A method for directed evolution of nanobodies based on intracellular mutagenesis and surface display of escherichia coli

Abstract

The application discloses a kind of nanobody directional evolution methods based on Escherichia coli intracellular mutagenesis and surface display, belong to antibody engineering technical field.The application adopts double plasmid system, pMut Mutagenesis plasmid realizes the mutation of nanobody on pDsiplay display plasmid by base editor;The double plasmid is co-transformed into escherichia coli to obtain engineering bacteria, and the expression of base editor is induced to realize nanobody mutation, to construct mutation library, and nanobody is displayed on bacterial surface;Mutated nanobody is captured by affinity using magnetic bead fixed antigen, and bacteria carrying high affinity antibody are enriched by washing, elution recovery;The positive bacteria obtained are subjected to multiple rounds of iterative mutation-magnetic bead enrichment screening, and finally sequencing, structure simulation and binding activity verification are carried out, and nanobody with improved affinity is screened.The application relies on intracellular in situ mutation to eliminate the cumbersome steps of in vitro library construction, combines bacterial whole-cell surface display and solid-phase antigen magnetic bead screening, the whole process is simple to operate, the screening cycle is short, the mutation site is gradually enriched in the key region of antigen binding with iteration, without expensive large equipment, greatly reduces the cost of nanobody affinity maturation, and can be widely used for in vitro diagnostic test paper, biosensing, high-affinity nanobody preparation for targeted reagent.

Description

Technical Field

[0001] To address the problems of cumbersome operation, long cycle, low mutation efficiency, and insufficient screening specificity in existing nanobody directed evolution technology, this invention provides a nanobody directed evolution method based on intracellular mutagenesis and surface display in Escherichia coli, which enables rapid, efficient, and low-cost screening of high-affinity nanobodies. Background Technology

[0002] Nanobodies (such as alpaca antibody VHH and shark antibody VNAR) or antibody analogs (such as affibody and monobody) have advantages such as small molecular weight, high stability, good water solubility, and ease of genetic engineering modification, and have wide applications in immunoassay, bioimaging, targeted therapy, and test strip development. High affinity is the core indicator for nanobodies to achieve high-sensitivity recognition, and natural nanobodies often need to achieve affinity maturation through directed evolution.

[0003] Existing methods for directed evolution of nanobodies mainly include error-prone PCR in vitro mutagenesis, phage display, and yeast display. In vitro mutagenesis requires cumbersome library construction and transformation, resulting in limited library capacity; phage display requires auxiliary phage infection, leading to a long operation cycle; yeast display has low transformation efficiency and slow expression induction, making it difficult to achieve high-throughput rapid screening. Furthermore, these methods generally cannot achieve an in-situ closed loop of "mutation-expression-display-screening," resulting in low mutation efficiency, long screening cycles, and high costs, making it difficult to meet the demand for rapidly obtaining high-affinity nanobodies.

[0004] E. coli surface display systems offer advantages such as high transformation efficiency, rapid culture, and simple induction, allowing target proteins to be displayed on the bacterial surface for direct whole-cell screening. If in situ continuous mutagenesis of nanobody genes can be achieved within E. coli cells and combined with surface display and antigen enrichment using magnetic beads, the directed evolution process can be significantly simplified, improving mutation efficiency and screening specificity. However, currently, there is no mature technology that combines intracellular deaminase-mediated mutagenesis, bacterial surface display, and iterative enrichment using magnetic beads for the directed evolution of nanobodies. Summary of the Invention

[0005] The purpose of this invention is to provide a method for the directed evolution of nanobodies based on the combined use of intracellular mutagenesis and surface display in *E. coli*, addressing the problems of cumbersome in vitro mutagenesis operations, complex library construction, low mutagenesis efficiency, and high cost. It also overcomes the shortcomings of phage display and yeast display technologies, such as long cycles, low transformation efficiency, insufficient screening throughput, and difficulty in achieving in situ continuous evolution. By achieving in situ deaminase-mediated mutagenesis of nanobodies within *E. coli* cells and organically coupling it with bacterial surface display and antigen magnetic bead enrichment screening, an integrated closed-loop screening system of "in vivo mutagenesis—surface display—directed enrichment—iterative evolution" is constructed. This enables the rapid preparation of high-affinity nanobodies without in vitro library construction, complex purification steps, or large-scale equipment. Furthermore, multiple rounds of iterative screening are used to improve the effective mutation ratio, enhance the binding specificity and affinity of nanobodies to antigens, and obtain high-affinity nanobodies with higher stability and stronger recognition capabilities.

