FT-IR spectroscopy for the study of bacterial membrane stress induced by recombinant protein production

Abstract

BackgroundMicroorganisms respond to environmental stresses regulating their membrane fluidity by changing the lipid fatty acid composition. In particular, in bacterial cells the alteration of membrane lipid composition plays an important role in response to heat and toxic stresses [1,2]. It is known that at increasing temperatures the regulation of membrane fluidity occurs through the incorporation of more saturated fatty acids, longer acyl chains and, in some cases, by changing the unsaturated fatty acids from cis to trans conformation. Among different stresses, production of heterologous proteins is of particular interest for its applications in biotechnology. ResultsIn this work we show the potential of FT-IR microspectroscopy to monitor changes in membrane composition during recombinant protein production. In particular, we studied the infrared absorption of E. coli strains expressing several proteins with different amount of soluble and aggregated fractions. In addition, as a control experiment, we have studied E. coli strains expressing beta galactosidase, as reporter gene, under the chaperone IbpB promoter [3].In all model systems examined, an increase in the band intensities at 2850 cm -1 and at 2925 cm -1 was observed in presence of a high level of stress [4]. As these two bands are due to CH 2 stretching vibrations [5], this spectral behaviour may indicate that longer acyl chain and /or more saturated fatty acids are incorporated in cell membranes. The protein expression analysis indicates that the enzymes that lead to the accumulation of Acetyl Co-A (precursor of fatty acids) are strongly accumulated whereas the phospholipide degradation seems being inhibited.Indeed, when the protein is expressed also in a soluble form -as indicated by the FT-IR protein response in the amide I region and confirmed by the SDS-PAGE analysis -the two CH 2 bands are more intense than in the case of IB formation. This behaviour seems to indicate that the presence of the soluble recombinant protein induces a stress in the cell, suggesting that IBs could limit the toxicity of overexpressed foreign proteins. ConclusionThis work highlights the potential of FT-IR spectroscopy not only to study protein aggregation in IBs [6][7][8], but also to monitor the membrane response induced by the heterologous production.

Biochemical and Mechanical Analysis of Occlusal and Proximal Carious Lesions

“…This is due to the higher absorbance of amide III than CH2-CH- bands in the CID zone, as shown in Figure 2 , with statistically no significant difference between CAD and sound dentin ( p > 0.05). This is referred to as the increased aliphatic side-groups of various amino acid residues in the infected tissues in these lesions which promote the demineralization process [ 37 ]. The difference between the occlusal and proximal lesions indicate that this ratio cannot be applied for all carious studies due to the variation in collagen denaturation in various tissues and regions.…”