Lemon protein disulfide isomerase: cDNA cloning and biochemical characterization

Botanical Studies
Yu-Ting ChenChi-Tsai Lin

Abstract

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 KM, kcat and kcat/KM values were 8.3 × 10(-3) μM, 3.0 × 10(-5) min(-1), and 3.6 × 10(-1) min(-1...Continue Reading

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Datasets Mentioned

BETA
AY063059
AAC60578

Methods Mentioned

BETA
protein folding
PCR
PCRs
Protein Assay

Software Mentioned

SPDBV
BLAST
DeepView PdbViewer
BLASTP
SWISS
MODEL

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