Macrocyclic peptide libraries displayed on surface of yeast cells

Abstract

The present invention relates to the generation of a library of cysteine-enriched macrocyclic peptide sequences expressed on the surface of yeast cells, methods for their preparation and a population of yeast cells genetically modified to express a plurality of disulfide tethered macrocyclic peptide ligands having different structures and amino acid sequences on the surface of yeast cells.

Description

[0001] Invention Field

[0002] This invention relates to the generation of libraries of cysteine-enriched cyclic peptide sequences expressed on the surface of yeast cells. More specifically, this invention provides a yeast cell-based macrocyclic peptide display system, its preparation method, and a yeast cell population that is genetically modified to express multiple disulfide-tethered macrocyclic peptide ligands with different structures and amino acid sequences on its cell surface. Background of the Invention

[0004] Macrocyclic peptides are increasingly recognized as a valuable molecular form for drug development (Ji, Nielsen, and Heinis 2024). Currently, there are approximately 80 peptide therapeutics on the global market, of which more than 40 are macrocyclic peptides; and the number of macrocyclic peptide drugs approved annually is increasing year by year (Muttenthaler et al., 2021; Fetse et al., 2023). Macrocyclic peptides possess many desirable properties, making them a highly attractive modulus for therapeutic drug development (Zhang and Chen 2022). They can bind to large molecular targets with high affinity and selectivity. Furthermore, they often exhibit good proteolytic stability and, in some cases, even membrane permeability. Moreover, they typically exhibit low inherent toxicity or antigenicity. In addition, macrocyclic peptides can be efficiently prepared through chemical synthesis and are easily modified. Furthermore, their modular structure, coupled with the readily available availability of hundreds of commercially available amino acid building blocks, simplifies the development of various macrocyclic peptide variants with specific customized properties. All these properties put macrocyclic peptides in an advantageous position to bridge the gap between small molecule drugs and large biologics such as antibodies (Zhang and Chen 2022).

[0005] Although many macrocyclic peptides still originate from the exploration and development of naturally occurring peptides, recent technological advancements and major breakthroughs in molecular biology have paved the way for the development of "de novo synthesis" peptides with specific desirable properties. de novoMacrocyclic peptide ligands paved the way (Li, Craven, and Levine 2022). In fact, the advent of powerful combinatorial techniques such as phage display (GPSmith and Petrenko 1997; Deyle, Kong, and Heinis 2017) and mRNA display has greatly accelerated the generation of macrocyclic peptide ligands, enabling them to target a wide range of proteins for which no natural peptide ligands have yet been discovered. All these in vitro directed evolution methods rely on the physical link between the "phenotype" (expressed macrocyclic peptide) and the "genotype" (encoding DNA or RNA sequences). By applying these techniques, macrocyclic peptide ligands with desired properties typically evolve following a similar scheme involving the generation of a large, random library of gene-encoded macrocyclic peptides, and multiple rounds of iterative selection, amplification, and diversification cycles. In recent years, the development of new technologies for post-translational chemical and enzymatic modifications has become increasingly important. The development of new strategies has significantly expanded the number of macrocyclic peptide forms that can be examined using the aforementioned combined techniques, in addition to classic disulfide-tethered peptides (Passioura et al., 2014; Sharma et al., 2023; Jaroszewicz et al., 2022; Dotter et al., 2021). These advances, coupled with recent technological advancements in screening procedures and automation, the emergence of novel reagents and tools, and the maturity of next-generation sequencing technologies, have made it possible to construct larger libraries and isolate macrocyclic peptide ligands with excellent binding properties, enabling these ligands to bind to increasingly challenging targets (TP). Smith et al., 2023; Stellwagen et al., 2019; Sloth et al., 2023). Examples of macrocyclic peptides successfully discovered using in vitro display technology include Merck & Co.'s oral PCSK9 inhibitor MK-0616 (Kingwell 2023) for the treatment of atherosclerotic cardiovascular disease, and UCB's recently FDA-approved zilucoplan (Mullard 2023) for the treatment of anti-acetylcholine receptor antibody-positive adults with generalized myasthenia gravis.

[0006] While these techniques have proven capable of generating and screening extremely large libraries of sequence and structural diversity, enabling rapid identification of macrocyclic peptide ligands for virtually any target, they still rely on rather difficult-to-control procedures because it is challenging to monitor the performance of individual screening clones or populations during high-throughput screening. Typically, the success of a screening activity is only seen after several weeks of work, when the isolated macrocyclic peptide molecules are identified. Furthermore, biophysical characterization of the selected ligands often requires chemical synthesis or subcloning and recombinant production, followed by purification; all these additional steps ultimately slow down downstream processes, making them more complex and costly (Jaroszewicz et al., 2022; Vodnik et al., 2011; Wang and Liu, 2011). Finally, the number of selected macrocyclic peptide ligands that can be synthesized, purified, and characterized is typically limited to 10–100 molecules.

[0007] This paper proposes a novel method based on yeast surface display technology to address the aforementioned issues. Since its invention, yeast surface display technology has proven to be a transformative tool for the direct evolution of various immunoglobulin and non-immunoglobulin proteins (Boder and Wittrup 1997; Angelini et al., 2015). This technology was initially shown to enhance the binding affinity of existing antibody fragments, but it has since proven highly effective in isolating de novo proteins with well-regulated binding affinity and specificity for multiple targets from original combinatorial libraries (Cherf and Cochran 2015; Lopez-Morales, Vanella, Appelt et al., 2023). Combining yeast surface display technology with fluorescence-activated cell sorting (FACS) offers the advantage of real-time monitoring of the iterative screening process for large combinatorial libraries containing various macrocyclic peptide ligands. By applying flow cytometry, information on the affinity, specificity, stability, and enrichment of individual yeast clones encoding different macrocyclic peptide ligands can be monitored continuously and quantitatively through successive rounds of sorting (Angelini et al., 2015; Teymennet-Ramírez, Martínez-Morales, and Trejo-Hernández 2022). Furthermore, by applying different labeling strategies (including balanced binding, kinetic binding, and competitive binding), the combination of yeast display technology and flow cytometry enables precise control over the stringency of screening; this not only facilitates rapid and precise epitope localization but also allows for the quantitative differentiation of individual macrocyclic peptide variants directly in a fusion-expressed state on the cell surface, eliminating the need for chemical synthesis or recombinant expression and purification (Cherf and Cochran 2015; Lopez-Morales, Vanella, Appelt et al., 2023; Lopez-Morales, Vanella, Kovacevic et al., 2023).

[0008] Yeast display has previously been used to screen peptide libraries, but primarily for engineering naturally occurring linear and cysteine-bound peptides (Linciano et al., 2019) or for the affinity maturation or binding characteristics of existing molecules (van Rosmalen et al., 2017; Ishii et al., 2012). Recently, the first studies reported the de novo generation of cyclic peptide ligands using yeast display technology, such as generating conjugates targeting lysozyme and human interleukin-17, where the dissociation constant (… K DThe values ​​ranged from 300 nM to 3 μM (Bacon et al., 2020). Similarly, screening of post-translational enzymatically modified octapeptide libraries yielded cyclic peptide ligands capable of binding to two different domains of the yes-associated protein (YAP), in which epigenetic... K D Values ​​ranged from 700 nM to 1.5 μM (Bowen et al., 2021). However, the size of these libraries remains limited, and the structural diversity of peptides is limited to monocyclic peptides of 7 and 8 amino acids.

[0009] Therefore, this study aimed to fully explore the potential of yeast surface display technology in isolating and characterizing macrocyclic peptide ligands with finely bound properties. To this end, we generated a large and structurally diverse original combinatorial library encoding macrocyclic peptide ligands with different topologies. To provide a more robust approach, we turned to disulfide-bonded cyclized peptide ligands. Spontaneous intramolecular oxidation of cysteine ​​residues present in gene-encoded peptide sequences is significantly easier and more reliable than post-translational cyclization. In this regard, we anticipate that eukaryotic expression mechanisms in yeast cells will be able to support precise disulfide isomerization of cysteine-enriched sequences, ultimately enabling the generation of unique disulfide-tethered patterns that are often difficult to achieve with other expression systems (Angelini et al., 2015; Teymennet-Ramírez, Martínez-Morales and Trejo-Hernández 2022; Linciano et al., 2019).

[0010] Existing technology

[0011] Linciano S. et al ('Molecular evolution of peptides by yeast surfacedisplay technology', Medicinal Chemistry Communications (2019, 10, 1569-1580) describes the recent development and use of yeast display technology for the engineering of peptide molecules, highlighting its differences from other existing in vitro display systems.

[0012] Angelini A. et al. ('Protein Engineering and Selection Using Yeast Surface Display', Methods in Molecular Biology (2015, 1319, 3-36) describes a protocol for modifying and isolating protein variants from combinatorial libraries and their subsequent characterization using yeast display technology.

[0013] Van Deventer JA et al ('A platform for constructing, evaluating, and screening bioconjugates on the yeast surface', Protein Engineering Design & Selection (2016, 29, 485-493) describes the site-specific incorporation of non-classical amino acids into proteins expressed on the surface of yeast cells and their post-translational chemical modifications.

[0014] Stieglitz JT et al ('A robust and quantitative reporter system to evaluate noncanonical amino acid incorporation in yeast', ACS Synthetic Biology (2018, 7, 2256-2269) describes a display-based system in yeast that involves incorporating non-classical amino acids into proteins via an orthogonal translation system (OTS).

[0015] Van Rosmalen, M. et al. ('Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutation Scanning', Journal of Biological Chemistry (2017, 292, 1477–1489) describes a combination of yeast display and deep mutation scanning techniques to improve the affinity of a known cyclic peptide called the mediatope for the therapeutic antibody cetuximab.

[0016] Ishii, J. et al. ('Cell Wall Trapping of Autocrine Peptides for Human G-Protein-Coupled Receptors on the Yeast Cell Surface', PLoS One (2012, 7, e37136) describes a yeast-based cell wall autocrine peptide capture (CWTrAP) system for selecting agonist peptides of human GPCRs.

[0017] Bacon, K. et al ('Isolation of Chemically Cyclized Peptide BindersUsing Yeast Surface Display', ACS Combinatorial Science (2020, 22, 519–532) describes a method for generating cyclic peptides displayed on the surface of yeast cells by crosslinking two primary amine groups with bis(succinimide) glutarate.

[0018] Bowen, J. et al. ('Screening of Yeast Display Libraries of EnzymaticallyTreated Peptides to Discover Macrocyclic Peptide Ligands', International Journal of Molecular Sciences (2021, 22, 1634) describes a method for producing cyclic peptides displayed on the surface of yeast cells by using transglutaminase.

[0019] US 9134309B2 describes a method for generating and expressing a peptide library displayed on the surface of bacterial cells.

[0020] EP 2535346A1 describes a method for generating and screening peptide libraries displayed on the surface of bacterial cells to identify enzyme ligands.

[0021] US 5922545A describes a general method for generating multiribosomal libraries displaying nascent peptides and antibody derivatives suitable for affinity interaction screening, based on the E. coli S30 translation system for efficient mRNA translation.

[0022] WO2007109823A2 describes a method for generating a vector library containing a variety of different eukaryotic secretory vectors, each encoding an extracellular soluble fusion polypeptide.

[0023] WO2020236146A1 describes a method for generating and screening libraries of cyclic peptides displayed on the surface of phage particles. Peptide cyclization occurs via cysteine ​​and non-classical amino acids. Attached Figure Description

[0024] Figure 1This work presents a schematic diagram of the yeast surface-displaying disulfide-tethered macrocyclic peptide system. The target cysteine-enriched macrocyclic peptide (MP) sequence (black) is expressed as an N-terminus (left) or C-terminus (right) fusion of a cell surface protein (CSP, white). A long flexible linker (FL) is positioned between the MP and the immunofluorescent tag (IFT, light gray) to ensure sufficient distance and minimize potential steric hindrance to the detection reagent. Connecting a single cysteine ​​pair via a disulfide bridge generates a macrocyclic peptide ligand with a “monocyclic” and unique topology. Connecting two or more cysteine ​​pairs via a disulfide bridge generates macrocyclic peptide ligands with “bicyclic” or “multicyclic” structures and different topologies, which can be sampled for binding to target protein sites.

[0025] Figure 2 Generate a yeast-encoded cysteine-rich macrocyclic peptide library with high main-chain diversity. a) Generate a cysteine-rich macrocyclic peptide library with large sequence diversity, encoding macrocyclic peptide topologies fused to the N-terminus of cell surface anchoring proteins expressed on the yeast cell surface via monocyclic or bicyclic disulfide bonds. Insert fragments were created by appending randomized DNA sequences encoding macrocyclic peptides (MPs) to the 5' end of DNA encoding flexible adapters using forward degenerate primers with partial annealing to the 3' of the gene encoding the cell surface anchoring protein (white) and constant reverse primers. MPs can have randomized amino acid sequences and lengths. The final construct includes the following genetic elements from the 5' to 3' ends: an inducible promoter (P), a segment encoding a secretion signal (SS, gray), a segment encoding a random cysteine-rich macrocyclic peptide sequence (MP, black), a segment encoding a long and flexible spacer region (FL, light gray), a segment encoding an immunofluorescence detection tag (IFT, dark gray), and a gene encoding a cell surface anchoring protein (CSP, white).

[0026] (b) A cysteine-enriched macrocyclic peptide library was generated, exhibiting vast sequence diversity encoding "single-loop" or "double-loop" topologies fused to the C-terminus of cell surface anchoring proteins expressed on the surface of yeast cells. Insert fragments were created by appending randomized DNA sequences (black) encoding MP sequences of varying amino acid sequences and lengths to the 3' end of DNA sequences encoding flexible adapters using a reverse degenerate primer with partial 5' annealing to the gene encoding the cell surface anchoring protein (white) and a constant forward primer. The final DNA construct included the following elements from 5' to 3': an inducible promoter (P), a segment encoding a secretion signal (SS, gray), a gene encoding the cell surface anchoring protein (CSP, white), a segment encoding an immunofluorescence detection tag (IFT, dark gray), a segment encoding a long and flexible spacer region (FL, light gray), and a segment encoding a randomized cysteine-enriched macrocyclic peptide sequence (MP, black).

[0027] Figure 3 An exemplary "monocyclic" cysteine-enriched macrocyclic peptide library (X) was designed and analyzed. n C(X) m C(X) t The image is displayed on the surface of yeast cells. a) An exemplary “monocyclic” macrocyclic peptide library, called CX7C, was generated, containing two cysteine ​​residues (C) at fixed positions between seven random amino acids (X). The experimental size diversity of the library was reported. The random amino acid X is encoded by the “NNK” codon. Cysteine-enriched macrocyclic peptide libraries were constructed using a homologous recombination-based approach (Van Deventer and Wittrup 2014; Angelini et al., 2015). The quality of the original CX7C library was assessed by next-generation sequencing (NGS). Analysis of the library confirmed that the encoded disulfide-tethered macrocyclic peptide ligands exhibited the expected number of cysteines, amino acid frequencies, and topological diversity. d) Bar plots showing the number of cysteines (b) and the normalized frequency (%) of the “TAG” amber-stop codon (c) determined by NGS analysis. Individual amino acids (20) are represented by single-letter codes. Amber stop codons are represented by the letter "Z". The height of each band is correlated with the frequency ratio. The experimental / theoretical ratio of enriched amino acids in the original library is >1, while that of depleted amino acids is <1.

[0028] Figure 4 An exemplary "bicyclic" cysteine-enriched macrocyclic peptide library (X) was designed and analyzed. n C(X) m C(X) t C(X) yThe image is displayed on the surface of yeast cells. a) An exemplary “bicyclic” macrocyclic peptide library, named CX3CX9C, was generated, containing three fixed-position cysteine ​​residues (C) and 12 random amino acids (X). The experimental size diversity of the library was reported. The random amino acid X is encoded by the “NNK” codon. Cysteine-enriched macrocyclic peptide libraries were constructed using a homologous recombination-based approach (Van Deventer and Wittrup 2014; Angelini et al., 2015). The quality of the original CX3CX9C library was assessed by next-generation sequencing (NGS). Analysis of the library confirmed that the encoded disulfide-tethered macrocyclic peptide ligands exhibited the expected number of cysteine ​​residues, amino acid frequencies, and topological diversity. d) Bar plots showing the number of cysteine ​​residues (b) and the frequency of the “TAG” amber stop codon (c) as determined by NGS analysis. Individual amino acids (20) are represented by single-letter codes. Amber stop codons are represented by the letter "Z". The height of each band is correlated with the frequency ratio. The experimental / theoretical ratio of enriched amino acids in the original library is >1, while that of depleted amino acids is <1.

[0029] Figure 5 An exemplary "bicyclic" cysteine-enriched macrocyclic peptide library (X) was designed and analyzed. n C(X) m C(X) t C(X) j C(X) yThe image is displayed on the surface of yeast cells. a) An exemplary “bicyclic” macrocyclic peptide library, named CX4CX4CX4C (SEQ ID NO:70), was generated, containing four fixed-position cysteine ​​residues (C) and 12 random amino acids (X). The experimental size diversity of the library was reported. The random amino acid X was encoded by the “NNK” codon. Cysteine-enriched macrocyclic peptide libraries were constructed using a homologous recombination-based approach (Van Deventer and Wittrup 2014; Angelini et al., 2015). The quality of the original CX4CX4CX4C library was assessed by next-generation sequencing (NGS). Analysis of the library confirmed that the encoded disulfide-tethered macrocyclic peptide ligands exhibited the expected number of cysteine ​​residues, amino acid frequencies, and topological diversity. Bar charts showing the number of cysteine ​​residues (b) and the frequency of the “TAG” amber stop codon (c) determined by NGS analysis; and bar charts showing the experimental frequency of each individual amino acid determined by NGS analysis relative to the theoretical expected value of the “NNK” codon. Individual amino acids (20) are represented by single-letter codes. Amber stop codons are represented by the letter “Z”. The height of each band is correlated with the frequency ratio. Amino acids enriched in the original library showed an experimental / theoretical ratio >1, while depleted amino acids showed an experimental / theoretical ratio <1. Detailed Implementation

[0030] This invention relates to the design and generation of large libraries of cysteine-enriched macrocyclic peptides, which have been optimized for recombinant expression on the surface of yeast cells. Figure 1 ).

[0031] A random DNA sequence encoding a cysteine-enriched macrocyclic peptide variant has been cloned into a vector for recombinant expression on yeast cells. The vector has been designed and optimized to allow the generation of cysteine-enriched macrocyclic peptide (MP) sequences, which are expressed on the yeast cell surface as N-terminal or C-terminal fusions (CSPs) of cell surface proteins. Figure 2 ).

[0032] The MP sequence may include multiple fixed cysteine ​​residues varying between 2 and 4. The number of random amino acid “X” can vary between 3 and 21. Random amino acid “X” can be native (or “classical amino acid” or cAA) or non-native amino acid (or “non-classical amino acid” or NcAA). In summary, the length of the macrocyclic molecule generated herein can range from 5 to 25 amino acid residues.

[0033] Yeast cell surface anchoring proteins (CSPs) may have: i) an intracellular terminal (N-terminus or C-terminus) and an extracellular terminal (C-terminus or N-terminus), the latter being covalently linked to a target cysteine-enriched macrocyclic peptide sequence; ii) an extracellular N-terminus and a C-terminus, one of which (N-terminus or C-terminus) is covalently linked to the target cysteine-enriched macrocyclic peptide sequence; iii) an N-terminus or C-terminus not linked to the target cysteine-enriched macrocyclic peptide sequence that is covalently or non-covalently bound to the membrane.

[0034] The vector also includes gene segments encoding one or more immunofluorescent tags (IFTs) located near cell surface anchoring proteins (CSPs). IFTs are used to identify and / or isolate yeast cells expressing MP-CSP (or CSP-MP) fusions on their cell surfaces using various biochemical techniques, such as flow cytometry, fluorescence-activated cell sorting, magnetic beads, fluorescence microscopy, confocal microscopy, ELISA, microfluidic devices, and systems. Furthermore, the vector includes a long, flexible linker (FL) positioned between the cysteine-rich peptide sequence and the cell surface anchoring protein to minimize potential steric hindrance to the cell surface anchoring protein and the immunofluorescent tag detection reagent. Once inserted into cells, the vector can recombinantly express multiple copies (10^6) of the protein. 4 -10 6 The unique disulfide-tethered macrocyclic peptide is fused to an anchoring protein displayed on the surface of yeast cells.

[0035] The cysteine-enriched peptide library of the present invention encodes "monocyclic" or "polycyclic" macrocyclic peptide topologies, wherein the sequence diversity ranges from 10. 6 Up to 10 9 Monocyclic macrocyclic peptide ligands contain one disulfide bridge, bicyclic macrocyclic peptide ligands contain two disulfide bridges, and polycyclic macrocyclic peptide ligands contain more than two disulfide bridges. We designed yeast-encoded (X) n C(X) m C(X) t peptide library in the form of (LIB ID NO:1, Figure 3 In this study, all macrocyclic peptide sequences primarily contain two fixed cysteine ​​residues (C), where X = any amino acid, including classical (cAA) and non-classical (NcAA) amino acids, with the final peptide sequence length ranging from 5 to 23 amino acids. Similarly, we created yeast-encoded (X) sequences. n C(X) m C(X) t C(X) y (LIB ID NO:2, Figure 4 ) and (X)n C(X) m C(X) t C(X) j C(X) y (LIB ID NO:3, Figure 5 A peptide library in the form of a ) where all macrocyclic peptide sequences primarily contain three or four constant cysteine ​​residues (C) separated by different random amino acids (X), where X = any amino acid, including classical (cAA) and non-classical (NcAA) amino acids, and the final length of the peptide sequence is 7 to 25 amino acids. (X) n C(X) m C(X) t C(X) y In the (LIB ID NO:2) form, the theoretical probability of encountering an additional cysteine ​​residue is estimated at 37%. The first disulfide bond can form between any two of the three fixed cysteines, and the second disulfide bridge can form between the third and fourth cysteines appearing at any random position. A total of up to 3 ( l – 4)! / 2( l – 6)! different kinds of lengths l The topological structure of macrocyclic peptides with a high number of amino acids (Kong et al., 2020) should increase the likelihood of identifying polycyclic macrocyclic peptides with complementary shapes to the target protein surface. Next-generation sequencing (NGS) analysis of cysteine-enriched primitive macrocyclic peptide libraries confirmed that the peptide sequences exhibited the expected number of cysteine ​​residues, amino acid frequencies, and topological diversity. We demonstrate that yeast exhibiting disulfide-tethered macrocyclic peptide ligands with high structural diversity and fine binding properties against a diverse set of protein targets can be rapidly identified using a quantitative flow cytometry-based screening method. This technique appears capable of effectively screening and enriching rare disulfide-tethered macrocyclic peptide ligands with different motifs and topologies for each test target.

[0036] In a first aspect, the present invention provides a yeast cell containing an expression vector encoding a fusion protein (I): (NH2- or COOH) – [MP] – [FL] – [IFT] – [CSP] – (COOH or NH2) (I) in: (i) (NH2 or COOH) represents the amino or carboxyl terminus of the target cysteine-enriched macrocyclic peptide [MP], and (COOH or NH2) represents the corresponding carboxyl or amino terminus of the protein [CSP] expressed on the surface of yeast cells, wherein the first terminus is located extracellularly; (ii) [MP] represents macrocyclic peptide (X)n C(X) m C(X) t Two cysteine ​​residues, "C", form a disulfide bond; n , m , t It is 0 or an integer, where n = 0-5; m = 3-21; t = 0-5, the condition is n + m + t = 3-21 or [MP] indicates macrocyclic peptide (X) n C(X) m C(X) t C(X) y Two cysteine ​​residues, "C", form a disulfide bond; n , m , t and y It is 0 or an integer, where n = 0-5; m = 0-22; t = 0-22; y = 0-5, the condition is n + m + t + y = 4-22; or [MP] indicates macrocyclic peptide (X) n C(X) m C(X) t C(X) j C(X) y Each pair of Cys residues "C" forms a disulfide bond; n , m , t , j and y It is 0 or an integer, where n = 0-5, m = 0-21, t = 0-21, j = 0-21, y = 0-5, the condition is n + m + t +j + y = 3-21; “X” represents any natural (cAA) or non-natural (NcAA) amino acid, where: - Natural amino acids (cAA) are encoded by any one of 61 naturally occurring codons, or by a selection of degenerate codons from the following: "NNN" and the TAG, TAA, TGA stop codons encoding all 20 natural amino acids; "NNK" and the TAG stop codon encoding all 20 natural amino acids; "NNS" and the TAG stop codon encoding all 20 natural amino acids; "NDT" encoding 12 natural amino acids: R, N, D, C, G, H, I, L, F, S, Y, and V; "DBK" encoding 12 natural amino acids: A, R, C, G, I, L, M, F, S, T, W, and V; "NRT" encoding 8 natural amino acids: R, N, D, C, G, H, S, and Y; and "NRT" encoding 15 natural amino acids: F, S, Y, C, L, P, H, R, I, T, N, ... "NNC" encodes V, A, D, and G; "NWW" and the TAA stop codon encodes 11 natural amino acids: D, E, F, H, I, K, L, N, Q, V, and Y; "RVK" encodes 10 natural amino acids: A, D, E, G, H, K, N, R, S, and T; "DVT" encodes 8 natural amino acids: A, C, D, G, N, S, T, and Y; "NVT" encodes 10 natural amino acids: C, D, G, H, N, P, R, S, T, and Y; "VVC" encodes 9 natural amino acids: A, D, G, H, N, P, R, S, and T; "RST" encodes 4 natural amino acids: A, G, S, and T; "NTT" encodes 4 natural amino acids: F, I, L, and V; "TDK" and the TAG stop codon encodes 5 natural amino acids: C, F, L, W, and Y. The condition is that at least one random amino acid exists in the sequence of the target macrocyclic peptide. Preferably, 20% to 50% of the amino acids in the sequence are random, and more preferably, >50% of the amino acids are random. - Non-natural amino acids (NcAA) are encoded by TAG, TAA, or TGA stop codons present within the coding sequence of the target peptide in the expression vector; (iii) [FL] represents a spacer sequence containing 1 to 25 amino acids, preferably selected from the following sequences: (GGGGS) n (SEQ ID NO:71), where n The range is 1 to 5; (G) n (SEQ ID NO:72), where n Integers from 1 to 25; (SEQ ID NO:78), where n Integers from 1 to 25; (P) n (SEQ ID NO:73), where n Integers from 1 to 25; (EAAAK) n(SEQ ID NO:74), where n Integers from 1 to 5; (A(EAAAK)4ALEA(EAAAK)4A) n (SEQ ID NO:75), where n An integer between 1 and 2; (XP) n (SEQ ID NO:77), where n It is an integer from 1 to 12, and "X" can be any natural amino acid, preferably alanine (A), lysine (K) and glutamic acid (E); (iv) [IFT] represents a sequence suitable for detecting and / or isolating yeast cells expressing MP-CSP or CSP-MP fusion on the surface of yeast cells; (v) [CSP] represents a protein expressed on the surface of yeast cells, which may be fused to the N-terminus or C-terminus of a macrocyclic peptide [MP] sequence and contains: (a) at least one extracellular portion; (b) Transmembrane portion; or portion bound to the membrane and / or wall, which is covalently or non-covalently anchored to the molecules of the membrane and / or wall.

[0037] In one embodiment, the [CSP] is an Aga1 protein; an Aga2 protein; a member of the glycosylphosphatidylinositol (GPI) binding protein family, preferably Cwp1p, Cwp2p, Tip1p, Flop1p, Sed1p, YCR89, Tir1p; or a synthetic, non-naturally occurring protein that can be stably anchored to the yeast membrane and / or wall through covalent or non-covalent interactions.

[0038] In another embodiment, the yeast cell contains one or more vectors encoding an orthogonal translation system (OTS), which consists of aminoacyl-tRNA synthetase (aaRS) and corresponding transfer RNA (tRNA) capable of inserting non-natural amino acids (NcAA) at the TAG, TAA, or TGA stop codons present in the expression vector encoding the fusion protein.

[0039] In other embodiments, the non-natural amino acid (NcAA) present in the macrocyclic peptide [MP] is encoded by stop codons (TAG, TAA, TGA) present in the macrocyclic peptide DNA sequence. NcAA can be inserted into the macrocyclic peptide sequence at the level of stop codons (TAG, TAA, TGA) by means of natural amino acid deletion or by the action of a bioorthogonal translation system (OTS) comprising aminoacyl-tRNA synthetase (aaRS) and the corresponding transfer RNA (tRNA). The bioorthogonal aaRS / tRNA system is encoded by a plasmid inserted into yeast cells along with a vector encoding the target cysteine-enriched macrocyclic peptide. NcAA is defined as a chemical molecule composed of carbon atoms bonded to hydrogen atoms, carboxyl groups, amino groups, and non-naturally present side chains. NcAA can undergo selective post-translational modifications.

[0040] In other embodiments, [IFT] represents a sequence suitable for detecting and / or isolating yeast cells expressing MP-CSP (or CSP-MP) fusions on their cell surface using various biochemical techniques, such as flow cytometry, fluorescence-activated cell sorting, magnetic beads, fluorescence microscopy, confocal microscopy, ELISA, microfluidic devices, and systems. Examples of IFT sequences that can be used include

[0041] c-myc-tag (EQKLISEEDL_SEQ ID NO:1), HA-tag (YPYDVPDYA_SEQ ID NO:2), Flag-tag (DYKDDDK_SEQ ID NO:3), His-tag (HHHHHH_SEQ ID NO: 4), Strep-tag (WSAPGFEK_SEQ ID NO:5), TC-tag (CCPGCC_SEQ ID NO:6), S-tag (KETAAAKFERQHMDS_SEQ ID NO:7), AviTag (GLNDIFEAQKIEWH_SEQ ID NO:8), Strep-tag2 (AWRHPQFGG_SEQ ID NO:9).

[0042] In other embodiments, [CSP] represents a protein expressed on the surface of yeast cells that contains: (c) at least one extracellular portion; (d) Transmembrane portion; or portion bound to the membrane and / or wall, which is covalently or non-covalently anchored to the membrane and / or wall molecules, such as fatty acids, sugars, chemically synthesized molecules or recombinantly synthesized biomolecules.

[0043] Preferably, the cell surface protein [CSP] that can fuse with the N-terminus or C-terminus of the target macrocyclic peptide [MP] sequence includes, but is not limited to, Aga1 protein; Aga2 protein; or members of the glycosylphosphatidylinositol (GPI) binding protein family, preferably Cwp1p, Cwp2p, Tip1p, Flop1p, Sed1p, YCR89, Tir1p; or synthetic, non-naturally occurring proteins that can be stably anchored to the yeast membrane and / or wall through covalent or non-covalent interactions.

[0044] In another embodiment, the present invention provides an expression vector encoding the fusion protein (I) disclosed herein.

[0045] Preferably, as defined above (I), the yeast cell may contain one or two expression vectors, an immunofluorescence (IFT) tag, and a spacer region (FL), said vector encoding a cysteine-enriched macrocyclic peptide (MP) sequence fused to the amino (N-) or carboxyl (C-) terminus of a protein (CSP) expressed on the surface of the yeast cell. The vector is preferably a plasmid suitable for replication and expression in yeast cells; more preferably, it further comprises an inducible promoter (P) functionally linked to the fusion protein (I). In a preferred aspect, the vector further comprises a secretion signal (SS) located at the 5' position relative to the fusion protein (I).

[0046] Preferably, the secretion signal [SS] is a sequence selected from the following: Aga2 protein secretion signal (MQLLRCFSIFSVIASVLA; SEQ ID NO: 10); α-mating factor secretion signal (MRFPSIFTAVLFAASSALAAPANTTTEDETAQIPAEAVIDYSDLEGDFDAAALPLSNSTNGLSSTNTTIASIAAKEEGVQLDKREA; SEQ ID NO: 11); bacteriophage secretion signal (MKYLLPTAAAGLLLLAAQPAMA; SEQ ID NO:12); Chicken lysozyme (CLY) secretion signal (MRSLLILVLCFLPLAALG; SEQ ID NO:13); CLY-L8 (MRLLLLLLPLAALG; SEQ ID NO:14); Saccharomyces cerevisiae ( S. cerevisiae Invertase (SUC2) (MLLQAFLLAGFAAKISA; SEQ ID NO: 15); MF-α (Prepro) (MRFPSIFTAVLFAASSALAAPVNTTTEEGVSLEKR; SEQ ID NO: 16); MF-α (Pre)-apv (MRFPSIFTAVLFAASSALAAPV; SEQ ID NO:17); MF-α(Pre)-apv-SLEKR(MRFPSIFTAVLFAASSALAAPVSLEKR; SEQ ID NO:18); MF-α(Prepro)-(EA)3(MRFPSIFTAVLFAASSALAAPVNTTTE-EGVSLEKREAEAEA; SEQ ID NO: 19); αF peptide signal (MRFPSIFTAVLFAASSALAAPVNTTTE-DETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNGLNGLLFINTTIASIAAKEEGVSLEKR; SEQ ID NO: 20); KILM1 peptide signal (MTKPTQVLVRSVSILFFITLLHLVVALNDVAGPAETAPVSLLPR; SEQ ID NO:21); repressible acid phosphatase (PHO1) peptide signal (MFSPILSLEIILALATLQSVFA; SEQ ID NO:22); GOX (Aspergillus niger) A. niger Peptide signal (MQTLLVSSLVVSLAAALPHYIR; SEQ ID NO:23); S. occidentalis Glucosylamylase (GAM1) gene peptide signal (MIFLKLIKSIVIGLGLVSAIQA; SEQ ID NO:24); Human serum albumin (HSA) signal peptide containing the pro sequence (MKWVTFISLLFLFSSAYSRGVFRR; SEQ ID NO:25); Human serum albumin (HSA) signal peptide without the pro sequence (MKWVTFISLLFLFSSAYS; SEQ ID NO:26); Human serum albumin (HSA) signal peptide ISN (MALWMRLLPLLALLALWGPDPAA; SEQ ID NO:27); Human serum albumin (HSA) signal peptide IFN (MKYTSYILAFQLCIVLGSLGCDLP; SEQ ID NO:28); HGH signal peptide (MAADSQTPWLLTFSLLCLLWPQEPGA; SEQ ID NO:29); Phytohemagglutinin (PHA) (MKKNRMMMIWSVGVWMLLLVGGSYG; SEQ ID NO:30); Silkworm lysozyme (MQKLIIFALVVLCVGSEA; SEQ ID NO:31); Human lysozyme (LYZ1) (MKALIVLGLVLLSVTVQG; SEQ ID NO:32); Type I activin receptor (MVDGVMILPVLIMIALPSPS; SEQ ID NO:33); Type II activin receptor (MGAAAKLAVFLISCSSG; SEQ ID NO:34); Immunoglobulin-binding protein Pichia pastoris ( P. pastoris (PpBiP) immunoglobulin-binding protein (MLSLKPSWLTLAALMYAMLLVVPFAKPVRA; SEQ ID NO:35); and human antibody 3D6 light chain leader (MDMRVPAQLLGLLWLPGAKC; SEQ ID NO:36) (Felgenhauer, Kohl and Rüker 1990; Damasceno et al., 2007; Daly and Hearn 2006; Koganesawa et al., 2001; Raemaekers et al., 1999; Gellissen et al., 2005; OKA et al., 1999; Hashimoto, Koyabu and Imoto 1998; Kingwell 2023; Mullard 2023).

[0047] In other respects, the present invention provides a method for preparing the expression vector disclosed herein, wherein the fusion protein (I) has the following orientation: (HOOC)-[CSP]-[IFT]-[FL]-[MP]-(NH2) (I), The method includes the following steps: (1) Provide an expression vector, wherein the vector comprises the following constructs from 5' to 3': 5'-[ip] – [ss] – [re] – [sr] – [fl] – [ift] – [csp]-3’ in: [ip] indicates an induced promoter (e.g., GAL1-10). [ss] represents the DNA sequence encoding the secretion signal as defined above regarding expression vectors; [re] represents a DNA sequence that can be specifically and uniquely recognized by a restriction enzyme, wherein the restriction enzyme is preferably selected from, but not limited to, a subset of, the restriction enzyme. Bam HI、 Nhe I, EcoR I, Xho I, Sal I, Nde I, Nco I, Sfi I, Sfo I, Bgl II. Kpn I, Hind III. Xba I, No tI, wherein the site is located inside the DNA spacer region [sr] and / or at the 5' end and / or the 3' end; (iv)[sr] indicates a spacer region containing 6 to 3000 nucleotides; (v)[fl] represents the DNA sequence encoding the flexible linker [FL] as defined above for the fusion protein (I); (vi) [ift] represents the DNA sequence encoding the immunofluorescence detection tag [IFT] as defined above for fusion protein (I); (vii) [csp] represents a DNA sequence encoding a cell surface anchoring protein [CSP] as defined above for fusion protein (I), and contains at least one restriction site as defined in (iii) at its interior and / or 5' end and / or 3' end.

[0048] (2) The sequence encoding the target macrocyclic peptide was inserted into the vector via PCR using the following primer pairs: (i) A reverse primer that pairs with the antisense strand of a sequence encoding a target macrocyclic peptide [MP], the pairing region being located approximately 10 to 60 base pairs downstream of a restriction enzyme [re] cleavage site contained within or at the end of a cell surface anchor protein encoding gene (csp); preferably, the reverse primer is 5 to 60 base pairs in length, wherein the pairing temperature range is 45°C to 80°C, preferably 55°C to 70°C, and more preferably 60°C to 65°C; (ii) A forward primer that pairs with the sense strand of a sequence encoding the target macrocyclic peptide [MP], wherein the sense strand encodes from the 5' to the 3' end: 5 ' -[Area paired with ss]-[Target mp]-[Area paired with sr]-3 ', in: (a) The region that pairs with the secretion signal (ss) is located about 10 to 60 base pairs upstream of the restriction enzyme (re) cleavage site between the secretion signal and the DNA linker, wherein the annealing temperature is higher than 20°C, preferably higher than 30°C, and more preferably higher than 40°C; (b) The length of the region that pairs with the spacer region (SR) is 5 to 60 base pairs, and the pairing temperature range is 45°C to 80°C, preferably 55°C to 70°C, and more preferably 60°C to 65°C.

