[Proteomic analysis of contaminants in recombinant membrane hemeproteins expressed in E. coli and isolated by metal affinity chromatography].

Contaminating proteins have been identified by "shotgun" proteomic analysis in 14 recombinant preparations of human membrane heme- and flavoproteins expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Immobilized metal ion affinity chromatography of ten proteins was performed on Ni2+-NTA-sepharose 6B, and the remaining four proteins were purified by ligand affinity chromatography on 2',5'-ADP-sepharose 4B. Proteomic analysis allowed to detect 50 protein impurities from E. coli. The most common contaminant was Elongation factor Tu2. It is characterized by a large dipole moment and a cluster arrangement of acidic amino acid residues that mediate the specific interaction with the sorbent. Peptidyl prolyl-cis-trans isomerase SlyD, glutamine-fructose-6-phosphate aminotransferase, and catalase HPII that contained repeating HxH, QxQ, and RxR fragments capable of specific interaction with the sorbent were identified among the protein contaminants as well. GroL/GroS chaperonins were probably copurified due to the formation of complexes with the target proteins. The Ni2+ cations leakage from the sorbent during lead to formation of free carboxyl groups that is the reason of cation exchanger properties of the sorbent. This was the putative reason for the copurification of basic proteins, such as the ribosomal proteins of E. coli and the widely occurring uncharacterized protein YqjD. The results of the analysis revealed variation in the contaminant composition related to the type of protein expressed. This is probably related to the reaction of E. coli cell proteome to the expression of a foreign protein. We concluded that the nature of the protein contaminants in a preparation of a recombinant protein purified by immobilized metal ion affinity chromatography on a certain sorbent could be predicted if information on the host cell proteome were available.

[Analysis of serum lipid profiles in guinea pigs for early detection of changes in metabolism under exposure to environmental contaminants].

The exposure to anthropogenic factors has a multifaceted impact on the body of humans and animals. Due to their complex influence the detection of negative effects of the certain factors is a rather complicated task. Metabolomic methodology which permits to overcome these difficulties, has been applied in the evaluation of the nature and degree of the impact of potash fertilizers production waste on lipid profiles of experimental animals after intranasal inoculation with potassic fertilizer production waste and consumption of drinking water obtained from sources located in the zone of potential action of potassic fertilizer production. Isolation of lipids from serum was performed with the help of specially developed technique based on solid-phase extraction of samples which allows to remove cholesterin from the samples. Each sample was subjected to HPLC -MS analysis, after which the obtained chromatograms were treated with the use of the method of principal component analysis and cluster analysis. The developed technique allows to efficiently separate hydrophobic metabolites in blood serum. There was established serum lipid profile of experimental animals, in particular the content of phospholipids and oxysteroids, and there were found differences in the metabolic processes of the test and control animals. It is shown that in the serum of experimental animals, there is observed an increased concentration of hydroxysteroid as compared with the control group.