[0006] The method of this invention is simple to operate, has a short screening cycle, strong reproducibility, and wide applicability. It can significantly improve the efficiency of directed evolution of nanobodies and reduce experimental costs. It provides an efficient, low-cost, and easy-to-promote high-affinity nanobody preparation technology for fields such as immunoassay, biosensing, rapid diagnostic test strip development, and targeted molecule screening. It has important scientific research value and practical application prospects.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution: (1) Constructing display plasmids Based on the nanobody gene sequence and the E. coli surface display vector backbone, a specific display plasmid was designed and constructed. This plasmid carries the nanobody encoding gene, E. coli surface display elements, resistance genes, and low-copy replicons such as pSC101, enabling the targeted expression and display of nanobody proteins on the E. coli cell membrane surface while ensuring the spatial structural integrity of the nanobody antigen recognition domain, providing a basic expression vector for subsequent mutant screening. After plasmid construction, sequence accuracy was verified by sequencing to ensure the vector is mutation-free and its elements are intact, ready for subsequent experimental operations.

[0008] (2) Construction of mutagenic plasmids A base-editing mutagenesis plasmid capable of intracellular mutagenesis in *E. coli* was constructed. The plasmid carries an MmP1 phage RNA polymerase fusion base editor, requiring no in vitro mutagenesis and specifically mediating random gene base mutations within the bacterial cell. After plasmid construction, restriction enzyme digestion and sequencing verification were performed to ensure the integrity of functional elements, the correctness of regulatory sequences, and its ability to induce site-specific random mutagenesis within the cell.

[0009] (3) Construction of a mutant library by inducing the expression of a base editor The constructed nanobody display plasmid and intracellular mutagenesis plasmid were co-transformed into competent *E. coli* cells to obtain engineered strains carrying both plasmids. Positive transformants were screened for resistance to ensure that the engineered bacteria carried both the complete display and mutagenesis plasmids. The selected dual-plasmid engineered bacteria were inoculated into liquid culture medium, and a specific inducer was added to the culture system to precisely activate the expression and mutagenesis function of the intracellular base editor, resulting in efficient and random base mutations in the *E. coli* nanobody gene. This end-to-end intracellular mutagenesis approach avoids the cumbersome steps of traditional in vitro PCR mutagenesis, enzyme digestion, ligation, and in vitro library construction, directly completing nanobody gene mutations within *E. coli*, ultimately constructing a diverse and rich in vivo mutant library of nanobodies.

[0010] (4) Preparation of antigen-immobilized magnetic beads Functionalized antigen magnetic beads were prepared by coupling streptavidin magnetic beads with pre-prepared and purified biotinylated antigen through incubation. First, the streptavidin magnetic beads were thoroughly mixed, and an appropriate amount of beads was washed to remove impurities from the bead preservation solution. Then, a measured amount of biotinylated antigen was added, and the mixture was incubated in a constant-temperature shaker in the dark. Utilizing the specific and efficient binding properties of streptavidin and biotin, the antigen was stably immobilized on the surface of the magnetic beads. After incubation, the magnetic beads were washed using magnetic field separation technology to remove unbound free antigen and impurities, ultimately obtaining functionalized magnetic beads with intact active antigen immobilized on their surface. These beads were then stored at low temperature for subsequent affinity screening experiments.

[0011] (5) Mutation display surface screening A pre-constructed E. coli in vivo nanobody mutant library was induced and cultured, and a cell surface display system was initiated to express and stably display nanobody mutants with different base mutations on the surface of E. coli cells. The E. coli cell system displaying the mutant nanobodies was then thoroughly mixed and incubated with a prepared antigen-fixed magnetic bead system. Utilizing the specific immune recognition between the nanobodies and their corresponding antigens, the effective mutants on the cell surface precisely bound to the antigens on the magnetic beads, achieving preliminary specific capture of positive mutant cells and completing the surface-targeted screening process for mutants.

[0012] (6) Enrichment After incubation, the mixed system was screened and enriched using a magnetic field separation system. Antigen beads bound to positive bacteria were immobilized by magnetic adsorption, while unbound or weakly bound negative E. coli cells and impurities remained free in the supernatant. The negative bacteria were removed by discarding the supernatant. After thorough washing, the specifically bound positive mutant bacteria were recovered from the magnetic bead surface, achieving single-round affinity enrichment of high-affinity nanobody mutants and significantly increasing the proportion of positive mutants in the system.

