The list of the predicted Asp-N endoproteinase digested native TRP47 peptides from <i>E. chaffeensis</i>.

<p>The identified peptides are shown in bold letters. aa, amino acids; Da, Dalton; and (TR), tandem repeat.</p

Immunoprecipitation of TRP47 with anti-pTyr antibody.

<p>Whole cell lysates from uninfected (THP-1) and <i>E. chaffeensis-</i>infected THP-1 cells (ECH) probed with anti-TRP47 antibody [lanes 1 and 2]. ECH whole cell lysates immunoprecipitated with, mouse anti-pTyr antibody (pTyr-IP, lane 3), normal mouse IgG (IgG-IP, lane 4) and detected with TRP47 antibody.</p

Molecular characteristics of native TRP47 and recombinant full-length, N- and C-terminal fragments.

<p>EDC, molecular mass post 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide treatment in SDS-PAGE; ¹, aspartic acid + glutamic acid; and ², arginine + lysine.</p

The list of the predicted trypsin digested native TRP47 peptides from <i>E. chaffeensis</i>.

<p>The identified peptides are shown in bold letters and TR-containing region is italicized. aa, amino acids; Da, Dalton.</p

Separation and purification of <i>E. chaffeensis</i> secreted major immunoreactive proteins by one-dimensional and two-dimensional gel electrophoresis (2-DE).

<p>(<b>A</b>) Cell-free supernatant collected from <i>E. chaffeensis</i> infected DH82 cells was precipitated with 20% ammonium sulfate before separation by SDS-PAGE. The TRP32, TRP47, TRP120, and Ank200 were major immunoreactive proteins as determined by Western immunoblotting with canine anti-<i>E. chaffeensis</i> serum. (<b>B</b>) Western immunoblot and (<b>C</b>) silver stained gel of <i>E. chaffeensis-</i>secreted proteins collected from cell-free <i>E. chaffeensis-</i>infected DH82 cells resolved by 2-DE. The approximate pH and molecular mass standards of the proteins are shown on top and left side of the Western immunoblot and silver stained gel images. The <i>E. chaffeensis</i> 2-DE gel resolved proteins were detected by anti-<i>E. chaffeensis</i> serum. The arrow indicates the three acidic, major immunoreactive proteins separated by 2-DE according to their respective pI and larger-than-the-predicted molecular mass (TRP120, TRP47, and TRP32). TRP120, −47, and −32 spots detected with protein-specific antibodies are shown as insets, from top to bottom on right side of the image (B).</p

MALDI-TOF mass spectra of <i>E. chaffeensis</i> native TRP47 and native TRP32.

<p>(<b>A</b>) For native TRP47, the mass-spectrum was recorded within the range of m/z 5,000 to 50,000 illustrates a peak of TRP47 singly charged ion (33,104.5). (<b>B</b>) For native TRP32, the mass-spectrum recorded within the range of m/z 5,000 to 40,000 illustrates a peak of the TRP32 singly charged ion (22,736.8). The relative intensities of the ions are shown on the <i>y</i> axis; the mass to charge ratios are shown on the <i>x</i> axis.</p

EDC modification of native and recombinant <i>E. chaffeensis</i> TRP47.

<p>(<b>A</b>) Western immunoblot of native TRP47 detected with rabbit anti-TRP47 serum. Lane 1, molecular mass standards; lane 2, unmodified native TRP47 (2.5 µg); lane 3, EDC-modified TRP47 (2.5 µg). (<b>B</b>) Schematic representation of TRP47 showing amino-terminal (N), tandem repeats (TR), and carboxy-terminal (C) regions with predicted molecular weight and pI of native protein, also represented are the recombinant GST-NterTRP47 and GST-CterTRP47. (<b>C</b>) Coomassie blue staining of proteins resolved by SDS-PAGE, 1, molecular mass standards; lane 2, unmodified GST-only (2.5 µg); lane 3, EDC-modified GST-only (2.5 µg); lane 4, unmodified GST-TRP47 (2.5 µg); lane 5, EDC-modified GST-TRP47 (2.5 µg); lane 6, unmodified GST-NterTRP47 (1.5 µg); lane 7, EDC-modified GST-NterTRP47 (1.5 µg); lane 8, unmodified GST-CterTRP47 (2.5 µg); lane 9, EDC-modified GST-CterTRP47 in excess of ethanolamine. (−), unmodified; (+), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)-modified; arrow, indicates the EDC modified protein band; aa, amino acids; MW, molecular weight; and pI, isoelectric potential.</p

Sedimentation velocity profiles of TRP120-1TR and TRP120-2TR proteins.

<p>The molecular weights of TRP120-1TR and -2TR proteins were determined by sedimentation velocity experiments. Sedimentation coefficient distribution <i>c(s)</i> profile for TRP120-1TR and TRP120-2TR show one major species, which corresponds to the molecular weight of 12.3 or 22.2 kDa, respectively. The observed molecular weights indicate that both TRP120 constructs are monomers in solution.</p

TR units of TRP120.

<p>(A) Domain organization of TRP120 and sequences of each TR unit. The primary sequences of TR units are shown with residues that differ between TR units highlighted in yellow (top). The sequence was analyzed by secondary structure (middle) and disorder (bottom) predictions. Predicted α-helical and β-strand regions are indicated by H and E, respectively. (B) SDS-PAGE of purified TRP120-1TR and TRP120-2TR proteins. Molecular weights for TRP120-1TR and -2TR proteins are 11.5 and 20.3 kDa, respectively, but the proteins migrate at twice their expected size.</p

DNA probes used in NMR titrations.

<p>DNA probes used in NMR titrations.</p