[0049] In other respects, the present invention provides a method for preparing an expression vector as defined herein, wherein the fusion protein (I) has the following orientation: (NH2)-[CSP]-[IFT]-[FL]-[MP]-(COOH) (I), The method includes the following steps: (1) Provide an expression vector containing the following genetic elements from 5' to 3': 5'-[ip] – [ss] – [csp] – [ift] – [fl] – [sr] – [re]-3’ Wherein: [ip] represents the inducible promoter as defined above for fusion protein (I), [ss] represents the DNA sequence encoding the secretion signal as defined above for fusion protein (I), [csp] represents the DNA sequence encoding the cell surface anchoring protein [CSP] as defined above for fusion protein (I), [ift] represents the DNA sequence encoding the immunofluorescence detection tag IFT as defined above for fusion protein (I), [fl] represents the DNA sequence encoding the flexible linker FL as defined above for fusion protein (I), [sr] represents the spacer region as defined above for fusion protein (I), and [re] represents the DNA sequence as defined above for fusion protein (I) that can be specifically and uniquely recognized by restriction enzymes.

[0050] (2) The coding sequence of the target macrocyclic peptide was inserted into the vector via PCR using the following primer pairs: (i) A forward primer that pairs with the sense strand of a sequence encoding the target peptide [MP], wherein the pairing region is located 10-60 base pairs upstream of the restriction enzyme (re) cleavage site at the 5' end or inside the cell surface anchor protein encoding gene (csp); preferably, the length of the forward primer is 5 to 60 base pairs, and the pairing temperature range is 45°C to 80°C, more preferably 55°C to 70°C, and even more preferably 60°C to 65°C; (ii) A reverse primer that pairs with the antisense strand of the sequence encoding the target peptide and encodes from the 5' to the 3' end: 5 ' -[Main Chain Pairing Area]-[Target MP]-[Area Paired with SR]-3 ', in: (a) The main chain pairing region is paired with a plasmid sequence located 10 to 50 base pairs downstream of a restriction site (re) at or inside the end of the linker sequence (fl), wherein the pairing temperature is above 50°C; (b) The length of the region that pairs with the spacer region (sr) is 5 to 50 base pairs, and the pairing temperature range is 45°C to 80°C, preferably 55°C to 70°C, more preferably 60°C to 65°C; The condition is that the temperature difference between the two primers must be less than 20°C, preferably less than 10°C, and more preferably less than 5°C.

[0051] Preferably, the PCR reaction is performed according to the following procedure: 1) Initial denaturation at 95-98℃ (3 min) 2) 30 cycles consisting of three steps: 95℃ (30 sec), 60-55℃ (30 sec), and 72℃ (1-5 min). 3) Final extension at 72℃ (10 min).

[0052] The method of the present invention allows: - Generate cysteine-enriched macrocyclic peptide libraries fused to the C-terminus or N-terminus in various yeast cell display systems, including but not limited to Aga1-Aga2, Cwp1p, Cwp2p, Tip1p, Flop1p, Sed1p, YCR89, Tir1p, GPL-1, GPL-1 peptide derivatives, synthetic artificial proteins, or fusion proteins. Broad compatibility is achieved by introducing homologous regions that are universal and translatable across different genetic vectors. - Generate highly diverse libraries of cysteine-enriched macrocyclic peptides fused at the C-terminus or N-terminus in different yeast cell display systems, including but not limited to Aga1-Aga2, Cwp1p, Cwp2p, Tip1p, Flop1p, Sed1p, YCR89, Tir1p, GPL-1, GPL-1 peptide derivatives, synthetic proteins or fusion proteins; - Direct next-generation sequencing analysis of the original library and selected libraries.

[0053] The PCR conditions are based solely on the paired region, which depends only on the sequence of the selected starting vector, and not on the sequence, structure, or length of the designed cysteine-enriched macrocyclic peptide region. This procedure can be used to create both completely and / or partially random sequences, as well as to clone peptides with defined amino acid sequences.

[0054] In other respects, the present invention provides a cell population consisting of multiple yeast cells, wherein each single cell expresses a unique target cysteine-enriched macrocyclic peptide [MP] on its surface, the sequence of which is different throughout the cell population. Different cells within the cell population may express different target cysteine-enriched macrocyclic peptides [MP], preferably expressing different peptides for each cell, wherein the target peptide is selected from: (X)n C(X) m C(X) tt (X) n C(X) m C(X) t C(X) y (X) n C(X) m C(X) t C(X) j C(X) y .

[0055] Each yeast cell can express multiple copies (10) on its cell surface. 4 -10 6 The unique cysteine-enriched macrocyclic peptide [MP] sequence is fused with a cell surface protein selected from a previously reported library (LIB ID NO: 1-3).

[0056] In a preferred embodiment, the diversity of the yeast cell population of the present invention can be at least 1 with respect to the sequence of the target macrocyclic peptide. 10 6 Preferably higher than 1 10 8 More preferably higher than 1 10 9 .

[0057] The term "diversity" refers to the number of yeast cells with different genetic modifications obtained after creating the library. "Diversity" can be calculated using the following methods: i ) Inoculate with serially diluted yeast cell transformants and count the colonies of successfully transformed cells containing a carrier encoding a cysteine-enriched macrocyclic peptide fused to a cell surface protein; and / or ii Large-scale parallel next-generation sequencing (NGS) analysis was performed on yeast-extracted DNA vectors encoding cysteine-enriched macrocyclic peptide sequences.

[0058] This invention allows for the efficient de novo generation and isolation of high-affinity and specific disulfide-bonded macrocyclic peptide ligands from a large and topologically diverse combinatorial library expressed on the surface of yeast cells. Yeast surface display technology can also be used to enhance the properties of naturally occurring macrocyclic peptide ligands and / or enhance those previously achieved using other techniques (“from seedling to leader”). hit-to- leadThe properties of macrocyclic peptide ligands have been developed through the process of yeast surface display. Furthermore, yeast surface display can be combined with other in vitro directed evolution tools (such as phage and mRNA display) to further leverage their unique advantages, enabling previously untapped applications. These applications include evaluating and improving binding and stability, as well as rapid and precise epitope localization. Moreover, based on the tremendous success achieved by yeast surface display technology in rapidly screening and evaluating the biophysical properties of hundreds of computationally designed proteins (Pan and Kortemme 2021; Linciano et al., 2019; Listov et al., 2024), this tool is also suitable for efficiently characterizing computationally designed proteins. in silico The gene-encoded macrocyclic peptide ligand was developed.

[0059] Another use of the macrocyclic peptide library according to the invention is to screen for relatively small disulfide-tethered macrocyclic peptide ligands (5-25 amino acid length) that are bioactive. The screening and selection of disulfide-tethered macrocyclic peptide ligands, capable of binding to a given target site (e.g., protein, DNA, RNA, cell), can be conveniently performed using magnetic bead (MB) separation and / or fluorescence-activated cell sorting (FACS). The identity of the isolated disulfide-tethered macrocyclic peptide ligands can be revealed by applying Sanger sequencing and next-generation DNA sequencing. Applications of the selected disulfide-tethered macrocyclic peptide ligands include, but are not limited to, the development of therapeutics for use alone or in combination with other pharmacologically active entities (e.g., radionuclides, chemotherapeutic agents, proteins, antibodies, cells, DNA, RNA, lipid nanoparticles). Disulfide-tethered macrocyclic peptide ligands discovered using yeast display technology can also potentially be used to develop diagnostic tools or for applications in the veterinary, botanical, and food industries.

[0060] Experimental Section

[0061] Generate a cysteine-enriched macrocyclic peptide library

[0062] 1. PCR reactions used to create cysteine-enriched macrocyclic peptide inserts that fuse with cell surface proteins.

[0063] The procedure for constructing the expression vector is disclosed above. This vector comprises the following elements: 5'-[ip] – [ss] – [re] – [sr] – [fl] – [ift] – [csp]-3’ or

[0064] 5'-[ip] – [ss] – [csp] – [ift] – [fl] – [sr] – [re]-3’

[0065] The PCR reaction for the insert fragment was performed in a 50 μL reaction volume containing degenerate forward primer F1 (500 nM), reverse primer R (500 nM), a dNTP mixture (200 μM each), 1 ng of pCT-CSP as template DNA, 10X DreamTaq buffer (1X), 1.25 U DreamTaq DNA polymerase (Thermo Fisher), and H2O milli-Q.

[0066] Perform the PCR reaction according to the following steps: 1) Initial denaturation at 95-98℃ (3 min) 2) 30 cycles consisting of three steps: 95℃ (30 sec), 60-55℃ (30 sec), and 72℃ (1-5 min). 3) Final extension at 72℃ (10 min).

[0067] The PCR conditions are based solely on the paired region, which depends only on the sequence of the selected starting vector, and not on the sequence, structure, or length of the designed cysteine-enriched macrocyclic peptide region. This procedure can be used to create random sequences as well as to clone known amino acid sequences.

[0068] The backbone vector DNA is linearized using 1 to 5 different enzymes, preferably 2 to 3 restriction enzymes, more preferably 3 restriction enzymes. Digestion is carried out in a reaction volume of 20 to 200 μL, with an incubation time of 1 to 48 hours, preferably 12 to 32 hours, more preferably 16 to 30 hours. The digested product is purified by ethanol precipitation and resuspended in sterile H2O milli-Q at a concentration of 0.5-3 μg / μL, preferably 1-2 μg / μL. The absorbance ratio 260 / 280 is in the range of 1.4-2.0, preferably higher than 1.6, and the ratio 260 / 230 is in the range of 1.6-2.5, preferably higher than 1.8.

