Fourier transform infrared spectroscopy analysis of the conformational quality of recombinant proteins within inclusion bodies.
The solubility of recombinant proteins produced in bacterial cells is considered a key issue in biotechnology as most overexpressed polypeptides undergo aggregation in inclusion bodies, from which they have to be recovered by solubilization and refolding procedures. Physiological and molecular strategies have been implemented to revert or at least to control aggregation but they often meet only partial success and have to be optimized case by case. Recent studies have shown that proteins embedded in inclusion bodies may retain residual structure and biological function and question the former axiom that solubility and activity are necessarily coupled. This allows for a switch in the goals from obtaining soluble products to controlling the conformational quality of aggregated proteins. Central to this approach is the availability of analytical methods to monitor protein structure within inclusion bodies. We describe here the use of Fourier transform infrared spectroscopy for the structural analysis of inclusion bodies both purified from cells and in vivo. Examples are reported concerning the study of kinetics of aggregation and structure of aggregates as a function of expression levels, temperature and co-expression of chaperones.
Doglia
Silvia Maria
SM
Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Milano, Italia.
Ami
Diletta
D
Natalello
Antonino
A
Gatti-Lafranconi
Pietro
P
Lotti
Marina
M
D004591:Electrophoresis, Polyacrylamide Gel; D002479:Inclusion Bodies / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D012995:Solubility; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
D011994:Recombinant Proteins