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PfaR Purification Strategy and Structure Analysis
Author(s) -
OrtizRosario Melissa Cristina,
RullánLind Carlos,
PérezOquendo Mabel,
MoralesLozada Yermary,
GonzálezMéndez Ricardo,
BaergaOrtiz Abel
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.lb52
Subject(s) - gene , biology , circular dichroism , conserved sequence , regulator , escherichia coli , biochemistry , computational biology , genetics , peptide sequence
The biosynthesis of polyunstaurated fatty acids (PUFA) in certain bacterial species, is regulated by processes that are not fully understood. One possible factor in the modulation of gene expression in PUFA biosynthesis is pfaR, a putative transcriptional regulator whose gene is encoded directly upstream of pfaA in some bacterial species but entirely absent in others. Here we report the expression of pfaR in Escherichia coli together with its purification by Ni 2+ affinity chromatography. Our results show that the majority of pfaR is produced as an insoluble protein that can be reconstituted from inclusion bodies. After inclusion body reconstitution, pfaR remains amenable for affinity purification. Additional bioinformatics analyses indicate the presence of a conserved winged helix‐turn‐helix (wHTH) domain flanked by a number of conserved motifs which include a predicted protein‐protein interaction site. Sequence alignments and motif structure analyses reveal evolutionary divergence in this family of transcriptional regulators, consistent with the likely DNA‐binding function of pfaR. Currently, we are in the process of analyzing pfaR secondary structure using circular dichroism. Future work will include functional analysis using systematic evolution of ligands by exponential enrichment. Support or Funding Information This work was supported by the National Institutes of Health [R25 GM061838, R25GM061151‐ 15, 5T34GM007821‐37, T36‐GM‐095335]; and the NationalScience Foundation [CHE0953254]. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .