Efficient Phage Display with Multiple Distinct Non‐Canonical Amino Acids Using Orthogonal Ribosome‐Mediated Genetic Code Expansion

Abstract Phage display is a powerful approach for evolving proteins and peptides with new functions, but the properties of the molecules that can be evolved are limited by the chemical diversity encoded. Herein, we report a system for incorporating non‐canonical amino acids (ncAAs) into proteins displayed on phage using the pyrrolysyl‐tRNA synthetase/tRNA pair. We improve the efficiency of ncAA incorporation using an evolved orthogonal ribosome (riboQ1), and encode a cyclopropene‐containing ncAA (CypK) at diverse sites on a displayed single‐chain antibody variable fragment (ScFv), in response to amber and quadruplet codons. CypK and an alkyne‐containing ncAA are incorporated at distinct sites, enabling the double labeling of ScFv with distinct probes, through mutually orthogonal reactions, in a one‐pot procedure. These advances expand the number of functionalities that can be encoded on phage‐displayed proteins and provide a foundation to further expand the scope of phage display applications.

Phage display is apowerful approach for selecting peptides and proteins,f rom diverse libraries,w ith high affinity for am olecular target [1] and has been extensively used to select therapeutic peptides and proteins. [2] Several adaptations of phage display have expanded its scope for diverse applications,including:the selection of bicyclicpeptides constrained by covalent tethering to as mall molecule, [3] the evolution of catalytic function, [4] thes ynthesis of new materials and nanowires, [5] and the profiling of cell-surface proteomes. [6] Classical phage display is limited to encoding proteins containing the 20 canonical amino acids,a nd this limits the range of functions that can be accessed in phage-displayed peptides and proteins.Selenocysteine can be incorporated but this approach is limited in chemical scope and the sequences that may be encoded. [7] Close analogues of methionine can be incorporated by selective pressure incorporation;h owever, this leads to insertion of the analogues in response to all Met codons. [8] Advances in genetic-code expansion [9] have enabled the site-specific co-translational incorporation of non-canonical amino acids (ncAAs) into proteins displayed on phage. [4b, 10] These approaches have primarily used variants of the Menthanococus janaschii (Mj)t yrosyl-tRNAs ynthetase (MjTy rRS)/tRNA CUA pair, which is orthogonal in Escherichia coli. [10] This system has been used to label proteins expressed on phage through azide-alkyne cycloadditions [4b, 10a] and Staudinger ligations; [10b] it has also been used to evolve single-chain antibody variable fragments (ScFvs) with chemical warheads [10c, d] and proteins that chelate metal ions. [10f] Despite these important advances,c urrent approaches have significant limitations.F irst, the MjTy rRS/tRNA CUA pair has been exclusively used to incorporate amino acids derived from phenylalanine;t his precludes the genetic encoding of diverse aliphatic ncAAs.Second, each phage generated using this approach only incorporates as ingle type of ncAA in response to asingle amber codon in the gene of interest;this precludes the incorporation of multiple distinct ncAAs on as ingle phage,w hich may facilitate ar ange of applications, including the selective double labeling of displayed proteins.
Herein, we report ap hage display system for incorporating ncAAs into proteins displayed on phage that takes advantage of the pyrrolysyl-tRNAsynthetase (PylRS)/tRNA pair (Figure 1a). This pair has been extensively developed for incorporating diverse aliphatic ncAAs into proteins, [11] and read-through of stop codons in gene 3ofM13 phage using this pair has been demonstrated as part of acontinuous evolution strategy. [12] However,aphage display system that takes advantage of PylRS/tRNAh as not been characterized. Here we demonstrate the site-specific incorporation of ac yclopropene-containing ncAA (CypK, N e -[((2-methylcycloprop-2-en-1-yl)methoxy)carbonyl]-l-lysine) into proteins displayed on phage using the PylRS/tRNApair. Phage-displayed proteins incorporating CypK are labeled through ar apid inverse electron-demand Diels-Alder reaction with tetrazine derivatives. [13] We show that the efficiencyo fd isplaying proteins containing ncAAs is substantially improved by translation of the displayed protein fusion from an orthogonal ribosome binding site using an evolved orthogonal ribosome, and that the optimized system can be used to incorporate CypK at diverse sites on an ScFv,i nr esponse to both amber and quadruplet codons.F inally,w ed emonstrate that PylRS/ tRNA UACU and an evolved MjTy rRS/tRNA CUA pair can be used to incorporate both CypK and p-propargyloxy-l-phenylalanine (PrpF) on an ScFv.This enables the double labeling of the displayed ScFv with distinct probes,t hrough mutually orthogonal reactions,inaone-pot procedure.
We first developed aphage display system for incorporating ncAAs into phage-displayed proteins using the PylRS/ tRNAp air. This phagemid-based system is composed of two plasmids (System 1i nF igure S1 in the Supporting Information). ThePylRS/tRNA CUA pair is expressed from ahigh copy pAux plasmid [14] (Supporting Information, Figure S2), while ap hagemid vector is used to encode the gene of interest (herein an anti-Her2 ScFv) containing an amber codon fused to the p3 gene (g3). We initially introduced an amber codon at position 252 in the scFv (118 EU numbering), creating an ScFv(252TAG)-g3 gene;this site is commonly used to modify antibodies without affecting antigen binding. [15] Production of phage from cells bearing phagemid-based systems is dependent on helper phage infection to provide phage coat proteins. [1b, 2b] All [10] but one [4b] report of using MjTy rRS/tRNA CUA derivatives to incorporate ncAAs rely on ah elper phage in which g3 has been deleted (dM13);t his makes the production of phage particles dependent on expression of the full length p3 fusion from the phagemid vector and phage titers are ncAA dependent in these systems.
We observed CypK-dependent production of phage upon addition of dM13 to cells bearing our two-plasmid system with Abstract: Phage displayi sapowerful approach for evolving proteins and peptides with new functions,but the properties of the molecules that can be evolved are limited by the chemical diversity encoded. Herein, we report asystem for incorporating non-canonical amino acids (ncAAs) into proteins displayed on phage using the pyrrolysyl-tRNAs ynthetase/tRNAp air.W e improve the efficiency of ncAA incorporation using an evolved orthogonal ribosome (riboQ1), and encode ac yclopropenecontaining ncAA (CypK) at diverse sites on adisplayed singlechain antibody variable fragment (ScFv), in response to amber and quadruplet codons.CypK and an alkyne-containing ncAA are incorporated at distinct sites,e nabling the double labeling of ScFv with distinct probes,t hrough mutually orthogonal reactions,i naone-pot procedure.T hese advances expand the number of functionalities that can be encoded on phagedisplayed proteins and provide afoundation to further expand the scope of phage displaya pplications.
We hypothesized that the decreased titer of the phage incorporating CypK in ScFv(252TAG)-g3, with respect to phage produced from scFv-g3 without an amber stop codon, was ar esult of sub-optimal read-through of the amber stop codon. We have previously shown that the efficiency of ncAA incorporation can be enhanced by translating the message of interest, under the control of an orthogonal ribosome binding site,using an evolved orthogonal ribosome (riboQ1). [16] We therefore created as econd-generation system (System 2i nFigure S1 in the Supporting Information) in which riboQ1 was used to translate O-ScFv(252TAG)-g3 (a phagemid in which ScFv(252TAG)-g3 is downstream of an orthogonal ribosome binding site). Using this system with dM13 helper phage led to CypK-dependent phage production, with titers an order magnitude higher than in our initial system (Figure 1b). Moreover,w hen using System 2w ith dM13 for CypK incorporation, the phage titer from the O-ScFv(252TAG)-g3 fusion was comparable to that for the amber-codon-free gene fusion translated from ac anonical ribosome binding site (Figure 1b). We suggest that this system maximizes titers by providing sufficient ScFv-p3 containing the ncAA.
Thenew system allowed us to easily detect the displayed anti-Her2 scFv,p roviding an ELISA signal at least 10-fold above the initial system, and comparable to that from control experiments with amber-codon-free ScFv-g3 fusions (Figure 1c). System 2a lso allowed us to easily detect labeling of CypK incorporated into the ScFv-p3 fusion (expressed from O-ScFv(252TAG)-g3), providing alabeling signal at least 10fold higher than the initial system (Figure 1d,e). As expected, both the ELISA and labeling signal were strongly CypK dependent. Moreover,a tl east 84 AE 6% of phage displayed reactive CypK as quantified through abiotin-capture assay [17] (Supporting Information, Figure S3). We conclude that System2/dM13 enables the specific incorporation of CypK with titers of phage displaying ncAAs that are comparable to the parental system with no amber codon.
Next, we tested an interference-resistance helper phage (CM13) in combination with System 1. CM13 provides acopy of g3, such that the production of infectious phage particles is not dependent on the phagemid-encoded p3 fusion. Theu se of interference-resistant helper phage commonly leads to the production of high phage titers and facilitates monovalent display. [18] As expected, we found that phage titers with CM13 helper phage were excellent (10 10 cuf mL À1 ), and independent of the amber stop codon in ScFv,o rt he addition of CypK (Figure 1b). Using the amber codon variant of System 1 (Supporting Information, Figure S1) with CM13 we detected the displayed ScFv and the labeling of CypK (Figure 1c-e).
