This journal is©The Royal Society of Chemistry 2018 Chem. Commun.

Cite this:DOI: 10.1039/c8cc03819h

A noncanonical amino acid-based relay system forsite-specific protein labeling†

Yuda Chen, a Axel Loredo, a Aviva Gordon,a Juan Tang,a Chenfei Yu,a

Janett Ordoneza and Han Xiao *ab

Genetically site-specific introduction of noncanonical amino acids

(ncAAs) for protein conjugation generally requires incorporation

through exogenous feeding of chemically synthesized ncAAs. We

developed a p-amino-phenylalanine (pAF)-based relay system that

enables site-specific functionalization of proteins without chemical

synthesis of the building blocks. pAF was biosynthesized under optimized

conditions, followed by site-specific incorporation into a specific protein

residue. The resulting protein was ready for functionalization using

an oxidative conjugation reaction. We demonstrated the use of this

relay system by preparing a fluorophore-labeled anti-HER2 single-

chain variable fragment antibody for fluorescent imaging.

Proteins conjugated at a specific site with fluorophores, drugs,and polymers, are required for many biological and therapeuticapplications.1,2 To carry out this precise protein labeling, it isessential to introduce bioorthogonal handles into a definedposition of protein. Both biosynthetic and semisynthetic strategieshave been developed to site-specifically modify proteins withbioorthogonal functional groups; however, semisynthetic methodscan be challenging with larger proteins and cell-based labeling.3

To overcome these limitations, noncanonical amino acids (ncAAs)have been genetically incorporated into proteins in response toa nonsense or a frameshift codon using an engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pair that is orthogonal to theexpression host.4–7 Using this technology, ncAAs with bio-orthogonal chemical reactivities (e.g., ketones, azides, alkynes,tetrazine, bicyclo[6.1.0]non-4-yn-9-ylmethanol, and cyclooctyne)have been site-specifically incorporated into proteins, whichenables the preparation of diverse protein conjugates.8 How-ever, the genetic introduction of these bioorthogonal handlesrequires adding exogenously synthesized ncAAs as a mediumsupplement, which restricts the utility of this approach.

To address this issue, several existing synthetic gene clusters forncAAs from distinct species have been identified and employed toproduce and introduce amino acids with an aniline9 or diol10 sidechain into proteins in Escherichia coli. Among these noncanonicalside chains, aniline has been identified as a bioorthogonalhandle for precision protein modification. Francis and co-workersdeveloped an oxidative reaction that enables conjugation of aniline-containing proteins with o-quinone moieties with a short reactiontime and excellent chemoselectivity.11 To demonstrate the utility ofthis reaction, bioreagents, including imaging probes, epidermalgrowth factors, and DNA aptamers have been conjugated tosynthetic MS2 viral capsids for positron emission tomographyimaging or cancer cell targeting.12 However, the aniline moieties onthe synthetic MS2 viral capsid were introduced either by modifyingcysteines using maleimide derivatives or by genetically incorporating

Fig. 1 A relay system to site-specifically label proteins. Together withE. coli aminotransferase, the pAF gene cluster from S. venezuelae couldefficiently convert chorismate to pAF. The biosynthesized pAF was thenincorporated into anti-HER2–pAF with an orthogonal pAFRS/tRNATyr pair.The resulting anti-HER2–pAF could be labeled with a 2-amino-4-methylphenol fluorescent derivative to yield an anti-HER2–pAF conjugatefor HER2-positive cell detection.

a Department of Chemistry, Rice University, 6100 Main Street, Houston, Texas,

77005, USA. E-mail: han.xiao@rice.edub Department of Biosciences, Rice University, 6100 Main Street, Houston, Texas,

77005, USA

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8cc03819h

Received 11th May 2018,Accepted 4th June 2018

DOI: 10.1039/c8cc03819h

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exogenously fed p-amino-phenylalanine ( pAF). Chemical preparationof pAF requires a multistep synthesis and the use of a metal catalyst,which is not cost-effective or environmentally friendly.13 Thus, thedevelopment of a platform spanning the biosynthesis, site-specificincorporation, and bioconjugation of aniline-containing ncAAswould significantly facilitate site-specific protein functionalization(Fig. 1). Here, we report a pAF-based relay system for the biosynthesisand incorporation of pAF into proteins followed by a site-specificfunctionalization using oxidative bioorthogonal reactions.

