Lemon protein disulfide isomerase: cDNA cloning and biochemical characterization

Yu-Ting Chen; Lisa Wen; Kuo-Chuan Ho; Rong-Huay Juang; Chi-Tsai Lin

doi:10.1186/1999-3110-54-34

Lemon protein disulfide isomerase: cDNA cloning and biochemical characterization

Bot Stud. 2013 Dec;54(1):34. doi: 10.1186/1999-3110-54-34. Epub 2013 Sep 16.

Authors

Yu-Ting Chen¹, Lisa Wen², Kuo-Chuan Ho³, Rong-Huay Juang⁴, Chi-Tsai Lin⁵

Affiliations

¹ Institute of Genomics and Bioinformatics, Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan.
² Department of Chemistry, Western Illinois University, 1 University Circle, Macomb, IL, 61455-1390, USA.
³ Institute of Bioscience and Biotechnology and Center of Excellence for the Oceans, National Taiwan Ocean University, 2 Pei-Ning Rd, Keelung, 202, Taiwan.
⁴ Department of Biochemical Science and Technology, National Taiwan University, Taipei, 106, Taiwan.
⁵ Institute of Bioscience and Biotechnology and Center of Excellence for the Oceans, National Taiwan Ocean University, 2 Pei-Ning Rd, Keelung, 202, Taiwan. B0220@mail.ntou.edu.tw.

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.

Results: 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.

Conclusions: 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.

Keywords: Citrus limonum; Protein disulfide isomerase (PDI); Scrambled RNase A (sRNase A); Three-dimension structural model (3-D structural model).