Protein solubility and differential proteomic profiling of recombinant Escherichia coli overexpressing double-tagged fusion proteins.

Background: Overexpression of recombinant proteins usually triggers the induction of heat shock proteins that regulate aggregation and solubility of the overexpressed protein. The two-dimensional gel electrophoresis (2-DE)-mass spectrometry approach was used to profile the proteome of Escherichia coli overexpressing N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), both fused to glutathione S-transferase (GST) and polyionic peptide (5D or 5R).

Results: Overexpression of fusion proteins by IPTG induction caused significant differential expression of numerous cellular proteins; most of these proteins were down-regulated, including enzymes connected to the pentose phosphate pathway and the enzyme LuxS that could lead to an inhibition of tRNA synthesis.

Proteomic changes in the hypothalamus and retroperitoneal fat from male F344 rats subjected to repeated light-dark shifts.

Chronic circadian desynchronization induced by repeated 12 h light-dark cycle shifts conducted twice weekly resulted in elevated food intake, body weight gain, and retroperitoneal fat mass in male F344 rats. Using a proteomic approach, we found that repeated light-dark shifts caused changes in expression levels of five hypothalamic (four upregulated) and 22 retroperitoneal fat (13 upregulated) 2-DE protein spots. Proteins involved in carbohydrate metabolism and in the citric acid cycle were upregulated, indicating a positive energy balance status.

Production of N-acetyl-D-neuraminic acid using two sequential enzymes overexpressed as double-tagged fusion proteins.

Background: Two sequential enzymes in the production of sialic acids, N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), were overexpressed as double-tagged gene fusions. Both were tagged with glutathione S-transferase (GST) at the N-terminus, but at the C-terminus, one was tagged with five contiguous aspartate residues (5D), and the other with five contiguous arginine residues (5R).

Results: Both fusion proteins were overexpressed in Escherichia coli and retained enzymatic activity.

Chromatographic characterization of molecularly imprinted polymers.

Recent efforts in the investigation of chromatographic characterization of molecularly imprinted polymers (MIPs) have focused mainly on the nature of heterogeneous binding sites. More data on the thermodynamics than on the kinetic features of MIP columns have been published. The present article addresses the sources of peak broadening and tailing, which are the main drawbacks often associated with imprinted polymers in chromatography for practical applications.