Application of combined reagent solution to the oxidative refolding of recombinant human interleukin 6

被引:4
作者
Harada, T
Kurimoto, E
Moriyama, Y
Ejima, D
Sakai, T
Nohara, D
Kato, K
机构
[1] Nagoya City Univ, Grad Sch Pharmaceut Sci, Mizuho Ku, Nagoya, Aichi 4678603, Japan
[2] Ajinomoto Co Inc, Pharmaceut Res Lab, Kawasaki Ku, Kawasaki, Kanagawa 2108681, Japan
[3] Gifu Univ, Fac Engn, Gifu 5011193, Japan
关键词
protein refolding; interleukin; 6; combined refolding solution; disulfide bond;
D O I
10.1248/cpb.49.1128
中图分类号
R914 [药物化学];
学科分类号
100701 ;
摘要
Human interleukin 6 (hIL-6), which is a cytokine involved in diverse biological activities, consists of a four-helix bundle with two disulfide bonds. For the clinical use of hIL-6 in cancer therapy, designing of commercial-scale production systems of recombinant hIL-6 (rhIL-6) expressed by E. coli has been attempted. Since rhIL-6 has been produced as inclusion bodies in the expression systems reported to date, establishment of a strategy to achieve a high yield of refolding of this recombinant protein is quite desirable. It has been reported that oxidation of rhIL-6 under a completely denaturing condition suppresses aggregation during the refolding process [Ejima et al., Biotechnol. Bioeng., 62. 301-310 (1999)]. In this protocol, however, small but significant amounts of unidentified by-products unavoidably arose, which might be problematic in the therapeutic use of rhIL-6. In the present study, detailed characterization of the individual by-products has been performed on inspection of peptide maps, and the by-products found to originate from improperly formed disulfide bonds, most of which are disulfide-linked dimers. In order to minimize these by-products, combined solutions of urea and LiCl were used for oxidative refolding of rhIL-6. It was demonstrated that combined use of 1-2 m urea and 1-3 m LiCl effectively suppresses the formation of the by-products as well as aggregates. We propose that the use of the combined reagents can be an alternative method for refolding of rhIL-6 for clinical purposes.
引用
收藏
页码:1128 / 1131
页数:4
相关论文
共 18 条
[1]   INORGANIC SALT DENATURANTS STABILIZE RIBONUCLEASE AGAINST DENATURATION BY UREA [J].
AHMAD, F ;
BIGELOW, CC .
CANADIAN JOURNAL OF BIOCHEMISTRY, 1978, 56 (11) :1003-1005
[2]  
BRAKENHOFF JPJ, 1987, J IMMUNOL, V139, P4116
[3]  
Ejima D, 1999, BIOTECHNOL BIOENG, V62, P301, DOI 10.1002/(SICI)1097-0290(19990205)62:3<301::AID-BIT6>3.3.CO
[4]  
2-N
[5]   FORMATION OF THE INTRACHAIN DISULFIDE BOND IN THE CONSTANT FRAGMENT OF THE IMMUNOGLOBULIN LIGHT CHAIN [J].
GOTO, Y ;
HAMAGUCHI, K .
JOURNAL OF MOLECULAR BIOLOGY, 1981, 146 (03) :321-340
[6]   PURIFICATION TO HOMOGENEITY AND CHARACTERIZATION OF HUMAN B-CELL DIFFERENTIATION FACTOR (BCDF OR BSFP-2) [J].
HIRANO, T ;
TAGA, T ;
NAKANO, N ;
YASUKAWA, K ;
KASHIWAMURA, S ;
SHIMIZU, K ;
NAKAJIMA, K ;
PYUN, KH ;
KISHIMOTO, T .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1985, 82 (16) :5490-5494
[7]   CLEAVAGE OF STRUCTURAL PROTEINS DURING ASSEMBLY OF HEAD OF BACTERIOPHAGE-T4 [J].
LAEMMLI, UK .
NATURE, 1970, 227 (5259) :680-+
[8]   DIFFERENCE BETWEEN GUANIDINIUM CHLORIDE AND UREA AS DENATURANTS OF GLOBULAR-PROTEINS - THE POSSIBILITY OF APPLICATION TO IMPROVED REFOLDING PROCESSES [J].
MATSUBARA, M ;
NOHARA, D ;
SAKAI, T .
CHEMICAL & PHARMACEUTICAL BULLETIN, 1992, 40 (02) :550-552
[9]   OVEREXPRESSION AND STRUCTURE-FUNCTION ANALYSIS OF A BIOENGINEERED IL-2 IL-6 CHIMERIC LYMPHOKINE [J].
ROCK, F ;
EVERETT, M ;
KLEIN, M .
PROTEIN ENGINEERING, 1992, 5 (06) :583-591
[10]   ROLES OF DISULFIDE BONDS IN RECOMBINANT HUMAN INTERLEUKIN-6 CONFORMATION [J].
ROCK, FL ;
LI, XM ;
CHONG, PL ;
IDA, N ;
KLEIN, M .
BIOCHEMISTRY, 1994, 33 (17) :5146-5154