Expression, purification, and characterization of rat interferon-β, and preparation of an N-terminally PEGylated form with improved pharmacokinetic parameters

被引：36

作者：

Arduini, RM ^{[1
]}

Li, ZF ^{[1
]}

Rapoza, A ^{[1
]}

Gronke, R ^{[1
]}

Hess, DM ^{[1
]}

Wen, DY ^{[1
]}

Miatkowski, K ^{[1
]}

Coots, C ^{[1
]}

Kaffashan, A ^{[1
]}

Viseux, N ^{[1
]}

Delaney, J ^{[1
]}

Domon, B ^{[1
]}

Young, CN ^{[1
]}

Boynton, R ^{[1
]}

Chen, LL ^{[1
]}

Chen, LQ ^{[1
]}

Betzenhauser, M ^{[1
]}

Miller, S ^{[1
]}

Gill, A ^{[1
]}

Pepinsky, RB ^{[1
]}

Hochman, PS ^{[1
]}

Baker, DP ^{[1
]}

机构：

[1] Biogen Idec Inc, Cambridge Ctr 14, Cambridge, MA 02142 USA

来源：

PROTEIN EXPRESSION AND PURIFICATION | 2004年 / 34卷 / 02期

关键词：

unmodified and PEGylated rat interferon-beta;

D O I：

10.1016/j.pep.2003.11.004

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

To identify potential new clinical uses and routes of administration for human interferon-beta-1a (IFN-beta-1a), we have developed an expression and purification procedure for the preparation of highly purified rat interferon-beta (IFN-beta) suitable for testing in rat models of human disease. An expression vector containing the rat IFN-beta signal sequence and structural gene was constructed and transfected into Chinese hamster ovary (CHO) cells. The protein was purified from CHO cell conditioned medium and purified to >99.5% purity using standard chromatographic techniques. Analytical characterization indicated that the protein was a heavily glycosylated monomeric protein, with two of the four predicted N-glycosylation sites occupied. Analysis of the attached oligosaccharides showed them to be a complex mixture of bi-antennary, tri-antennary, and tetra-antennary structures with a predominance of sialylated tri-antennary and tetra-antennary structures. Peptide mapping, N-terminal sequencing, and mass spectrometry confirmed the identity and integrity of the purified protein. The purified protein had a specific activity of 2.1 x 10(8) U/mg when assayed on rat RATEC cells, which is similar in magnitude to the potencies observed for murine IFN-beta and human IFN-beta-1a assayed on murine and human cells, respectively. We also prepared an N-terminally PEGylated form of rat IFN-beta in which a 20 kDa methoxy polyethylene glycol (PEG)propionaldehyde was attached to the N-terminal alpha-amino group of Ile-1. The PEGylated protein, which retained essentially full in vitro antiviral activity, had improved pharmacokinetic parameters in rats as compared to the unmodified protein. Both the unmodified and PEGylated forms of rat IFN-beta will be useful for testing in rat models of human disease. (C) 2004 Elsevier Inc. All rights reserved.

引用

页码：229 / 242

页数：14

共 49 条

[1] Characterization of a soluble ternary complex formed between human interferon-β-1a and its receptor chains [J].

Arduini, RM ;

Strauch, KL ;

Runkel, LA ;

Carlson, MM ;

Hronowski, XP ;

Foley, SF ;

Young, CN ;

Cheng, WJ ;

Hochman, PS ;

Baker, DP .

PROTEIN SCIENCE, 1999, 8 (09) :1867-1877

[2] Rational design of a potent, long-lasting form of interferon:: A 40 kDa branched polyethylene glycol-conjugated interferon α-2a for the treatment of hepatitis C [J].

Bailon, P ;

Palleroni, A ;

Schaffer, CA ;

Spence, CL ;

Fung, WJ ;

Porter, JE ;

Ehrlich, GK ;

Pan, W ;

Xu, ZX ;

Modi, MW ;

Farid, A ;

Berthold, W .

BIOCONJUGATE CHEMISTRY, 2001, 12 (02) :195-202

[3]

Baker D E, 2001, Rev Gastroenterol Disord, V1, P87

[4] Regulation of STAT protein synthesis by c-Cbl [J].

Blesofsky, WA ;

Mowen, K ;

Arduini, RM ;

Baker, DP ;

Murphy, MA ;

Bowtell, DDL ;

David, M .

ONCOGENE, 2001, 20 (50) :7326-7333

[5] Emerging protein delivery methods [J].

Cleland, JL ;

Daugherty, A ;

Mrsny, R .

CURRENT OPINION IN BIOTECHNOLOGY, 2001, 12 (02) :212-219

[6]

COHEN B, 1995, MOL CELL BIOL, V15, P4208

[7] DIRECT BINDING TO AND TYROSINE PHOSPHORYLATION OF THE ALPHA-SUBUNIT OF THE TYPE-I INTERFERON RECEPTOR BY P135(TYK2) TYROSINE KINASE [J].

COLAMONICI, O ;

YAN, H ;

DOMANSKI, P ;

HANDA, R ;

SMALLEY, D ;

MULLERSMAN, J ;

WITTE, M ;

KRISHNAN, K ;

KROLEWSKI, J .

MOLECULAR AND CELLULAR BIOLOGY, 1994, 14 (12) :8133-8142

[8]

COLAMONICI OR, 1994, J BIOL CHEM, V269, P3518

[9]

CONRADT HS, 1987, J BIOL CHEM, V262, P14600

[10] Integrin α1β1 and transforming growth factor-β1 play distinct roles in Alport glomerular pathogenesis and serve as dual targets for metabolic therapy [J].

Cosgrove, D ;

Rodgers, K ;

Meehan, D ;

Miller, C ;

Bovard, K ;

Gilroy, A ;

Gardner, H ;

Kotelianski, V ;

Gotwals, P ;

Amatucci, A ;

Kalluri, R .

AMERICAN JOURNAL OF PATHOLOGY, 2000, 157 (05) :1649-1659

← 1 2 3 4 5 →