Impact of different cultivation and induction regimes on the structure of cytosolic inclusion bodies of TEM1-beta-lactamase.
The enzyme TEM1-beta-lactamase has been used as a model to study the impact of different cultivation and induction regimes on the structure of cytosolic inclusion bodies (IBs). The protein has been heterologously expressed in Escherichia coli in fed-batch cultivations at different temperatures (30, 37, and 40 degrees C) as well as induction regimes that guaranteed distinct product formation rates and ratios of soluble to aggregated protein. Additionally, shake flask cultivations at 20, 30, and 37 degrees C were performed. IBs were sampled during the whole bioprocess and structural analysis was performed by attenuated total reflectance Fourier transform infrared (ATR-FT-IR) spectroscopy. This work clearly demonstrates that the tested production regimes and rates had no impact on the IB structure, which was characterized by decreased alpha-helical and increased and modified beta-sheet contents compared to the native protein. Moreover, aggregates formed during refolding of IBs by solubilization and simple dilution showed very similar FT-IR spectra suggesting (i) the existence of only one critical folding step from which either aggregation (IB formation) or native folding branches off, and (ii) underlining the important role of the specific amino acid sequence in aggregation. The findings are discussed with respect to the known structure of TEM1-beta-lactamase and the reported kinetics of its (un)folding as well as contradictory data on the effect of cultivation regimes on IB structure(s) of other proteins.
Margreiter
Gerd
G
Department of Biotechnology Division of Biochemistry, BOKU-University of Natural Resources and Applied Life Sciences, Vienna, Vienna, Austria.
Schwanninger
Manfred
M
Bayer
Karl
K
Obinger
Christian
C
D003600:Cytosol / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D002479:Inclusion Bodies / Q000201:enzymology*; D011485:Protein Binding; D017510:Protein Folding; D017550:Spectroscopy, Fourier Transform Infrared; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism
D029968:Escherichia coli Proteins; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1