ИСТИНА

The use of hybrid protein constructs as a means for production of biotechnological products is wide-spread in biopharmaceutical industry. This technology is effectively used to improve the expression, solubility and production of biologically active proteins. The human enteropeptidase light chain (L-HEP) is one of the enzymes that are used for the hybrid constructs cleavage due to their high specificity to the Lys (Arg) – X peptide bond, as a part of the sequence Asp4Lys (Arg) – X sequence. Therefore, this enzyme can be used for selective cleavage of hybrid proteins containing the L-HEP recognition site. The aim of our work is to develop the processes for producing L-HEP as part of a hybrid construct. Escherichia coli BL21/ (Trx/ DDDDK/ L-HEP) - strain was constructed and cultured to produce a hybrid protein in the form of inclusion bodies, consisting of thioredoxin (Trx), a specific L-HEP recognition site (DDDDK) and L-HEP itself. Inclusion bodies were purified by detergents, and the hybrid protein contained in them was extracted and renatured. The renatured hybrid protein was cleaved by a cascade reaction that was triggered by adding a small amount of human enteropeptidase light chain. It was shown that after proper protein folding, the enzyme eteropeptidase should not be additionally added, since the process was autocatalytically initiated. The reaction mixture containing L-HEP was used without additional purification to produce purotoxin-6 (PT-6) and interferon alpha-2b from hybrid proteins consisting of thioredoxin, L-HEP cleavage site and the relevant target proteins. The high performance liquid chromatography (HPLC) and mass spectrometry analysis of the PT-6 hybrid protein hydrolysis products, that were catalyzed by L-HEP showed that the cleavage yields only the target product PT-6 in a wide range of conditions, whereas, in contrast cleavage of the hybrid interferon alpha protein 2 resulted in a high yield of the target product only under the optimum conditions. However, the experimental evidence suggests that the most critical aspect in the success of the research digestion is the nature of substrate construct. Thus, our researches resulted in the development of a simple L-HEP production process and demonstrated the possibility of using this enzyme for the production of purotoxin-6 and interferon alpha-2b. Hence, we conclude that producing L-HEP in own laboratory is an efficient alternative to others commercial enzymes.