To site-specifically incorporate pAF into defined positions ofproteins, a modified Methanococcus janaschii tyrosyl-tRNA synthetase(MjTyrRS)/tRNATyr pair was recently developed.9 We first evaluatedthe efficiency of this pAFRS/tRNATyr pair to incorporate biosynthe-sized pAF into proteins using a super-folding green fluorescentprotein (sfGFP) reporter assay. The suppressor pDule plasmid(pDule-pAF) encoding this pAFRS/tRNATyr pair was used to expressa sfGFP variant containing a C-terminal His6 tag and an ambercodon replacing Tyr150 (encoded on reporter plasmid pET28a-sfGFP*). Compared to the expression in the absence of pAF, a20-fold improvement in suppression efficiency was observed withthe use of 1 mM exogenously fed pAF (Fig. S1, ESI†). To improvethe incorporation efficiency of biosynthesized pAF, the expressionlevels of the suppressor tRNA and corresponding aaRS wereoptimized. We subcloned the pAFRS into the recently reportedpUltra vector. The resulting pUltra-pAF encodes strong promoter(lacI)-driven pAFRS and proK-driven Mj-tRNATyr cassettes. Toevaluate the suppression efficiency of this new plasmid, pUltra-pAF was transformed into BL21(DE3) cells containing pET28a-sfGFP*. Protein expression was carried out in M9-glucose minimalmedium in the presence or absence of 1 mM pAF. Compared withthe pDule-pAF plasmid, the expression level of pAF-containingsfGFP using pUltra-pAF was two-fold higher (Fig. S1, ESI†).Furthermore, without adding pAF into the culture medium, asimilar level of background incorporation was observed for theexpression using either the pUltra-pAF or pDule-pAF plasmid.Thus, pUltra-pAF was used in following experiments.

Novel pAF synthetic gene clusters from Streptomyces venezuelae(SvPapABC)9 and Pseudomonas fluorescens (PfPapABC)14 have beenidentified that allow for the production of pAF in E. coli (Fig. 1).Three genes, 4-amino-4-deoxychorismate synthase (PapA), 4-amino-4-deoxychorismate mutase (PapB), and 4-amino-4-deoxyprephenatedehydrogenase (PapC), can convert chorismate to 4-aminophenyl-pyruvate, which can undergo a transamination to yield pAF usingE. coli endogenous aminotransferases. Under optimized condi-tions, highly-efficient production of pAF (up to 4.4 g L�1) wasachieved using a bacterial fermentation platform.14 Next, we exam-ined the efficiencies of different reported bio-metabolic systems toproduce pAF. The screening was carried out by growing BL21(DE3)cells containing the suppressor plasmid (pUltra-pAF), reporterplasmid (pET28a-sfGFP*), and different pAF biosynthesis plasmidsin M9-glucose minimal medium. The highest GFP expressionlevels were observed using the pAF synthetic gene clusters fromS. venezuelae under an lpp promoter (encoded on plasmidpLASC-lppPW) (Fig. 2a). Introduction of plasmid pLASC-lppPWinto E. coli was reported to generate 0.7 mM cellular pAF.9 Wealso optimized the expression conditions of proteins containing

biosynthesized pAF. As shown in Fig. S1 and S2 (ESI†), thehighest expression level was achieved after 24 h and the expressionof pAF-containing protein at 30 1C was significantly higher than at37 1C. However, the shaking speed did not have a significant effecton the expression of proteins with the biosynthesized pAF (Fig. S3,ESI†). Under the optimized conditions, 1 mg L�1 of sfGFP containingbiosynthesized pAF was produced, compared with the 4.5 mg L�1

yield when pAF was added exogenously (Fig. 2b). As reportedpreviously, a certain level of tyrosine incorporation was also observedin the absence of 1 mM pAF due to the plasticity of the pAFRS.9

However, the pAFRS/tRNATyr pair has reported to selectively incorpo-rate the ncAA in the presence of pAF.

