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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/37721

Title: Lemon protein disulfide isomerase: cDNA cloning and biochemical characterization
Authors: Yu-Ting Chen;Lisa Wen;Kuo-Chuan Ho;Rong-Huay Juang;Chi-Tsai Lin
Contributors: 國立台灣海洋大學:生命科學系
Keywords: Citrus limonum;Protein disulfide isomerase (PDI);Scrambled RNase A (sRNase A);Three-dimension structural model (3-D structural model)
Date: 2013
Issue Date: 2016-05-02T01:47:01Z
Publisher: An International Journal
Abstract: Abstract:Background
Protein disulfide isomerases (PDIs), a family of structurally related enzymes, aid in protein folding by catalyzing disulfide bonds formation, breakage, or isomerization in newly synthesized proteins and thus.

A ClPDI cDNA (1828 bp, GenBank accession HM641784) encoding a putative PDI from Citrus limonum was cloned by polymerase chain reaction (PCR). The DNA sequence encodes a protein of 500 amino acids with a calculated molecular mass of 60.5 kDa. The deduced amino acid sequence is conserved among the reported PDIs. A 3-D structural model of the ClPDI has been created based on the known crystal structure of Homo sapiens (PDB ID: 3F8U_A). The enzyme has two putative active sites comprising the redox-active disulfides between residues 60–63 and 405–408 (motif CGHC). To further characterize the ClPDI, the coding region was subcloned into an expression vector pET-20b (+), transformed into E. coli Rosetta (DE3)pLysS, and recombinant protein expressed. The recombinant ClPDI was purified by a nickel Sepharose column. PDI’s activity was assayed based on the ability of the enzyme to isomerize scrambled RNase A (sRNase A) to active enzyme. The K M , k cat and k cat/K M values were 8.3 × 10-3 μM, 3.0 × 10-5 min-1, and 3.6 × 10-1 min-1 mM-1. The enzyme was most active at pH 8.

The advantage of this enzyme over the PDI from all other sources is its low K M. The potential applications of this PDI in health and beauty may worth pursuing.
Relation: 54(34)
URI: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/37721
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