[0069] The reported library format was obtained using the homologous recombination method described in the literature (Angelini et al., 2015). To increase the diversity of the library, the method reported in previous literature was modified as follows: - Penicillin and streptomycin were lacking in the culture medium during all passages; - OD in the range of 0.25-0.3 600 Initially incubate cells; - The digested vector and PCR product are pre-incubated on ice for 5-15 minutes to form a DNA mixture. The volume of the DNA mixture is 5-20 μL, preferably 8-15 μL. The ratio of insert fragment to vector in the mixture is 50:1, and the total amount of DNA is 5-20 μg, preferably 10-15 μg, more preferably 12-14 μg. - Pre-incubate the DNA mixture with yeast cells on ice for 5-30 minutes, then perform electroporation; - Resuscitate electroporated yeast cells using YPD or YPD:sorbitol solution (1:1); - Wash the electroporated yeast cells and incubate them at 30°C in fresh SD-CAA medium free of penicillin and streptomycin.

[0070] The library was obtained by performing 8 to 32 electroporations, preferably 10 to 20, and its diversity ranges from 10. 8 -10 9 Preferred values ​​are above 10 9 .

[0071] 2. Deep sequence analysis of a macrocyclic peptide library enriched with cysteine ​​displayed on the surface of yeast cells.

[0072] To gain a better understanding of the genetic diversity and abundance of the original library of cysteine-enriched macrocyclic peptides, next-generation sequencing (NGS) analysis was performed. The following analysis presents NGS data from the original library: 1) Divide according to the barcode sequence; 2) Translation of cysteine-enriched macrocyclic peptide sequences; 3) Quantification of TAG codons; 4) Statistical analysis of amino acid frequencies.

[0073] The amplicon was sequenced using a NovaSeq6000 (Illumina, IgaTech) to obtain 150-base-pair paired-end reads.

[0074] 3. Extracting plasmid DNA from yeast cells of a polyclonal library

[0075] 1) Culture yeast cells encoding the original library at 30°C with shaking (250 rpm) for 16-24 hours, ensuring the cell number is at least 10 times the estimated diversity of the library, and the concentration is within the range of 0.1. 10 7 -10 8 cells / mL (preferably 1-3) 10 7 (cells / mL); 2) Dilute the yeast cells encoding the original library to 10⁻⁶. 7A concentration of cells / mL was achieved by using cells that were 2–10 times more diverse than the estimated original library. 3) Culture yeast cells encoding the original library to grow to 2-3 [units of length]. 10 7 Concentration per cell / mL; 4) Extract plasmid DNA from yeast cells encoding the original library using a commercial kit (e.g., Zymoprep yeast plasmid miniprep II, Zymo Research); 4. NGS amplicon was generated by performing two rounds of consecutive PCR reactions. The first round of PCR was performed in a 30 μL reaction volume, which contained: - 400 nM universal forward primers per vector; - 400 nM universal reverse primers per vector; - 250 μM dATP; - 250 μM dCTP; - 250 μM dTTP; - 250 μM dGTP; - 30% DMSO; - 10 μL of yeast-extracted plasmid DNA is equivalent to a total plasmid DNA volume of 0.1–100 ng (DNA molecule count range of 10). 7 – 10 10 ); - Taq DNA polymerase buffer, 1X working concentration; - 0.2 – 2 U Taq DNA polymerase; - Sterile H2O milli-Q.

[0076] The forward primers for the first round of PCR have the following 5'-3' structure: (a) An additional region that is identified and annealed to the 3' end of the second forward primer used during the second NGS-PCR. The length of this region can vary from 1 to 50 base pairs; b) A pairing region of 5 to 50 base pairs in length, with a pairing temperature range of 45 to 80°C, preferably 70 to 55°C, and more preferably 68 to 63°C. The primer pair is located in a region of 1 to 200 base pairs, preferably 1 to 100 base pairs, and more preferably 1 to 50 base pairs upstream of the 5' end of the DNA encoding the target cysteine-rich macrocyclic peptide sequence.

[0077] The reverse primers for the first round of PCR have the following 5'-3' structure: (a) Random regions, with a length of 0 to 50 base pairs; b) A pairing region, 5 to 50 base pairs in length, with a pairing temperature range of 45 to 80°C, preferably 70 to 55°C, and more preferably 68 to 63°C. The primer pair is preferably located 10 to 200 base pairs downstream of the 3' end of the DNA encoding the target macrocyclic peptide.

[0078] The products from the first round of PCR can be purified by ethanol precipitation, gel extraction, silica gel column chromatography, or any commercially available kit suitable for purifying DNA fragments amplified by PCR. The DNA products are eluted in sterile H2O milli-Q or a commercially recommended buffer at a concentration ranging from 0.01 to 1 μg / μL, with an absorbance ratio of 260 / 280 of 1.4 to 2.0, preferably greater than 1.6, and an absorbance ratio of 260 / 230 of 1.6 to 2.5, preferably greater than 1.8.

[0079] The second round of PCR was performed in a 50 μL reaction volume, which contained: - 400 nM forward primer containing the barcode region; - 400 nM reverse primer; - 250 μM dATP; - 250 μM dCTP; - 250 μM dTTP; - 250 μM dGTP; - 30% DMSO; - 10 ng of first-round PCR product; - High-fidelity DNA polymerase buffer, 1X working concentration; - 0.2-2 U Phusion high-fidelity DNA polymerase; - Sterile H2O milli-Q The forward primers for the second round of PCR have the following 5'-3' structure: a) A region containing the nucleotide sequence of the Illumina forward adaptor; b) A barcode nucleotide sequence of 4 to 20 base pairs in length, preferably 8 to 15 base pairs, with a C and G content of 20 to 70%, preferably 40 to 60% (examples of barcodes used are shown in Table 1); c) A pairing region, 5 to 50 base pairs in length, with a pairing temperature range of 45 to 80°C, preferably 70 to 55°C, and more preferably 68 to 63°C. The primer pair is located in a region 1 to 200 base pairs, preferably 1 to 100 base pairs, and more preferably 1 to 50 base pairs upstream of the 5' end of the DNA encoding the target macrocyclic peptide.

[0080]

[0081] Table 1. Examples of DNA barcode sequences used for NGS analysis

[0082] The reverse primers for the second round of PCR have the following 5'-3' structure: a) A region containing the nucleotide sequence of the Illumina reverse adaptor; b) A pairing region, 5 to 50 base pairs in length, with a pairing temperature range of 45 to 80°C, preferably 70 to 55°C, and more preferably 68 to 63°C. The primer pair is located 1 to 200 base pairs downstream of the 3' end of the DNA sequence encoding the target macrocyclic peptide.

[0083] The PCR program set up for these two reactions is as follows: 1) Initial denaturation at 95-98℃ (1-3 min); 2) 30 cycles consisting of three steps: 95-98℃ (30 sec), 60-55℃ (30 sec), and 72℃ (1-5 min); 3) Final extension at 72℃ (2-10 min); The products from the second round of PCR were purified from agarose gels by gel extraction. The agarose content of the gel must be 0.8 to 2.5% w / v, preferably 1 to 2% w / v, more preferably 1.2 to 1.5% w / v. The products were eluted in sterile H2O milli-Q or a commercially recommended buffer with a concentration ranging from 0.01 to 1 μg / μL, an absorbance ratio of 260 / 280 of 1.4 to 2.0, preferably greater than 1.6, and an absorbance ratio of 260 / 230 of 1.6 to 2.5, preferably greater than 1.8.

[0084] 5. Generate a yeast-encoded cysteine-enriched macrocyclic peptide library, said macrocyclic peptide library having the ability to fuse to the cell surface. The structure of the N-terminus of a surface-anchored protein (X) n C(X) m C(X) t and (X) n C(X) m C(X) t C(X) y And it contains only classic amino acids. (cAA)

[0085] By forming intramolecular disulfide bridges between cysteine ​​residues, a large and structurally diverse library of cysteine-enriched macrocyclic peptides (5-25 amino acids) was generated and displayed on the surface of yeast cells. To ensure distance and minimize potential steric hindrance of the detection reagent, a flexible glycine-serine (G4S)3 linker (SEQ ID NO:76) was placed between the peptide sequence and the immunofluorescent tag (IFT). Detection of the IFT tag using fluorescently labeled antibody-bound flow cytometry not only allows for rapid quantification of the expression level of disulfide-tethered macrocyclic peptide ligands on yeast cells but also allows for normalization of the binding signal relative to surface expression.

[0086] We generated a cysteine-enriched peptide library that preferentially encodes "single-loop" or "bi-loop" macrocyclic topologies, with sequence diversity ranging from 10^6 to 10^6. 6 Up to 10 9 We designed a yeast-encoded (X) n C(X) m C(X) t A "monocyclic" macrocyclic peptide library in which all sequences predominantly contain two fixed cysteine ​​residues, where X = any amino acid, including classical (cAA) and non-classical (NcAA) amino acids, and n = 0-5, m = 3-21, and t = 0-5 (The final length of the "monocyclic" macrocyclic peptide sequence varies between 5 and 23 amino acids). Similarly, we created the yeast-encoded (X) n C(X) m C(X) t C(X) y A "bicyclic" macrocyclic peptide library in which at least 30% of the peptides contain four cysteine ​​residues separated by a varying number of random amino acids (X), where X = any amino acid, including classical (cAA) and non-classical (NcAA) amino acids, and n = 0-5, m =0-22, t = 0-22, y= 0-5 (The final length of the “bicyclic” macrocyclic peptide sequence varies between 7 and 25 amino acids). Random amino acid positions in all libraries are encoded by “NNK” codons. Next-generation sequencing (NGS) analysis of the original libraries confirmed that the macrocyclic peptides exhibited the expected number of amber stop codons (“TAG”; Table 2), amino acid frequencies, and topological diversity (Table 4). We demonstrate that yeast-displayed disulfide-tethered macrocyclic peptide ligands with high structural diversity and fine binding properties against a diverse set of protein targets can be rapidly identified using a quantitative flow cytometry-based screening method. This technique appears capable of efficiently screening and enriching rare disulfide-tethered macrocyclic peptide ligands with different motifs and topologies for each test target.

