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Site-Specific Protein Modifications Through Pyrroline-Carboxy-Lysine Residues

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Site-Specific Protein Modifications Through Pyrroline-Carboxy-Lysine Residues Site-specific protein modifications through pyrroline-carboxy-lysine residues Weijia Ou1, Tetsuo Uno1, Hsien-Po Chiu, Jan Grünewald, Susan E. Cellitti, Tiffany Crossgrove, Xueshi Hao, Qian Fan, Lisa L. Quinn, Paula Patterson, Linda Okach, David H. Jones, Scott A. Lesley, Ansgar Brock, and Bernhard H. Geierstanger2 Genomics Institute of the Novartis Research Foundation, 10675 John-Jay-Hopkins Drive, San Diego, CA 92121-1125 Edited* by Peter G. Schultz, The Scripps Research Institute, La Jolla, CA, and approved May 11, 2011 (received for review April 4, 2011) Pyrroline-carboxy-lysine (Pcl) is a demethylated form of pyrrolysine acids will face similar hurdles to achieve site-specificity without that is generated by the pyrrolysine biosynthetic enzymes when deleterious effects to the protein of interest. the growth media is supplemented with D-ornithine. Pcl is readily The most elegant way to generate homogenously, site-specifi- incorporated by the unmodified pyrrolysyl-tRNA/tRNA synthetase cally modified proteins is the in vivo incorporation of unnatural pair into proteins expressed in Escherichia coli and in mammalian amino acids (7–9). Over 70 unnatural amino acids featuring a cells. Here, we describe a broadly applicable conjugation chemistry wide array of functionalities can be incorporated at TAG codons that is specific for Pcl and orthogonal to all other reactive groups using specific tRNA/tRNA synthetase pairs engineered through on proteins. The reaction of Pcl with 2-amino-benzaldehyde or an in vivo selection process to be orthogonal to the cellular 2-amino-acetophenone reagents proceeds to near completion at machinery of the host cells. A set of reactive unnatural amino neutral pH with high efficiency. We illustrate the versatility of acids carrying keto (10), azido (11), and alkyne (12) moieties are the chemistry by conjugating Pcl proteins with poly(ethylene gly- most frequently used for bioconjugation through oxime forma- col)s, peptides, oligosaccharides, oligonucleotides, fluorescence, tion and “click-chemistry.” Although oxime formation requires and biotin labels and other small molecules. Because Pcl is geneti- acidic pH and some click-chemistry reactions require a copper cally encoded by TAG codons, this conjugation chemistry enables catalyst for efficient conjugation, unnatural amino acid technol- enhancements of the pharmacology and functionality of proteins ogy provides unprecedented opportunities for protein labeling through site-specific conjugation. and conjugation through chemical functionalities that are not available among the common 20 amino acids. protein engineering ∣ protein medicinal chemistry ∣ desmethylpyrrolysine ∣ Pyrrolysine (Pyl), the 22nd naturally encoded amino acid, was amber suppression discovered in several methyltransferases in methanogenic archaea (Fig. 1) (Selenocysteine being the 21st amino acid) ust as small molecule drug candidates are optimized through (13–17). In Methanosarcinae, the TAG codon, a canonical termi- Jmedicinal chemistry, protein biotherapeutics need to be opti- nation signal, is primarily repurposed to signal Pyl incorporation mized in terms of their pharmacological and physiochemical (18). Two of the three Pyl biosynthetic genes, pylC and pylD, along properties, selectivity, and immunogenicity (1–3). Such protein with the pyrrolysl-tRNA/tRNA synthetase pair can be transferred medicinal chemistry efforts may, for example, involve choosing to Escherichia coli (19) as well as mammalian cells (20), and an appropriate expression system to achieve a particular glycosy- facilitate the incorporation of pyrroline-carboxy-lysine (Pcl) lation state of the protein (2). Changes in the primary protein rather than Pyl when D-ornithine (D-Orn) is added to the growth sequence through recombinant DNA technology can enhance media (Fig. 1) (20, 21). Here we show that Pcl can be incorpo- solubility and stability or alter immunogenicity and selectivity rated into a broad range of target proteins at TAG codons in both of the protein drug. Only a few of the canonical 20 amino acids E. coli and mammalian cells, and we report a specific conjugation are commonly used to modify the properties of proteins through chemistry that enables coupling of synthetic compounds to Pcl in chemical derivatization. Conjugation of poly(ethylene glycol)s recombinant proteins. This combination provides a powerful (PEGs) for serum half-life extension, chemical haptenation to platform for site-specific modification of proteins. increase the immunogenicity of antigens, and other conjugations to effect the physicochemical and pharmacological properties of Results proteins drugs and vaccines are primarily performed through Pcl Protein Production in E. coli and Mammalian Cells. Pyl-containing lysines, cysteines, and the N terminus (4). Amine reactive re- proteins