title: Methods for preparation of recombinant cytokine proteins V. mutant analogues of human interferon-gamma with higher stability and activity.
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.
authors.affiliation: M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Ul. Miklukho-Maklaya, Moscow, GSP, Russia.
authors.forename: Sergey E
authors.initials: SE
authors.lastname: Pechenov
authors.forename: Roman V
authors.initials: RV
authors.lastname: Tikhonov
authors.forename: Lyudmila N
authors.initials: LN
authors.lastname: Shingarova
authors.forename: Vyacheslav G
authors.initials: VG
authors.lastname: Korobko
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Yakimov
authors.forename: Vadim E
authors.initials: VE
authors.lastname: Klyushnichenko
authors.forename: Alla A
authors.initials: AA
authors.lastname: Babajantz
authors.forename: Dmitriy L
authors.initials: DL
authors.lastname: Beliaev
authors.forename: Vladimir P
authors.initials: VP
authors.lastname: Kuznetzov
authors.forename: Vitaliy I
authors.initials: VI
authors.lastname: Shvetz
authors.forename: Andrey N
authors.initials: AN
authors.lastname: Wulfson
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry / Q000302:isolation & purification; D004355:Drug Stability; D004926:Escherichia coli; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D020673:Protein Renaturation; D011994:Recombinant Proteins; D013696:Temperature
chemical_list: D000998:Antiviral Agents; D011994:Recombinant Proteins; D007371:Interferon-gamma