High-Level Expression of TGF-beta 2 and the TGF-beta 2(414) Precursor in Chinese Hamster Ovary Cells

被引：22
|
作者：
Madisen, Linda [1 ]
Lioubin, Mario N. [1 ]
Marquardt, Hans [1 ]
Purchio, A. F. [1 ]
机构：
[1] ONCOGEN, 3005 1st Ave, Seattle, WA 98121 USA
来源：
GROWTH FACTORS | 1990年 / 3卷 / 02期
关键词：
TGF-beta; 2; in vitro expression; immunoblotting; protein sequencing;
D O I：
10.3109/08977199009108275
中图分类号：
Q2 [细胞生物学];
学科分类号：
071009 ; 090102 ;
摘要：
Chinese hamster ovary (CHO) clones secreting high levels of transforming growth factor-beta 2 (TGF-beta 2) were obtained after transfection with a cDNA clone coding for the 414-amino acid TGF-beta 2 precursor and subsequent amplification with methotrexate. The TGF-beta 2 was secreted in a latent form since acidification was necessary for detection of maximal levels of bioactivity. Amino- and carboxy-terminal sequencing of purified recombinant TGF-beta 2 indicated that correct processing of mature TGF-beta 2 had occurred. In addition to mature TGF-beta 2, the recombinant CHO clones secreted larger proteins having molecular weights of 85, 105, and 130 kD, which consisted of both mature and pro-region sequences when analyzed by immunoblotting using site-specific anti-peptide antibodies. Analysis of serumand cell-free media from recombinant CHO cells metabolically labeled with [H-3] glucosamine and [P-32] orthophosphateindicated that pro-TGF-beta 2 was glycosylated and phosphorylated. Two-dimensional electrophoretic analysis of acid hydrolysates showed that the P-32 was incorporated into mannose-6-phosphate.
引用
收藏
页码：129 / 138
页数：10
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