Optimized small tissue specific protein isolation trough complexed diafiltration technique.

Physiologically active and pure small tissue specific proteins are in demand in various fundamental and applied areas due to its bioactive properties. It is known that proteins can aggregate, as well as form a highly concentrated layer at the border of the filtration membrane or adhere to it, which significantly interferes with filtration. To eliminate these effects, protein-peptide extracts are recommended to be diluted, as well as using special agents, among them amino acids are more suitable for food industry. The purpose of the study is to develop the isolation and purification of proteins with molecular weight lower 50kDa from porcine hearts and aortas, using different amino acids and its combination. It was previously revealed, that family of fatty acid binding proteins (FABP) is one of the most stable and physiologically active protein in such raw materials. The final technology includes extraction, centrifugation, dilution of extract, diafiltration, lyophilization, dissolving in a small volume of distilled water, dialization and final drying. Concentration of 1.0% glycine and 0.1M proline in both diluted extract and exchange solution was revealed to be most effective to prevent protein aggregation. Dialysates were dried lyophilically and O'Farrell 2DE-electrophoresis with MALDI-TOF MS and MS/MS mass spectrometry identification were used. No proteins of 50kDa and bigger were detected. Proteins of lipid metabolism, peroxiredoxin 2, transgelins etc. with molecular weight lower 50kDa were found. The developed technology allows increasing cardiac and adipocytic isoforms of FABP family content. Cardiac isoform was previously identified in raw material, while adipocytic was newly detected. Adipocytic isoform content is lower than cardiac one; therefore, it was not detected in raw material, but application of complexed diafiltration technique led to increasing its content in dialysate. The developed technology can be used for separation and purification of animal proteins in native and stable forms. [ABSTRACT FROM AUTHOR]