ABSTRACT
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.
Subject(s)
Interferon-gamma/genetics , Protein Engineering/methods , Antiviral Agents/chemistry , Antiviral Agents/isolation & purification , Drug Stability , Escherichia coli , Humans , Interferon-gamma/chemistry , Interferon-gamma/isolation & purification , Mutation , Protein Folding , Protein Renaturation , Recombinant Proteins , TemperatureABSTRACT
The analysis has been made for the basic immunocorrection drugs, used in clinical practice. By their features, the drugs are divided into 6 groups: immunoactive structures of pathogens, polyelectrolites, inductors of IFN-alpha, thymic hormones and their analogs, bone marrow regulators (myelopeptides) and cytokines. The first 3 groups of drugs are characterized by the inductive mechanism of action, whereas thymic hormones, myelopeptides and cytokines by the substitutive mechanism. The substitutive immunomodulators are preferential drugs at the advanced stages of infection. Immunocorrection should be used by the adequate courses and under the control of clinical dynamics, immunogram or differential blood counts. Transplantation of heterologous tissues and organs, transfusion of blood cell fractions and the first trimester of gestation may be called as contraindications for administration of immunocorrection. The high clinical efficiency of immunocorrection is shown on the usage of leukinferon in an extremely severe human pathology, such as sepsis, complicated by syndrome of polyorgan insufficiency.
