Efficient site specific removal of a C-terminal FLAG fusion from a Fab′ using copper(II) ion catalysed protein cleavage

被引：24

作者：

Humphreys, DP ^{[1
]}

Smith, BJ ^{[1
]}

King, LM ^{[1
]}

West, SM ^{[1
]}

Reeks, DG ^{[1
]}

Stephens, PE ^{[1
]}

机构：

[1] Celltech Therapeut, Slough SL1 4EN, Berks, England

来源：

PROTEIN ENGINEERING | 1999年 / 12卷 / 02期

关键词：

copper (II); cupric; Fab '; Flag peptide; protein cleavage;

D O I：

10.1093/protein/12.2.179

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

The peptide sequence (DKTHC)-D-N was investigated as a site for efficient, specific cleavage of a fusion protein by cupric ions using a humanised gamma 1 Fab' as a model protein. The native upper hinge (DKTHC)-D-N sequence was mutated to create a site resistant to cleavage by cupric ions and a (DKTHC)-D-N sequence introduced between the hinge and a C-terminal FLAG peptide. Incubation of Fab' with Cu2+ at 62 degrees C at alkaline pHs resulted in removal of the FLAG peptide with efficiencies of up to 86%. Cleavage conditions did not detrimentally affect the Fab' protein. Use of the (DKTHC)-D-N sequence along with cupric ions may provide a cost-effective method for large scale proteolytic cleavage of fusion proteins.

引用

页码：179 / 184

页数：6

共 27 条

[1]

ADMAN ET, 1991, ADV PROTEIN CHEM, V42, P145

[2]

Allen G, 1996, INT J PEPT PROT RES, V48, P265

[3]

BRIZZARD BL, 1994, BIOTECHNIQUES, V16, P730

[4] ENGINEERING ENZYME SPECIFICITY BY SUBSTRATE-ASSISTED CATALYSIS [J].