Expression and purification of a mutant human growth hormone that is resistant to proteolytic cleavage by thrombin, plasmin and human plasma in vitro

被引:5
作者
Alam, KSM [1 ]
Morimoto, M [1 ]
Yoshizato, H [1 ]
Fujikawa, T [1 ]
Furukawa, K [1 ]
Tanaka, M [1 ]
Nakashima, K [1 ]
机构
[1] Mie Univ, Fac Med, Dept Biochem, Tsu, Mie 514, Japan
关键词
human growth hormone; mutation; proteolytic insensitivity; plasmin; thrombin; human plasma;
D O I
10.1016/S0168-1656(98)00113-8
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
The region having a sequence from amino acid 134 to 150 in human growth hormone (hGH) is known to be cleaved by proteases in human plasma, plasmin and thrombin. In this study, oligonucleotide primer-directed mutagenesis was used to produce recombinant mutant hGHs resistant to limited proteolysis by these proteases. Substitution of Arg(134) and Thr(135) of hGH with Asp(134) and Pro(135) yielded a thrombin-resistant hGH mutant: and substitution of Arg(134), Thr(135) and Lys(140) With Asp(134), Pro(135) and Ala(140) yielded a plasmin-resistant hGH mutant. The latter mutant hGH was also insensitive to in vitro proteolysis by human plasma incubated for 7 days. These alterations in amino acid residues of hGH did not disrupt its biological conformation and retained full growth promoting activities on rat Nb2 cells and human T-47D breast cancer cells. (C) 1998 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:183 / 190
页数:8
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