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.
Pechenov
Sergey E
SE
M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Ul. Miklukho-Maklaya, Moscow, GSP, Russia.
Tikhonov
Roman V
RV
Shingarova
Lyudmila N
LN
Korobko
Vyacheslav G
VG
Yakimov
Sergey A
SA
Klyushnichenko
Vadim E
VE
Babajantz
Alla A
AA
Beliaev
Dmitriy L
DL
Kuznetzov
Vladimir P
VP
Shvetz
Vitaliy I
VI
Wulfson
Andrey N
AN
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
D000998:Antiviral Agents; D011994:Recombinant Proteins; D007371:Interferon-gamma