Direct Production of Proteins with N-Terminal Cysteine for Site-Specific Conjugation

详细信息    查看全文

• 作者：Ian E. Gentle ; David P. De Souza ; and Manuel Baca
• 刊名：Bioconjugate Chemistry
• 出版年：2004
• 出版时间：May 2004
• 年：2004
• 卷：15
• 期：3
• 页码：658 - 663
• 全文大小：80K
• 年卷期：v.15,no.3(May 2004)
• ISSN：1520-4812

文摘

Proteins with N-terminal cysteine can undergo native chemical ligation and are useful for site-specificN-terminal labeling or protein semisynthesis. Recombinant production of these has usually been bysite-specific cleavage of a precursor fusion protein at an internal cysteine residue. Here we describea simpler route to producing these proteins. Overexpression in E. coli of several proteins containingcysteine as the second amino acid residue yielded products in which the intiating methionine residuehad been completely cleaved by endogenous methionine aminopeptidase. While secondary modificationof the terminal cysteine was a complicating factor, conditions were identified to eliminate or minimizethis problem. Recombinant proteins produced in this way were suitable for site-specific modificationof the amino terminus via native chemical ligation technology, as demonstrated by conjugation of athioester-containing derivative of fluorescein to one such protein. The ability to directly produce proteinswith N-terminal cysteine should simplify the application of native chemical ligation technology torecombinant proteins and make the technique more amenable to researchers with limited expertisein protein chemistry.