Constructs for Tsa1 Protein Expression

Constructs for expression of wt Tsa1 in yeast (p416-GPD) and bacteria (pET45b) have been reported previously.^{32 (link)} For yeast experiments, genes encoding wild-type Tsa1 or Tsa1 decamer interface variants (all with an N-terminal FLAG tag) were placed under the control of the GPD promoter, which allows for constitutive, high-level expression in yeast.^{31 (link),32 (link)} For bacterial over-expression, the Tsa1 gene was tagged at the N-terminus with a vector-encoded 6X-His tag. Codons for aromatic residues at the decamer interface in Tsa1 were mutated to Ala or Leu using the QuikChange mutagenesis protocol (Agilent) with the primers reported in Table S1 in the Supporting Information, as described previously.^{31 (link),32 (link)} All constructs containing mutations were verified by DNA sequencing.

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Loberg M.A., Hurtig J.E., Graff A.H., Allan K.M., Buchan J.A., Spencer M.K., Kelly J.E., Clodfelter J.E., Morano K.A., Lowther W.T, & West J.D. (2019). Aromatic Residues at the Dimer–Dimer Interface in the Peroxiredoxin Tsa1 Facilitate Decamer Formation and Biological Function. Chemical research in toxicology, 32(3), 474-483.

Publication 2019

QuikChange mutagenesis protocol

Bacterial Codons Gene Mutagenesis Mutations Primers Vector Yeast

Corresponding Organization : College of Wooster

Other organizations : The University of Texas Health Science Center at Houston, Wake Forest University

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Variable analysis

independent variables

Tsa1 gene variants (wild-type Tsa1 or Tsa1 decamer interface variants)

dependent variables

Not explicitly mentioned

control variables

N-terminal FLAG tag for yeast experiments
N-terminal 6X-His tag for bacterial over-expression
GPD promoter for constitutive, high-level expression in yeast

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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