title: Biophysical characterization of a recombinant aminopeptidase II from the thermophilic bacterium Bacillus stearothermophilus.
abstract: In the present study, the biophysical properties of His6-tagged Bacillus stearothermophilus aminopeptidase II (His6-tagged BsAmpII) are characterized in detail by gel-filtration, analytical ultracentrifugation, and various spectroscopic techniques. Using size-exclusion chromatography and analytical ultracentrifugation, we demonstrate that His6-tagged BsAmpII exists predominantly as a dimer in solution. The enzyme is active and stable at pHs ranging from 6.5 to 8.5. Far-UV circular dichroism analysis reveals that the secondary structures of His6-tagged BsAmpII are significantly altered in the presence of SDS, whereas the presence of 5-10% acetone and ethanol was harmless to the folding of the enzyme. Thermal unfolding of His6-tagged BsAmpII was found to be irreversible and led to the formation of aggregates. The native enzyme started to unfold beyond 0.6 M guanidine hydrochloride and had a midpoint of denaturation at 1.34 M. This protein remained active at concentrations of urea below 2.7 M but experienced an irreversible unfolding by >5 M denaturant. Taken together, this work lays a foundation for potential biotechnological applications of His6-tagged BsAmpII.
authors.affiliation: Department of Chemistry, National Cheng Kung University, Tainan City, 701, Taiwan, catalpa0905@gmail.com.
authors.forename: Tzu-Fan
authors.initials: TF
authors.lastname: Wang
authors.forename: Min-Guan
authors.initials: MG
authors.lastname: Lin
authors.forename: Huei-Fen
authors.initials: HF
authors.lastname: Lo
authors.forename: Meng-Chun
authors.initials: MC
authors.lastname: Chi
authors.forename: Long-Liu
authors.initials: LL
authors.lastname: Lin
mesh_terms: D000626:Aminopeptidases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D017510:Protein Folding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry; D012997:Solvents; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation; D014508:Urea / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012997:Solvents; D014508:Urea; D000626:Aminopeptidases