Recombinant Human Erythropoietin with Additional Processable Protein Domains: Purification of Protein Synthesized in Escherichia coli Heterologous Expression System.

Three variants of human recombinant erythropoietin (rhEPO) with additional N-terminal protein domains were obtained by synthesis in an Escherichia coli heterologous expression system. These domains included (i) maltose-binding protein (MBP), (ii) MBP with six histidine residues (6His) in N-terminal position, (iii) s-tag (15-a.a. oligopeptide derived from bovine pancreatic ribonuclease A) with N-terminal 6His. Both variants of the chimeric protein containing MBP domain were prone to aggregation under nondenaturing conditions, and further purification of EPO after the domain cleavage by enterokinase proved to be impossible. In the case of 6His-s-tag-EPO chimeric protein, the products obtained after cleavage with enterokinase were successfully separated by column chromatography, and rhEPO without additional domains was obtained. Results of MALDI-TOF mass spectrometry showed that after refolding 6His-s-tag-EPO formed a structure similar to that of one of native EPO with two disulfide bonds. Both 6His-s-tag-EPO and rhEPO without additional protein domains purified after proteolysis possessed the same biological activity in vitro in the cell culture.

Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) with Additional Protein Domain Synthesized in E. coli: In Vivo Osteoinductivity in Experimental Models on Small and Large Laboratory Animals.

Recombinant human bone morphogenetic protein-2 with an additional s-tag domain (s-tag-BMP-2) synthesized in E. coli is characterized by higher solubility and activity than the protein without additional s-tag domain, which increases the yield during purification and simplifies protein introduction into the osteoplastic materials. The high osteoinductivity of the demineralized bone matrix with s-tag-BMP-2 was shown on the model of regeneration of cranial defects of a critical size in mice and on the model of implantation of porous titanium matrix into defects of femoral and tibial bones in rabbits.