can only be produced in small amounts from E. coli. SCIENCES agents are broadly available, but conjugation results in heteroge- Pcl incorporation at a single site was, however, supported at nous mixtures with often substantially reduced activity relative to high yields in E. coli and mammalian cells when 1–5mM APPLIED BIOLOGICAL the unmodified protein. Homogenous products can be produced D-Orn was added to the growth media (20). So far, more than by optimizing reaction conditions such that only the N terminus is 350 Pcl mutants of over 30 different proteins have been produced, derivatized, but accessibility and effects on activity may limit this site-specific modification approach. Alternatively, a solvent ex- posed cysteine can be introduced through mutagenesis, but native Author contributions: W.O., T.U., S.E.C., S.A.L., A.B., and B.H.G. designed research; W.O., cysteines may need to be mutated to achieve derivatization at a T.U., H.-P.C., J.G., S.E.C., T.C., X.H., Q.F., L.L.Q., P.P., L.O., D.H.J., A.B., and B.H.G. performed research; W.O., T.U., H.-P.C., J.G., S.E.C., X.H., and A.B. contributed new reagents/analytic single desired site. Often, this type of mutagenesis is not a viable tools; W.O., T.U., H.-P.C., J.G., S.E.C., T.C., D.H.J., S.A.L., A.B., and B.H.G. analyzed data; option because additional cysteine residues can interfere with and W.O., T.U., H.-P.C., J.G., S.E.C., T.C., D.H.J., A.B., and B.H.G. wrote the paper. protein folding, causing aggregation or dimerization, and in pro- Conflict of interest statement: A patent application regarding the biosynthesis of teins expressed in mammalian cells, often form cysteine or glu- pyrroline-carboxy-lysine and site-specific protein modifications via chemical derivatization tathione adducts that need to be disrupted through reduction of pyrroline-carboxy-lysine and pyrrolysine residues has been filed. prior to conjugation with thiol-specific reagents (5). Site-specific *This Direct Submission article had a prearranged editor. bioorthogonal chemical or enzymatic derivatization can also 1W.O. and T.U. contributed equally to this work. be accomplished through a number of peptide tags (4, 6) but 2To whom correspondence should be addressed. E-mail: [email protected]. requires adding a stretch of nonendogenous sequence to the pro- This article contains supporting information online at www.pnas.org/lookup/suppl/ tein of interest. Any chemistry utilizing the standard 20 amino doi:10.1073/pnas.1105197108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1105197108 PNAS ∣ June 28, 2011 ∣ vol. 108 ∣ no. 26 ∣ 10437–10442 Downloaded by guest on September 27, 2021 O D-Orn NHH2N O strated by the improved yield of four hFAS-TE mutants with into media Pyl additional nucleotide changes at the þ1 codon (Fig. 2B). Incor- N OH poration efficiency cannot be predicted at this time but because Pyl Biosynthetic Genes Pcl of the ease of expression, many sites can quickly be scanned for H2N O each new application. Our observations for Pcl are consistent NH2 O NHH2N O with reports (18, 24, 25) that Pyl, unlike selenocysteine, incor- PylC H2N PylD H2N O O NH2 +L-Lys N N poration does not require a conserved mRNA element (22, 23). O H H N OH OH HO NH2 HO NH2 Expression of Pcl proteins has so far been tested in shake flask O O culture, small bioreactors and wavebags™ and feasibility of large Pcl PylS RS + PylT tRNA scale production needs to be evaluated in the future. Nascent protein Pcl Pcl-Specific Bioorthogonal Conjugation Chemistry. It has been sug- EF-TU gested previously that Pyl is chemically reactive (26). Previously, AUC Δ1 UAG mRNA 2-amino-benzaldehyde (2-ABA) was used to derivatize -pyrro- Ribosome AUC line-5-carboxylate, a molecule analogous to the pyrroline ring of E. coli or mammalian cell Pyl, in order to study its role in the biosynthesis of proline (27). Here, we show that Pcl (and Pyl) incorporated into proteins can Fig. 1. Biosynthetic generation and incorporation of Pcl into proteins with high efficiency and specificity be derivatized with 2-ABA or produced in E. coli or mammalian cells using the Pyl machinery from Metha- 2-amino-acetophenone (2-AAP) moieties (Fig. 3A, SI Appendix). nosarcinae maize and D-Orn added to the media (20). The reactions proceeded to near completion at pH 5 and pH 7.5 at molar ratios of 6∶1 of 2-ABA or 2-AAP using 17 μM Pcl including enzymes, membrane receptors, growth factors, Fc- protein (Fig. 3 B and C). Conjugation of 2-ABA to the Pcl residue fusion proteins, antibodies, and antibody fragments (Fig. 2, SI was confirmed by mass spectrometric (MS) analysis (SI Appendix: Appendix). Expression yields in E. coli were typically 25–40% Fig. S1). Additional conjugation events were only observed at very but some reached 80% of wild-type protein using a two-plasmid high 2-ABA concentration (>80 mM), both for the Pcl protein expression system (Fig. 2 A and B). Transient cotransfection of (Fig. 3D) and also for a control protein that did not contain HEK293 cells with the Pyl machinery regularly produced Pcl Pcl (Fig.
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