Conjugation of Proteins by Installing BIO-Orthogonally Reactive Groups at Their N-Termini Nagasundarapandian Soundrarajan1, Sriram Sokalingam1, Govindan Raghunathan1, Nediljko Budisa2, Hyun-Jong Paik1, Tae Hyeon Yoo3,4, Sun-Gu Lee1* 1 Department of Polymer Science and Chemical Engineering, Pusan National University, Busan, South Korea, 2 Department of Chemistry, Technische Universita¨t Berlin, Franklinstrasse, Berlin, Germany, 3 Department of Molecular Science and Technology, Ajou University, Suwon, South K oU4reDae,partment of Applied Chemistry and Biological Engineering, Ajou University, Suwon, South Korea Abstract N-terminal site-specific modification of a protein has many advantages over methods targeting internal positions, but it is not easy to install reactive groups onto a protein in an N-terminal specific manner. We here report a strategy to incorporate amino acid analogues specifically in the N-terminus of a protein in vivo and demonstrate it by preparing green fluorescent protein (GFP) having bio-orthogonally reactive groups at its N-terminus. In the first step, GFP was engineered to be a foldable, internal methionine-free sequence via the semi-rational mutagenesis of five internal methionine residues and the introduction of mutations for GFP folding enhancement. In the second step, the N-terminus of the engineered protein was modified in vivo with bio-orthogonally functional groups by reassigning functional methionine surrogates such as L- homopropargylglycine and L-azidohomoalanine into the first methionine codon of the engineered internal methionine-free GFP. The N-terminal specific incorporation of unnatural amino acids was confirmed by ESI-MS analysis and the incorporation did not affect significantly the specific activity, refolding rate and folding robustness of the protein. The two proteins which have alkyne or azide groups at their N-termini were conjugated each other by bio-orthogonal Cu(I)-catalyzed click chemistry. The strategy used in this study is expected to facilitate bio-conjugation applications of proteins such as N- terminal specific glycosylation, labeling of fluorescent dyes, and immobilization on solid surfaces. Citation: Soundrarajan N, Sokalingam S, Raghunathan G, Budisa N, Paik H-J, et al. (2012) Conjugation of Proteins by Installing BIO-Orthogonally Reactive Groups at Their N-Termini. PLoS ONE 7(10): e46741. doi:10.1371/journal.pone.0046741 Editor: Dafydd Jones, Cardiff University, United Kingdom Received May 16, 2012; Accepted September 4, 2012; Published October 8, 2012 Copyright: 2012 Soundrarajan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by Basic Science Program (NRF-2011-0021222) and Mid-career Research Program (NRF-2012016803) through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sungulee@pusan.ac.kr Introduction The N-terminus of a protein is an attractive target site for functionalization to afford protein modifications such as PEGyla- tion, glycosylation and fluorescent labeling; these modified proteins can be valuable sources for development of therapeutics and diagnostics [1–3]. Several chemical methods have been developed for N-terminal functionalization of a protein, but they are generally complicated and involve side reactions which yield heterogeneous products [4–6]. Biological methods for the N- terminal functionalization of a protein have recently been recognized as efficient ways to overcome the problems in chemical N-terminal modification. In particular, an approach based on the methionine (Met) residue substitution method allows the efficient production of proteins with an N-terminal specific functional group in vivo [7–9], which would pave the way to generate proteins with novel functions. The Met residue substitution method introduces unnatural Met analogues into a protein by reassigning the Met codon globally in a protein sequence [5,6]. The simple procedure using the Met auxotroph enables the production of a range of proteins with functional groups on a large scale. Bio-orthogonally reactive groups, such as L-homopropargylglycine (Hpg) and L-azidoho- moalanine (Aha), have been incorporated into the Met positions of proteins in vivo by adding the Met surrogates instead of Met because the wild-type Met-tRNA synthetase recognizes the unnatural amino acids [5,10]. In addition, engineering of the substrate specificity of Met-tRNA synthetase can expand the scope of this methodology [11,12]. Bacterial proteins are synthesized from Met and the removal process of the start Met can be suppressed by selecting the second residue next to the Met carefully [7,9]. Therefore, Met analogues can be incorporated into the N-termini of proteins using the Met residue substitution method. However, the presence of the internal Met codons in the target sequences limits the successful application of the Met residue substitution method for N-terminal specific functionalization due to the reassignment of unnatural Met surrogates to internal Met codons as well as to the first Met codon [7–9]. This problem can be overcome by engineering the protein sequence to be devoid of internal Met residues. Although this approach sometimes needs time-consuming protein engineer- ing work to find internal Met-free variants having original functions of proteins, to our knowledge, this approach is the only one that makes the N-terminal specific modification of a protein possible. Our previous report showed that a protein sequence could be engineered to be an internal Met-free using a consensus-based PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e46741