To demonstrate that proteins containing biosynthesized pAFwere ready for site-specific bioconjugation, the anti-humanepidermal growth factor receptor 2 (HER2) antibody was used.HER2 is a member of the epidermal growth factor family foundon breast and ovarian cancer cells.15 The anti-HER2–singlechain fragment variable (scFv) is a fusion protein, with variableheavy and light chains of immunoglobulins, that has been used todeliver bioactive molecules to HER2-expressing cancer cells.16 Toprepare the anti-HER2–scFv with pAF (anti-HER2–pAF) from itsbiosynthesis, we generated an anti-HER2–scFv plasmid (pET28a-anti-HER2–scFv*) that encodes an anti-HER2 scFv gene with TAG atGly113. This plasmid was co-transformed with pUltra-pAF andpLASC-lppPW in BL21(DE3) cells, and anti-HER2–pAF expressionwas carried out in M9-glucose minimal medium. To compare theexpression level of anti-HER2–pAF using biosynthesis with the levelfrom exogenous feeding, we also co-transformed pET28a-anti-HER2–scFv* with pUltra-pAF and expressed the mutant proteinsin the presence or absence of 1 mM pAF. All anti-HER2–pAFmutants were purified by Ni2+-NTA affinity chromatography andanalyzed by SDS-PAGE and ESI-MS. SDS-PAGE analysis revealedthat full-length anti-HER2–pAF was only expressed in the presenceof 1 mM pAF or when the biosynthesis of pAF was induced (Fig. 2b).The anti-HER2–pAF protein yield by biosynthesis was approxi-mately 30% of the 1 mg L�1 yield from exogenous feeding. Themass of the biosynthesized anti-HER2–pAF was 27 564 Da, which isconsistent with the mass of anti-HER2–pAF obtained by feedingpAF (27 563 Da) (Fig. S4, ESI†).

With its unique aromatic amino group, pAF is an idealcandidate for protein labeling via oxidative coupling; syntheticMS2 viral capsid protein with an aniline functional group

Fig. 2 Biosynthesis and incorporation of pAF. (a) Comparison of theefficiencies of the pAF gene clusters from S. venezuelae (SvPapABC) andP. fluorescens (PfPapABC) to produce pAF-containing proteins. (b) SDS-PAGE analysis of sfGFP and anti-HER2–scFv proteins in the presence (+) orabsence (�) of pAF or when inducing the pAF biosynthetic plasmid (bio).

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specifically reacts with o-aminophenol derivatives rapidly.17 Totest if proteins containing biosynthesized pAF could react witho-aminophenol under similar conditions, the anti-HER2–pAFmutant prepared above was coupled to a small molecule,2-amino-4-methylphenol, in pH 6.5 PBS buffer in the presenceof NaIO4 for 5 min. Upon reaction completion, the resultingconjugate was buffer-exchanged into PBS and further analyzedusing ESI-MS. The reaction between the biosynthesized anti-HER2–pAF mutant and 2-amino-4-methylphenol resulted in aproduct with a mass of 27 714 Da, which is consistent with theexpected mass (27 715 Da) (Fig. S4, ESI†).

After confirming the reactivity of anti-HER2–pAF from bio-synthesis, the utility of this relay system was demonstrated bygenerating an anti-HER2–pAF conjugated with a fluorophoreand examining its ability to detect HER2-positive cells. Theanti-HER2–pAF mutant was first coupled to Rhodamine Blabeled o-aminophenol in pH 6.5 PBS buffer, and the excessfluorophore was removed using a desalting column (Fig. 3a).After the reaction, the observed mass from ESI-MS analysis was28 253 Da, which was in agreement with the calculated mass ofthe conjugate (28 250 Da) (Fig. 3c). To demonstrate that thereaction was bioorthogonal, we also carried out the oxidativereaction using a wildtype anti-HER2–scFv and the Rhodamine

B-labeled o-aminophenol as a control. As expected, there was aband for both anti-HER2–pAF and wildtype anti-HER2–scFv onthe Coomassie stained SDS-PAGE gel, but a red fluorescentband was only observed for the anti-HER2–pAF (Fig. 3b).

