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Mutagenic studies on human protein disulfide isomerase by complementation of Escherichia coli dsbA and dsbC mutants
Author(s) -
Stafford Simon J,
Lund Peter A
Publication year - 2000
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)01728-7
Subject(s) - dsba , biochemistry , protein disulfide isomerase , cysteine , periplasmic space , complementation , histidine , escherichia coli , isomerase , active site , mutant , protein fragment complementation assay , chemistry , amino acid , enzyme , biology , gene
Protein disulfide isomerase (PDI) exhibits both an oxido‐reductase and an isomerase activity on proteins containing cysteine residues. These activities arise from two active sites, both of which contain pairs of redox active cysteines. We have developed two simple in vivo assays for these activities of PDI, based on the demonstration that PDI can complement both a dsbA mutation and a dsbC mutation when expressed to the periplasm of Escherichia coli . We constructed a variety of mutants in and around the active sites of PDI and analysed them using these complementation assays. Our analysis showed that the active site amino acid residues have a major role in determining the activities exhibited by PDI, particularly the N‐terminal cysteine of the N‐terminal active site. The roles of the histidine residue at position 38 and the glutamic acid residue at position 30 were also studied using these assays. The results show that these two in vivo assays should be useful for rapid screening of mutants in PDI prior to purification and detailed biochemical analysis.