Recombinant Human Erythropoietin Proteins Synthesized in Escherichia coli Cells: Effects of Additional Domains on the in vitro and in vivo Activities.

The aim of this work was to compare biological activities of three variants of bacterially expressed human recombinant erythropoietin (EPO) with additional protein domains: 6His-s-tag-EPO protein carrying the s-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) at the N-terminus and HBD-EPO and EPO-HBD proteins containing heparin-binding protein domains (HBD) of the bone morphogenetic protein 2 from Danio rerio at the N- and C-termini, respectively. The commercial preparation Epostim (LLC Pharmapark, Russia) produced by synthesis in Chinese hamster ovary cells was used for comparison. The EPO variant with the C-terminal HBD domain connected by a rigid linker (EPO-HBD) possesses the best properties as compared to HBD-EPO with the reverse domain arrangement. It was ~13 times more active in vitro (i.e., promoted proliferation of human erythroleukemia TF-1 cells) and demonstrated a higher rate of association with the erythropoietin receptor. EPO-HBD also exhibited the greatest binding to the demineralized bone matrix (DBM) and more prolonged release from the DBM among the four proteins studied. Subcutaneous administration of EPO-HBD immobilized on DBM resulted in significantly more pronounced vascularization of surrounding tissues in comparison with the other proteins and DBM alone. Therefore, EPO-HBD displayed better performance with regard to all the investigated parameters than other examined EPO variants, and it seems promising to study the possibility of its medical use.

Synthesis in Escherichia coli and Characterization of Human Recombinant Erythropoietin with Additional Heparin-Binding Domain.

Recombinant human erythropoietin (EPO) with additional N-terminal heparin-binding protein domain (HBD) from bone morphogenetic protein 2 was synthesized in Escherichia coli cells. A procedure for HBD-EPO purification and refolding was developed for obtaining highly-purified HBD-EPO. The structure of recombinant HBD-EPO was close to that of the native EPO protein. HBD-EPO contained two disulfide bonds, as shown by MALDI-TOF mass spectrometry. The protein demonstrated in vitro biological activity in the proliferation of human erythroleukemia TF-1 cell test and in vivo activity in animal models. HBD-EPO increased the number of reticulocytes in the blood after subcutaneous injection and displayed local angiogenic activity after subcutaneous implantation of demineralized bone matrix (DBM) discs with immobilized HBD-EPO. We developed a quantitative sandwich ELISA method for measuring HBD-EPO concentration in solution using rabbit polyclonal serum and commercial monoclonal anti-EPO antibodies. Pharmacokinetic properties of HBD-EPO were typical for bacterially produced EPO. Under physiological conditions, HBD-EPO can reversibly bind to DBM, which is often used as an osteoplastic material for treatment of bone pathologies. The data on HBD-EPO binding to DBM and local angiogenic activity of this protein give hope for successful application of HBD-EPO immobilized on DBM in experiments on bone regeneration.

Endonuclease Activity of MutL Protein of the Rhodobacter sphaeroides Mismatch Repair System.

We have purified the MutL protein from Rhodobacter sphaeroides mismatch repair system (rsMutL) for the first time. rsMutL demonstrated endonuclease activity in vitro, as predicted by bioinformatics analysis. Based on the alignment of 1483 sequences of bacterial MutL homologs with presumed endonuclease activity, conserved functional motifs and amino acid residues in the rsMutL sequence were identified: five motifs comprising the catalytic site responsible for DNA cleavage were found in the C-terminal domain; seven conserved motifs involved in ATP binding and hydrolysis and specific to the GHKL family of ATPases were found in the N-terminal domain. rsMutL demonstrated the highest activity in the presence of Mn2+. The extent of plasmid DNA hydrolysis declined in the row Mn2+ > Co2+ > Mg2+ > Cd2+; Ni2+ and Ca2+ did not activate rsMutL. Divalent zinc ions inhibited rsMutL endonuclease activity in the presence of Mn2+ excess. ATP also suppressed plasmid DNA hydrolysis by rsMutL. Analysis of amino acid sequences and biochemical properties of five studied bacterial MutL homologs with endonuclease activity revealed that rsMutL resembles the MutL proteins from Neisseria gonorrhoeae and Pseudomonas aeruginosa.

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.