Recombinant C1 Esterase Inhibitor Reduces Cytokine Storm in an Ex Vivo Whole Blood Model.

C1 esterase inhibitor (C1INH) is an abundant component of blood plasma (the average concentration is 250 mg/L); it is known to be involved in several biological processes, for instance, in the regulation of the coagulation system, adhesion of leukocytes on endothelial cells, and in the regulation of complement and kallikrein cascades. Lately, the role of C1INH in immunomodulation has gained considerable attention. We used an ex vivo whole blood model to examine the influence of C1INH and its mutated variants on the inflammatory cytokines interleukin (IL)-6, tumor necrosis factor-α (TNFα), and IL-1ß. The present study demonstrated for the first time that recombinant C1INH or its Seprin domain can downregulate bacterial endotoxin induced IL-6 release. We also observed that unstructured N-terminal domain of C1INH downregulates the release of IL-1ß and TNFα, but not IL-6. Our results suggest that C1INH may have therapeutic potential for treatment of inflammatory conditions.

A new hybrid protein for production of recombinant bacteriorhodopsin in Escherichia coli.

Unique properties of bacteriorhodopsin, namely, photochromism and high thermal stability, make this protein an attractive target for physico-chemical studies, as well as for various biotechnological applications. Using Mistic as a suitable carrier for insertion of recombinant membrane proteins into cytoplasmic membrane of Escherichia coli, we developed a system for overexpression of bacteriorhodopsin and worked out an efficient procedure for its purification and renaturation with the final yield of 120 mg/l of refolded protein, which is the highest value reported to date for bacteriorhodopsin produced in E. coli. Functional activity of recombinant bacteriorhodopsin was confirmed by spectroscopic and electrochemical assays.

Producing human mechano growth factor (MGF) in Escherichia coli.

MGF is a product of a unique muscle-specific splice variant of IGF1 gene (insulin-like growth factor). Its peculiar feature is a specific E-peptide, a 16 a.a. strand at the C-terminus. MGF increases cellular proliferation and inhibits terminal differentiation of myoblasts necessary for the secondary myotube formation. Previous analysis of physiological effects of MGF was performed using indirect methods such as RT-PCR based examination of the transcript contents in normal tissues, adenovirus-mediated DNA delivery and synthetic E-domain administration. Here, we describe isolation and purification of recombinant MGF thus allowing for the first time the possibility of direct examining MGF effects. The recombinant MGF of directly examining--was expressed in Escherichia coli as inclusion bodies (about 100-200mg/l), purified and refolded. Biological activity of refolded MGF was analyzed in vitro in proliferation assays with normal human myoblasts. As a result of our work, it has become possible to generate a standard MGF control with characterized activity and a ready-to use MGF test-system neither of which have been previously described. Our data open opportunities for the future works on MGF characterization and to the development of a powerful and highly specific therapeutic agent potentially applicable for muscle growth up-regulation, post-trauma muscle repair, age and hereditary myodystrophy mitigation and in sport medicine.

Recombinant human insulin IX. Investigation of factors, influencing the folding of fusion protein-S-sulfonates, biotechnological precursors of human insulin.

The peculiarities of molecular structures and the influence of reaction conditions on the folding efficiency of fusion proteins-biotechnological precursors of human insulin, expressed in Escherichia coli as inclusion bodies have been investigated. The fusion proteins contained proinsulin sequence with various leader peptides connected by an Arg residue to the insulin B-chain. The kind and the size of leader peptide do not have essential influence on folding efficiency. However, the efficiency of protein folding depends on the location of the (His)6 site, which is used for metal-chelating affinity chromatography. In our study the protein folding depends on the reaction medium composition (including additives), the presence of accompanied cell components, pH, temperature, concentrations of protein, and redox agents. A negative influence of nucleic acid and heavy metal ions on folding has been found. S-sulfonated fusion protein has proinsulin-like secondary structure (by CD-spectroscopy data) that is the key point for 95% efficient folding proceeding. Folded fusion proteins are transformed into insulin by enzymatic cleavage.

Methods for preparation of recombinant cytokine proteins V. mutant analogues of human interferon-gamma with higher stability and activity.

Mutant analogues of recombinant human immune interferon (IFN-gamma) with higher stability and biological activity were prepared. Depending on the analogue, protein structure modification might involve introduction of an intramonomer disulfide bond (through replacements of Glu7Cys and Ser69Cys), C-terminal shortening by 10 amino acid residues, as well as Gln133Leu substitution in truncated variant. Isolation, purification, and renaturation of the IFN-gamma analogues expressed in Escherichia coli as inclusion bodies were performed according to the scheme developed earlier for wild-type protein. The main idea of this scheme is to remove cellular impurities before recombinant protein renaturation. Folding kinetics of IFN-gamma was studied by reversed-phase HPLC. IFN-gamma and mutant proteins were characterized by their thermal stability and biological activity. Introduction of the intramolecular disulfide bond together with C-terminal shortening and replacement of C-terminal residue was shown to result in increasing the thermal stability by 19 degrees C and four times enhancement of biological activity compared with intact IFN-gamma molecule.