Fourier transform infrared spectroscopy analysis of the conformational quality of recombinant proteins within inclusion bodies

Abstract

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.

The Functional Quality of Soluble Recombinant Polypeptides Produced in Escherichia coli Is Defined by a Wide Conformational Spectrum

“…Thus, the specific biological activity rather than the presence of the protein species in the soluble cell fraction reveals the conformational quality of the product and, therefore, its biotechnological potential. In this regard, an increasing number of structural analyses reveal the coexistence, in the embedded protein species, of a cross-␤-sheet-based, amyloidlike organization (3) and also that of a native secondary structure (5). In inclusion bodies formed by enzymes, the associated enzymatic activity is sufficient for efficient in situ substrate processing.…”

Fourier Transform Infrared spectroscopy discloses different types of cell death in flow cytometrically sorted cells

“…One of the vibrational bands visibly altered in both dead HeLa -and Vero cells (but only significantly altered in Vero cells) was the appearance of the band at 1626 cm -1 . This band was assigned to intermolecular β-sheet protein-protein interactions suggestive of protein compactness and the degree of protein aggregation (Doglia et al, 2008). Zelig et al (2009) found that a decrease in α-helices and an increase in β-sheets were indicative of apoptosis and that the degree of β-sheets could be linked to the stage of apoptosis (Zelig et al, 2009).…”

Soluble Protein Expression in Bacteria

“…Spectral techniques such as Fourier transform infrared spectroscopy (FTIR) (1,2,16) and nuclear magnetic resonance (NMR) (3) revealed different folded forms in preparations from IBs. This indicates that there are differences in structure of proteins isolated from IBs compared to those expressed solubly (4).…”

“…Cell free growth (in lysates), or expression in yeast, insect cells or transfected mammalian cells are all possible alternatives. While expression systems in yeast (10,11) such as Pichia pastoris (12)(13)(14)(15) and the baker's yeast Saccharomyces cerevisiae (16) are inexpensive and well developed, eukaryotic or insect cell culture methods are significantly more expensive than bacterial. Stably transformed higher eukaryotic cell lines are given to variability, tests for expression tests are time consuming, and the scale-up can be quite difficult.…”