Lemon protein disulfide isomerase: cDNA cloning and biochemical characterization

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 K ^sub M^ , k ^sub cat^ and k ^sub cat^/K ^sub M^ values were 8.3 × 10^sup -3^ [mu]M, 3.0 × 10^sup -5^ min^sup -1^, and 3.6 × 10^sup -1^ min^sup -1^ mM^sup -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 ^sub M^. The potential applications of this PDI in health and beauty may worth pursuing.