[0087]

[0088] Table 2. Experimental probability of identifying additional amber stop codons (“TAG”) in two exemplary yeast-encoded macrocyclic peptide original libraries: (X) n C(X) m C(X) t A library of monocyclic macrocyclic peptides in the form of a single-ring macrocyclic peptide, in which n = 0, m = 7 and t =0; and (X) n C(X) m C(X) t C(X) y A bicyclic macrocyclic peptide library in which n = 0, m = 3 ，t = 9 and y = 0. The frequency of the “TAG” codon is 1.5 times higher than the expected theoretical value of “NNK”. One (1), two (2) and three (3) additional “TAG” codons occur at rates of approximately 32-39%, 9-14% and 1-3%, respectively.

[0089] As previously reported (Angelini et al., 2015), library diversity is calculated, and the range of library diversity is 1. 10 8 Up to 5 10 9 To verify the quality of the original library, the frequency of each amino acid was calculated from NGS data and compared with the theoretical frequencies of amino acids when using degenerate NNK codons (Tables 3 and 4) (Chen et al., 2013).

[0090]

[0091] Table 3. Theoretical probability of amino acid occurrence in the presence of degenerate codon NNK.

[0092] In a macrocyclic peptide sequence of length "n", the theoretical probability of having a given amino acid is defined by the following formula: probability ( n ) = Probability NNK n Some amino acids were found to be over-represented (cysteine, valine, tryptophan, and glycine) or under-represented (glutamine, histidine, proline, and threonine), with experimental / theoretical frequency ratios of 1.2 to 1.5 and 0.8 to 0.5, respectively. However, frequency variations did not introduce bias during selection operations because amino acid abundance was not correlated with the original abundance and was observed to be exclusively target-dependent (Table 4).

[0093]

[0094] Table 4. Ratio between experimental frequency (NGS data) and theoretical frequency (codon "NNK") of a single amino acid. Amino acids are represented by single-letter codes. Amber stop codons ("TAG") are shown as "Z". A ratio less than 1 indicates that the amino acid is underrepresented, while a ratio greater than 1 indicates that the amino acid is overrepresented.

[0095] 6. Generate a yeast-encoded structure with an N-terminus fused to the cell surface anchoring protein (X). n C(X) m C(X) t of Cysteine-enriched macrocyclic peptide libraries, wherein the macrocyclic peptide libraries contain non-classical amino acids referred to as Z at the TAG position in the sequence. (NcAA)

[0096] To test a procedure for generating a cysteine-enriched macrocyclic peptide library that is compatible with site-specific chemical modifications on non-classical amino acids and fused to the N-terminus of cell surface anchoring proteins, (X) was generated via homologous recombination as described above. n C(X) m C(X) t A library of monocyclic macrocyclic peptides in the form of a single-ring macrocyclic peptide, in which n = 0, m = 7 or 9 and t =0, and further transformed into yeast cells carrying plasmids encoding aminoacyl-tRNA transferase and E. coli biological ortholog tRNA. As previously reported (Angelini et al., 2015), library diversity was calculated, and the range of library diversity was 1. 108 Up to 1 10 9 Macrocyclic peptide libraries were designed to incorporate non-classical amino acids at specific positions, named with the letter Z, and consist of 7-amino acid rings (CXZX5C, CX3ZX3C, CX5ZXC, AZGCX7C) or 9-amino acid rings (CXZX7C, CX4ZX4C, CX7ZXC, AZGCX9C).

[0097] Yeast strain and culture medium preparation

[0098] The yeast strain used in this specific study had the following genotype: MATa AGA1::GAL1AGA1::URA3ura352 trp1 leu2 delta200 his3 delta200 pep4::HIS3 prbd1.6R can1 GAL. Angelini et al. described the culture media used for yeast growth and induction. All media were supplemented with penicillin / streptomycin (Corning 100X penicillin solution: streptomycin). Angelini et al. previously reported the preparation of uracil-free (Ura) and tryptophan-free (Trp) liquid media and yeast plates. Uracil-free (Trp), tryptophan-free (Trp), and leucine-free (Leu) yeast media were prepared using a deficient synthetic medium (Sigma Y1171) as the amino acid source. Each freshly prepared solution containing 50 mM of non-classical amino acids (NcAA) was added to the medium to a final concentration of 1 mM.

[0099] Library generation

[0100] A primitive library of cysteine-enriched macrocyclic peptides for display on the yeast cell surface was generated using a homologous recombination method in yeast cells. The disulfide-tethered macrocyclic peptides were displayed on the yeast surface as N-terminal fusions of cell surface anchoring proteins (CSPs). The example given here relates to the CSP protein Aga2, but can be applied to any CSP with a solvent-exposed extracellular N-terminus. The yeast surface display vector was based on a modified form of the pCT-CON backbone (Angelini et al., 2015), named pCT-CSP here. This vector contains a DNA sequence encoding a secretion leader sequence, Nhe The sequence consists of a restriction site, a long and flexible spacer region (G4S)3, a sequence encoding the hemagglutinin tag (HA; YPYDVPDYA), followed by a sequence encoding the CSP protein Aga2, which has been modified to include silencing. BamHI restriction site. The synthetic gene was codon-optimized for expression in yeast cells and obtained from Integrated DNA Technologies (Coralville, IA, USA). This was achieved by encoding a random peptide sequence C(X). 7 C. A raw library of yeast-encoded macrocyclic peptides was constructed by inserting DNA sequences of the flexible (G4S)3 linker, HA tag, and N-terminal portion of the CSP protein Aga2 into the pCT-CSP vector. The insert was created by appending DNA encoding random macrocyclic peptide sequences to the N-terminus of the (G4S)3-HA-Aga2 coding gene using forward degenerate primers F1-X (SEQ ID NO:47–54) and universal reverse primer R (SEQ ID NO:46) in a PCR reaction (30 cycles). Primers used for library construction were obtained from Integrated DNA Technologies and are shown in Table 5.

[0101] PCR of the insert fragment was performed in a 50 μL reaction volume containing degenerate forward primer F1 (500 nM), reverse primer R (500 nM), a mixture of dNTPs (200 μM each), 1 ng pCT-CSP template DNA, 10X DreamTaq buffer (1X), 1.25 U DreamTaq DNA polymerase (Thermo Fisher), and H2O milli-Q. The PCR program was set as follows: i) initial denaturation at 95°C for 3 min; ii) 30 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 1 min; and iii) final extension at 72°C for 10 min. Bam HI-HF (New England Lab) and Nhe I-HF (New England Lab) was used to linearize the vector backbone. Digestion was performed in a 50 μL reaction volume containing 10 μg pCT-CSP plasmid and 1 U... Nhe I-HF, 1U Bam HI-HF, 5X Cut Smart, and H2O milli-Q were used. Samples were incubated at 37°C for 24 hours. The linearized plasmid and PCR product were purified and concentrated to approximately 1 μg / μL by ethanol precipitation. The size and quality of the PCR insert and linearized plasmid were validated on a 1.2% w / v agarose gel.

[0102]

[0103] Table 5. Primer sequences used for macrocyclic peptide library generation.

[0104] NGS analysis of the original library

[0105] To determine the quality of the obtained library, NGS analysis was performed as described below.

[0106] Two PCR reactions were performed on DNA plasmids extracted from yeast cells to generate 280-base-pair NGS amplicones. For each polyclonal library, target-specific barcodes and Illumina sequencing tags were added during the second round of PCR. Primers used for library barcode tagging were obtained from Integrated DNA Technologies and are described in Table 6. The first round of PCR was performed in a 30 μL reaction volume containing 5×HF buffer (1X), DMSO (30% v / v), dNTPs (250 μM each), NGS-R primers (400 nM), NGS-F1 (400 nM), Phusion high-fidelity DNA polymerase (0.6 U; New England Biolabs), and 10 μL of DNA plasmid extracted from yeast. PCR amplicones were purified using the Zymoprep Clean and Concentrator kit (Zymo Research). The second round of PCR was performed in a 50 μL reaction volume containing 5X HF buffer (1X), DMSO (30% v / v), dNTPs (250 μM each), NGS-R2 primers (400 nM), NGS-2F-X primers (400 nM), Phusion high-fidelity DNA polymerase (0.6 U; New England Biolabs), and 10 ng of amplicons from the first round of PCR. Two PCR programs were set up as follows: i) initial denaturation at 98°C for 1 min; ii) 30 cycles of 98°C for 30 sec, 57°C for 30 sec, and 72°C for 1 min; and iii) final extension at 72°C for 5 min. The final PCR products were extracted from agarose gels (1.5% w / v) using a Qiagen Gel Extraction Kit (Qiagen, Hilden, Germany). The final DNA concentration and quality were evaluated using a BioPhotometer D30 UV spectrophotometer (Eppendorf, Hamburg, Germany). The amplicon was sequenced using a NovaSeq6000 (IGA Technology Services, Udine, Italy), yielding 150 bp paired-end reads. The raw data were processed using a MATLAB script adapted for this study (Rebollo et al., 2014).

