mengqing@dhu.edu.cn.

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.

KEYWORDS:

C-terminal labeling; ERp44; Intein; Protein trans-splicing; Segmental expression

PMID:
25907381
DOI:
10.1016/j.pep.2015.04.005
[Indexed for MEDLINE]

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