Direct Production of Proteins with N-Terminal Cysteine for Site-Specific Conjugation Ian E. Gentle, David P. De Souza, and Manuel Baca* The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria, 3050, Australia. Received February 3, 2004 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 intiating 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. INTRODUCTION Over the past decade, a number of new chemical methods have been devised which can be used to site- specifically modify the N-terminus of recombinant pro- teins (1-3). The most elegant of these, native chemical ligation (2), requires that the recombinant protein contain an N-terminal cysteine residue, which can then react in a specific manner with thioester-containing reagents. This method has been used to site-specifically label proteins with small molecules (4, 5), to generate semi- synthetic proteins via ligation of synthetic peptides (6) or to link together recombinantly produced protein frag- ments (7). While native chemical ligation has been extensively applied to chemically synthesized peptides (8), its application to recombinant proteins has been more limited. Current methods for producing N-terminal cys- teine proteins usually involves cleavage of a precursor fusion protein with a site-specific protease in order to expose an internal cysteine residue as the new N- terminus (4, 9). The drawbacks of this approach include possible cleavage at other sites within the protein and the need to repurify the cleaved product. Furthermore, the activity of site-specific proteases are not compatible with high levels of chaotropic agents which might oth- erwise be used to extract proteins from inclusion bodies and, if necessary, maintain them in solution. A preferable approach to the production of N-terminal cysteine proteins would be to take advantage of endog- enous methionine aminopeptidase (10) to cleave the initiating methionine from recombinantly expressed pro- teins containing cysteine as the second residue. As methionine excision is believed to occur cotranslationally (11-13), that is, before the completion of synthesis or folding of polypeptide chains, the expressed protein would be isolated with cysteine as the N-terminal residue. The specificity of methionine aminopeptidase is dependent upon the identity of the amino acid residue adjacent to the amino terminus. Methionine is efficiently removed from proteins when the second residue is a small side chain amino acid, even when these proteins are overex- pressed (14-16). However, methionine is not cleaved when a large side chain amino acid is the adjacent residue. Variable degrees of cleavage have been reported to occur when the second residue is an intermediate sized amino acid such as cysteine (14, 15). This appears to have limited attempts to produce N-terminal cysteine proteins by this route, due to a widely held belief that methionine excision will be incomplete (e.g. reference (4)), although at least one report has made use of this strategy (17). Given the usefulness of proteins with N-terminal cysteine for site-specific modification or protein semisyn- thesis, we have investigated the direct production of such proteins in E. coli by expression of constructs containing Met-Cys- amino terminii. We report conditions for opti- mizing the yield of the processed product and demon- strate its utility for site-specific modification of the N-terminus. EXPERIMENTAL PROCEDURES Construction of Protein Expression Vectors. For expression of proteins containing an N-terminal polyhis- tidine tag, a modified pET15b vector (Novagen) was used. Proteins produced corresponded to fragments from sup- pressor of cytokine signaling (SOCS)-7 (amino acids 378- 512) and SHP2 (amino acids 1-105 or 109-220). Each of the pET15b-based constructs encoded polypeptide products with either a MCSHHHHHHGARQ or MCRHH- HHHHGARQ N-terminal sequence, followed by the frag- ments defined above. For expression of proteins contain- ing a C-terminal polyhistidine tag, a pET22b vector (Novagen) was used. Proteins produced with this con- struct corresponded to fragments from SOCS7 (amino * Corresponding author: AMRAD Corporation, 576 Swan St., Richmond, Victoria 3121, Australia; Telephone: +613 9208 4371; fax: +613 9208 4100; e-mail: mbaca@amrad.com.au. 658 Bioconjugate Chem. 2004, 15, 658-663 10.1021/bc049965o CCC: $27.50 © 2004 American Chemical Society Published on Web 05/01/2004