Theamber codon variant of System 2displayed the ScFv at approximately 40 %o fthel evel of its no amber version, when using CM13 (Figure 1c). Monovalent display was confirmed (Supporting Information, Figure S4). Moreover, this system led to the highest level of CypK labeling signal (Figure 1d,e and Supporting Information, Figure S5), leading to a7 -fold increase in labeling with respect to the corresponding System 1experiment. At least 81 AE 8%of the phage displaying ScFv bears CypK ( Supporting Information, Figure S3). We decided to use System 2with CM13 helper phage for all further experiments.
Next, we demonstrated that System 2with CM13 enabled ncAA incorporation at diverse sites on phage-displayed proteins in response to amber or quadruplet codons.W e targeted residues far from the paratope (G128, 252) that may be derivatized without affecting binding,a sw ell as the complementarity-determining regions (CDRs) and their proximity (K161, N186, D233). We observed CypK-dependent production of the ScFv-p3 fusion protein for each amber mutant (Figure 2a), which was supported by ELISAs (Supporting Information, Figure S6). Labeling of the fusions confirmed that all the sites tested are accessible for the reaction (Figure 2a).
We were curious whether we could extend System 2t o enable ncAA incorporation in response to quadruplet codons. [16] We therefore replaced Pyl-tRNA CUA in System 2 with an evolved derivative bearing an extended anticodon and introduced the corresponding codons into the ScFv gene. Using this system, we observed CypK-dependent ScFv-p3 production and labeling at all positions tested (Figure 2b). These experiments demonstrated that the second-generation phage display system we have created enables ncAA incorporation using the PylRS/tRNAp air at diverse sites in response to quadruplet codons.
Next, we demonstrated that we could adapt System 2f or incorporation of ncAAs using ad ifferent orthogonal aaRS/ tRNApair. To achieve this,wereplaced PylRS/tRNA CUA with MjPrpRS/tRNA CUA that directs the incorporation of PrpF (Supporting Information, Figure S1). [19] ScFv-p3 synthesis was dependent on PrpF,a nd we labeled the incorporated PrpF with an azide fluorophore,v ia aC u I -catalyzed 3 + 2 cycloaddition (Figure 2c).
Finally,weasked whether we could combine our advances to facilitate the display and labeling of proteins containing multiple distinct ncAAs.W eh ave previously demonstrated that PylRS/tRNApairs and MjTy rRS/tRNA-derived pairs are mutually orthogonal [16a] and that we can selectively label encoded CypK and PrpF in proteins. [20] We created aversion of System 2t hat expresses PylRS/tRNA UACU and MjPrpRS/ tRNA CUA ,a nd an O-ScFv(127TAG,2 52AGTA)-g3 cassette (Supporting Information, Figure S1). Phage produced from this system in the presence of both CypK and PrpF were selectively labeled by both tetrazine-fluorophore conjugates and azide-fluorophore conjugates (Figure 3). Themajority of the phage displaying ScFv were labeled with both fluorophores and contain both ncAAs (Supporting Information, Figure 1 N8Z). CypK was labeled with tetrazine-sulfocyanine-5 (tetrazine-Cy5). b) Replacing TAGwith AGTAinthe phagemid and using aPyl-tRNA derivative with an extended (UACU) anticodon enables CypK incorporation via quadruplet decoding. c) Adding MjPrpFRS in pAux enables the incorporation of PrpF, which was labeled with azide-AlexaFluor 647 (azide-AF647). Black and white arrows indicate scFv-p3 and p3, respectively. IGF:i n-gel fluorescence. WB:w estern blot. In conclusion, we have developed an efficient phage display system for incorporating ncAAs into proteins displayed on phage using PylRS/tRNA. Since this pair can be used to encode multiple,structurally and functionally diverse ncAAs,our system expands the range of ncAAs that may be encoded in proteins displayed on phage.Using this system, we have encoded CypK, which enables the labeling of phagedisplayed proteins,through abioorthogonal metal-free cycloaddition, orders of magnitude faster than chemical reactions with functional groups that were previously displayed on phage. [4b, 10b] Thes econd-generation system we have developed takes advantage of translation by an evolved orthogonal ribosome to enhance the efficiency with which proteins containing ncAAs can be displayed on phage.T he wild-type, or near wild-type,d isplay levels we have achieved will facilitate directed evolution experiments (Supporting Information, Figure S4). Modular alterations to our system enable the incorporation of different classes of ncAAs,using distinct orthogonal aaRS/tRNApairs,and facilitate the incorporation of ncAAs in response to amber or quadruplet codons. Moreover,o ur approach enables the genetic encoding of multiple distinct ncAAs and enables the one-pot, site-specific, dual labeling of phage-displayed proteins.
Recent exciting advances in the generation and discovery of new mutually orthogonal synthetase/tRNAsystems [21] may enable additional functionalities to be encoded on phagedisplayed proteins.Weanticipate that increasing the chemical functionalities,a nd number of new building blocks,t hat can be encoded will further expand the scope of phage display to facilitate diverse applications.