[0107]

[0108] Table 6. Primer sequences used for NGS analysis of yeast-encoded macrocyclic peptide libraries.

[0109] 7. Generates proteins with C-terminus fusion with cell surface anchoring proteins (CSPs). (X) n C(X) m C(X) t yeast library of structure

[0110] To test a program for generating a cysteine-enriched macrocyclic peptide library fused to the C-terminus of a cell surface anchoring protein (CSP), a program with... (X) n C(X) m C(X) t A library of structures. The examples given here relate to the CSP protein Aga2, but can be applied to any CSP with a solvent-exposed extracellular C-terminus.

[0111] The encoding vector of the complex expressed on the yeast surface consists of the following: an inducible promoter, an α-mating factor secretion signal, a restriction enzyme, a sequence encoding the CSP protein Aga2 (which contains the restriction enzyme), and an influenza hemagglutinin epitope tag (HA-tag). N YPYDVPDYA C The library contains the spacer region (G4S)3 and macrocyclic peptide sequences. Library diversity was calculated as previously reported (Angelini et al., 2015). The range of library diversity determined per electroporation cycle was 10. 7 Up to 5 10 7 .

[0112] Yeast strain and culture medium preparation

[0113] The yeast strain used in this specific study had the following genotype: MATa AGA1::GAL1AGA1::URA3ura352 trp1 leu2 delta200 his3 delta200 pep4::HIS3 prbd1.6R can1 GAL. Angelini et al. described the culture media used for yeast growth and induction. All media were supplemented with penicillin / streptomycin (Corning 100X penicillin solution: streptomycin).

[0114] Library generation

[0115] The backbone used for library construction was generated by inserting a (G4S)3 adapter using the Gibson assembly protocol. The gene was optimized for expression in yeast cells using GeneArtGene Synthesis. Using the pCT-CSP backbone as a template, cysteine-enriched macrocyclic peptide sequences were incorporated downstream of the cell surface anchoring protein Aga2 via PCR. DNA encoding random cysteine-enriched macrocyclic peptide sequences was inserted using reverse primers, starting at the 5' end and containing: a paired homologous region on the plasmid backbone, a random macrocyclic peptide sequence, and a paired region at the (G4S)3 adapter (this paired region is common to macrocyclic peptide libraries fused to the N-terminus of binding proteins). Primers used for library construction were obtained from Integrated DNA Technologies and are shown in Table 7. Insertion PCR was performed in a 50 μL reaction volume containing constant LB-F forward primers (500 nM), degenerate reverse primer R (500 nM), a 200 μM dNTP mixture, 1 ng pCT-CSP template DNA, 10X DreamTaq buffer (1X), 1.25 U DreamTaq DNA polymerase, and H2O milli-Q. The PCR program was set as follows: i) initial denaturation at 95℃ for 3 min; ii) 30 cycles of 95℃ for 30 sec, 55℃ for 30 sec, and 72℃ for 1 min; and iii) final extension at 72℃ for 10 min. The results were obtained by using... EcoR I-HF (New England Lab) Bam HI-HF (New England Lab) and Nhe Triple digestion was performed using I-HF (New England Lab) to linearize the vector backbone. The vector was then processed with 10 μg of plasmid pCT-CSP and 1 U of... EcoR I-HF, 1 U Nhe I-HF, 1 U Bam Digestion was performed in 50 μL reaction volumes of H-HF, 5X Cut Smart, and H2Omilli-Q. The samples were incubated in a thermal cycler at 37°C for 24 hours. The linearized plasmids and PCR products were purified and concentrated to approximately 1 μg / μL by ethanol precipitation. The size and quality of the PCR insert and linearized plasmid were validated on 1.2% w / v agarose gels.

[0116]

[0117] Table 7. Cysteine-enriched macrocyclic peptide sequences C(X) used for fusion into cell surface anchoring proteins (CSPs) n Primers for C library construction

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Claims

1. A yeast cell containing an expression vector encoding a fusion protein (I): (NH2- or COOH) – [MP] – [FL] – [IFT] – [CSP] – (COOH or NH2) (I) in: (i) (NH2 or COOH) represents the amino or carboxyl terminus of the target cysteine-enriched macrocyclic peptide [MP] sequence, and (COOH or NH2) represents the corresponding carboxyl or amino terminus of the protein [CSP] expressed on the surface of yeast cells, wherein the first terminus is located extracellularly; (ii) [MP] represents macrocyclic peptide (X) n C(X) m C(X) t The sequence contains two cysteine ​​residues "C" that form a disulfide bond; n , m , t It is 0 or an integer, where n = 0-5; m = 3-21; t = 0-5, the condition is n + m + t = 3-21; or [MP] indicates macrocyclic peptide (X) n C(X) m C(X) t C(X) y The sequence contains two cysteine ​​residues "C" that form a disulfide bond; n , m , t and y It is 0 or an integer, where n = 0-5; m = 0-22; t = 0-22; y = 0-5, the condition is n + m + t + y = 4-22; or [MP] indicates macrocyclic peptide (X) n C(X) m C(X) t C(X) j C(X) y Each pair of cysteine ​​residues forms a disulfide bond; n , m , t , j , y It is 0 or an integer, where n = 0-5, m = 0-21, t = 0-21, j = 0-21, y = 0-5, the condition is n + m + t +j + y = 4-21; "X" represents any natural (cAA) or non-natural (NcAA) amino acid, where: - The natural amino acid (cAA) is encoded by any one of 61 naturally occurring codons, or is a random amino acid encoded by a degenerate codon selected from NNN, NNK, NNS, NDT, DBK, NRT, NNC, NWW, RVK; DVT, NVT, VVC, RST, NTT, TDK, provided that at least one random amino acid is present in the sequence of the target macrocyclic peptide. Preferably, 20% to 50% of the amino acids in the sequence are random, more preferably, >50% of the amino acids are random. - Non-natural amino acids (NcAA) are encoded by TAG, TAA, or TGA stop codons present in the coding sequence of the target peptide in the expression vector; (iii) [FL] represents a spacer sequence containing 1 to 25 amino acids, preferably selected from the following sequences: (GGGGS) n , where n ranges from 1 to 5; (G) n , where n is an integer from 1 to 25; (S) n , where n is an integer from 1 to 25; (P) n , where n is an integer from 1 to 25; (EAAAK) n Where n is an integer from 1 to 5; (A(EAAAK)4ALEA(EAAAK)4A) n , where n is an integer from 1 to 2; (XP) n , where n is an integer from 1 to 12, and "X" can be any natural amino acid, preferably alanine (A), lysine (K) and glutamic acid (E); (iv) [IFT] represents a sequence suitable for detecting and / or isolating yeast cells expressing MP-CSP or CSP-MP fusions on their cell surface; (v) [CSP] represents a protein expressed on the surface of yeast cells, which may be fused to the N-terminus or C-terminus of a macrocyclic peptide [MP] sequence and contains: -At least one extracellular portion; - Transmembrane portion; or portion bound to the membrane and / or wall, which is covalently or non-covalently anchored to the molecules of the membrane and / or wall.

2. The yeast cell according to claim 1, wherein the [CSP] is an Aga1 protein; an Aga2 protein; a member of the glycosylphosphatidylinositol (GPI) binding protein family, preferably Cwp1p, Cwp2p, Tip1p, Flop1p, Sed1p, YCR89, Tir1p; or a synthetic, non-naturally occurring protein that can be stably anchored to the yeast membrane and / or wall through covalent or non-covalent interactions.

3. The yeast cell according to claim 1, wherein the [IFT] is selected from... c-myc-tag (SEQ ID NO:1) HA-tag (SEQ ID NO:2), Flag-tag (SEQ ID NO:3) His-tag (SEQ ID NO: 4), Strep-tag (SEQ ID NO:5) TC-tag (SEQ ID NO:6) S-tag (SEQ ID NO:7) AviTag (SEQ ID NO:8) Strep-tag2 (SEQ ID NO:9).

4. The yeast cell according to claim 1, further comprising one or more vectors encoding an orthogonal translation system (OTS), said OTS being composed of aminoacyl-tRNA synthetase (aaRS) and corresponding transfer RNA (tRNA), capable of inserting non-natural amino acids at the TAG, TAA, or TGA stop codons present in the expression vector encoding the fusion protein (I).

5. An expression vector encoding a fusion protein (I) as defined in claims 1-4.

6. The expression vector according to claim 5, comprising a secretion signal located at the 5' position relative to the fusion protein (I).

7. The expression vector according to claims 5-6, further comprising an inducible promoter functionally linked to the fusion protein (I).

8. A cell population comprising a plurality of yeast cells, wherein each cell expresses a plurality of copies of a unique target macrocyclic peptide sequence as defined in claims 1-4 on its surface, the amino acid sequence of said macrocyclic peptide sequence being variable within the cell population.

9. The cell population of claim 8, wherein the sequence diversity of the target cysteine-enriched macrocyclic peptide is at least 10. 7 Preferred >10 8 More preferably >10 9 .

10. A macrocyclic peptide library comprising a yeast cell population according to claims 8 and 9, wherein different cells express different target macrocyclic peptides, preferably expressing different macrocyclic peptides for each cell, wherein the target macrocyclic peptides are selected from: (X) n C(X) m C(X) t (X) n C(X) m C(X) t C(X) y (X) n C(X) m C(X) t C(X) j C(X) y Among them, X, C, n, m, t, y, j As defined in claim 1.

11. The use of the macrocyclic peptide library according to claim 10 in screening bioactive macrocyclic peptide ligands.

Images