Biological Characterization of the Omp1-like Protein from Actinobacillus actinomycetemcomitans

Abstract

Actinobacillus actinomycetemcomitans is a gram-negative, nonmotile coccobacillus bacterium that is associated with several human diseases, including endocarditis, meningitis, osteomyelitis, subcutaneous abscesses and periodontal diseases. A full-length Omp1-like protein gene from A. actinomycetemcomitans was cloned into a pQE30 vector and overexpressed in Escherichia coli BL21(DE3) cells. The protein revealed sequence homologies to Seventeen kilodalton proteins (Skp) from Pasteurella multocida and E. coli that have been characterized as periplasmic chaperones. This soluble Omp1-like protein was successfully purified to homogeneity for further folding and functional studies. The purity, identity, and conformation of the protein were determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization mass spectrometry, circular dichroism, fluorescence spectroscopic, and differential scanning calorimetric studies. We showed that the protein formed an oligomer larger than a tetramer. We found, further, that it is comprised of mostly α-helices and boasts high thermal stability.

Interaction between IGFBP-5 and TNFR1

“…This matched the pH of the buffer used for elution. 30,31 The folding properties of both the IGFBP-5 and TNFR1 proteins were characterized by CD spectroscopy. To determine the natures of the secondary structural elements of IGFBP-5 and TNFR1, far-UV CD spectra were recorded and analyzed ( Fig.…”

Expression and pH-dependence of the Photosystem II Subunit S from Arabidopsis thaliana

“…5B). [16][17] DSC scans were performed for conditions under which the protein was irreversibly unfolded. The temperature for minimal heat absorption by the protein was -0.9 o C. In our study, the protein showed no thermal transitions or denaturation from 0 o C up to 90 o C. In the present study, we measured the stability and conformational changes of recombinantly purified PsbS protein using various biochemical and biophysical techniques, including CD, DSC, and fluorescence measurements.…”