With the fluorophore-labeled anti-HER2–pAF in hand, wethen tested if it retained its function after several chemicaltransformations. HER2-positive SK-BR-3 cells or HER2-negativeMDA-MB-468 cells were incubated with 30 nM RhodamineB-labeled anti-HER2–pAF (anti-HER2-RhB) for 30 min beforeimaging. Confocal fluorescent imaging indicated that cell-surface-associated red fluorescence was exhibited only by HER2-positiveSK-BR-3 cells, while HER2-negative MDA-MB-468 cells did notexhibit any associated fluorescence (Fig. 3d). These resultsindicated that the anti-HER2–pAF conjugate from the developedrelay conjugating system did not influence its biological activity.

In this study, we optimized the biosynthesis and incorporation ofpAF into various proteins. The resulting pAF-containing proteinscan be site-specifically functionalized using an oxidative coupling.The utility of this pAF-based relay system was demonstratedby generating a Rhodamine B-labeled anti-HER2–pAF andcharacterizing its ability to detect HER2-positive cells. The aboveenhanced pAF biosynthesis and incorporation system couldgreatly facilitate the large-scale production of pAF-containingproteins for industrial applications by bypassing the traditionalmultistep chemical synthesis of pAF. The preservation of theantigen-binding ability of anti-HER2–pAF after the oxidativereaction demonstrates the tolerability of conjugation by bioactivemolecules. This approach may be applied to further biomedicalapplications, such as the preparation of pAF-containing antibody–drug conjugates.

We thank Profs. Peter G. Schultz, Ryan Mehl, and NaokiTakaya for kindly providing the plasmids pDule-pAF, pBad-anti-HER2 scFv, pLASC-lppPW, pLASC-lacPW, pET-pfpapA, pCDF-pfpapBC, pET-pfpapBAC, and pAcYc-AroG4. This work wassupported by the Cancer Prevention Research Institute of Texas(CPRIT RR170014) and the Robert A. Welch Foundation (C-1970).

Conflicts of interest

There are no conflicts to declare.

Notes and references1 P. Agarwal and C. R. Bertozzi, Bioconjugate Chem., 2015, 26, 176–192.2 Y. Qi and A. Chilkoti, Curr. Opin. Chem. Biol., 2015, 28, 181–193.3 N. Krall, F. P. da Cruz, O. Boutureira and G. J. L. Bernardes, Nat.

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10 S. Kim, B. Hyun Sung, S. Chang Kim and H. Soo Lee, Chem.Commun., 2018, 54, 3002–3005.

Fig. 3 Preparation and characterization of the Rhodamine B-labeled anti-HER2–scFv. (a) Bioorthogonal reaction scheme for the fluorescent2-amino-4-methyl phenol analogue and anti-HER2–pAF. The reactionwith 30 mM anti-HER2–pAF and 10 mM Rhodamine B-labeled 2-amino-4-methylphenol proceeded for 50 min at RT, in the presence of 1 mM NaIO4.(b) SDS-PAGE analysis of the reaction product with the wt anti-HER2–scFvor anti-HER2–pAF, visualized by Coomassie staining (top) and red fluores-cence (bottom). (c) Mass spectra of the reaction product with anti-HER2–pAF (left) and the Rhodamine B-labeled 2-amino-4-methylphenol (right)(d) Binding of anti-HER2–pAF conjugates in SK-BR-3 and MD-MBA-468cells visualized by confocal microscopy. Cells were incubated with 30 nManti-HER2-RhB (red) in medium for 30 min at 37 1C and stained withDiOC18(3) (green) and Hoechst 33 342 nuclear stain (blue). Scale bar = 50 mm.

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