Direct production of proteins with N-terminal cysteine for site-specific conjugation.
Proteins with N-terminal cysteine can undergo native chemical ligation and are useful for site-specific N-terminal labeling or protein semisynthesis. Recombinant production of these has usually been by site-specific cleavage of a precursor fusion protein at an internal cysteine residue. Here we describe a simpler route to producing these proteins. Overexpression in E. coli of several proteins containing cysteine as the second amino acid residue yielded products in which the initiating methionine residue had been completely cleaved by endogenous methionine aminopeptidase. While secondary modification of the terminal cysteine was a complicating factor, conditions were identified to eliminate or minimize this problem. Recombinant proteins produced in this way were suitable for site-specific modification of the amino terminus via native chemical ligation technology, as demonstrated by conjugation of a thioester-containing derivative of fluorescein to one such protein. The ability to directly produce proteins with N-terminal cysteine should simplify the application of native chemical ligation technology to recombinant proteins and make the technique more amenable to researchers with limited expertise in protein chemistry.
Gentle
Ian E
IE
The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia.
De Souza
David P
DP
Baca
Manuel
M
D003545:Cysteine / Q000138:chemical synthesis / Q000737:chemistry*; D019793:Fluorescein; D008024:Ligands; D011506:Proteins / Q000138:chemical synthesis* / Q000302:isolation & purification; D013329:Structure-Activity Relationship
D008024:Ligands; D011506:Proteins; D003545:Cysteine; D019793:Fluorescein