Mutagenic studies on human protein disulfide isomerase by complementation of Escherichia coli dsbA and dsbC mutants

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Mutagenic studies on human protein disulfide isomerase by complementation of Escherichia coli dsbA and dsbC mutants. / Stafford, S J; Lund, P A.

In: FEBS Letters, Vol. 466, No. 2-3, 28.01.2000, p. 317-22.

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@article{c8b4ea604c3e4e41b09abcf9fde6d234,
title = "Mutagenic studies on human protein disulfide isomerase by complementation of Escherichia coli dsbA and dsbC mutants",
abstract = "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.",
keywords = "Mutagenesis, Site-Directed, Protein Disulfide-Isomerases, Humans, Molecular Sequence Data, Isoenzymes, Genetic Complementation Test, Escherichia coli, Amino Acid Sequence, Sequence Homology, Amino Acid, Binding Sites",
author = "Stafford, {S J} and Lund, {P A}",
year = "2000",
month = jan,
day = "28",
language = "English",
volume = "466",
pages = "317--22",
journal = "FEBS Letters",
issn = "0014-5793",
publisher = "Elsevier",
number = "2-3",

}

RIS

TY - JOUR

T1 - Mutagenic studies on human protein disulfide isomerase by complementation of Escherichia coli dsbA and dsbC mutants

AU - Stafford, S J

AU - Lund, P A

PY - 2000/1/28

Y1 - 2000/1/28

N2 - 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.

AB - 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.

KW - Mutagenesis, Site-Directed

KW - Protein Disulfide-Isomerases

KW - Humans

KW - Molecular Sequence Data

KW - Isoenzymes

KW - Genetic Complementation Test

KW - Escherichia coli

KW - Amino Acid Sequence

KW - Sequence Homology, Amino Acid

KW - Binding Sites

M3 - Article

C2 - 10682851

VL - 466

SP - 317

EP - 322

JO - FEBS Letters

JF - FEBS Letters

SN - 0014-5793

IS - 2-3

ER -