[0013] (7) Continuous screening Positive bacterial clones recovered from a single round of enrichment were inoculated into fresh liquid culture medium for resuscitation and amplification, serving as the starting material for the next round of screening. A complete experimental procedure was employed, involving cyclical repetition of intracellular base editing mutagenesis, surface display of nanobody mutants, specific binding of antigen magnetic beads, magnetic field separation, and elution and enrichment of positive bacterial cells, to conduct multiple rounds of iterative evolutionary screening. Through continuous rounds of screening and enrichment, mutants with low affinity and no binding activity were continuously eliminated, while high-affinity and high-specificity nanobody mutant strains were gradually enriched, continuously improving the abundance and purity of the target positive mutants, thus achieving in vitro directed evolution of nanobodies.

[0014] (6) Mutant identification and binding activity verification Single-colony positive clones obtained after multiple rounds of continuous screening were selected. The nanobody gene sequences were analyzed using gene sequencing technology to precisely locate mutated base sites and identify specific mutation types and numbers. Using bioinformatics structural simulation software, the spatial conformations of wild-type and mutant nanobodies were compared and analyzed to explore the impact of base mutations on the secondary and tertiary structures of antibody proteins, clarifying the structural mechanism by which mutations enhance antigen-binding ability. Simultaneously, ELISA (enzyme-linked immunosorbent assay) was used to quantitatively detect the binding titer and affinity of mutant nanobodies to target antigens. The binding activity data were compared with those of wild-type antibodies. Finally, positive nanobody mutants with significantly enhanced affinity and good specificity were screened and validated, completing the entire mutation screening and activity validation process. Detailed Implementation

[0015] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0016] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0017] Gene synthesis, vector construction Qingke Biotechnology Co., Ltd. Chloramphenicol Sangon Biotech (Shanghai) Co., Ltd. Kanamycin Sangon Biotech (Shanghai) Co., Ltd. E. coli competent cells Shanghai Vidi Biotechnology Co., Ltd. Streptavidin (SA) magnetic beads Sangon Biotech (Shanghai) Co., Ltd. PCR primers Qingke Biotechnology Co., Ltd. PCR Mix enzyme Background: Bestjet Biotechnology Co., Ltd. LB liquid medium, LB solid medium, agar powder Sangon Biotech (Shanghai) Co., Ltd. IPTG Sangon Biotech (Shanghai) Co., Ltd. PBS buffer Thermo Fisher Scientific Biotin Sangon Biotech (Shanghai) Co., Ltd. The instruments used in the specific embodiments are as follows: Clean bench, water bath, constant temperature incubator, constant temperature shaker, low temperature high speed centrifuge, developer, constant temperature water bath, ELISA reader, PCR gene amplification instrument, agarose gel electrophoresis instrument, gel imaging system, magnetic rack, autoclave. Attached Figure Description

[0018] Figure 1 The flowchart shows a method for directed evolution of nanobodies based on intracellular mutagenesis and surface display in Escherichia coli. Figure 2The pDisplay and pMut dual plasmid maps are shown.

[0019] Figure 3 Figure 1 shows the results of PCR amplification and agarose gel electrophoresis identification of characteristic fragments of two plasmids; Note: a) Amplification of characteristic fragments of pMut plasmid, product size approximately 745 bp; b) Amplification of characteristic fragments of pDisplay plasmid, product size approximately 400 bp.

[0020] Figure 4 Figure 1 shows the results of Western Blot detection and gray-scale quantitative analysis of eEGP fusion protein expression at different induction temperatures; Note: a) Western Blot results of whole bacteria binding to eEGFP at different induction temperatures, M is the protein marker, lanes 1-3 are the induction groups at 30℃, 37℃ and 42℃ respectively; b) Quantitative analysis of the gray-scale values ​​of specific bands; all samples were adjusted to the same total protein concentration using the BCA method to eliminate the influence of differences in sample loading.

[0021] Figure 5 This is a distribution map of mutation sites in an in vivo mutation library of antibody genes.

[0022] Figure 6 Figure 1 shows the PCR amplification and agarose gel electrophoresis verification results of the characteristic fragments of the two plasmids after the construction of the mutant library; Note: a) Amplification of the characteristic fragment of pMut plasmid, product size approximately 745 bp; b) Amplification of the characteristic fragment of pDisplay plasmid, product size approximately 400 bp. Example

[0023] 1. Constructing a carrier Plasmid synthesized by a bioengineering company: pDisplay displays the plasmid vector. The plasmid is shown below. Figure 2 As shown in figure a, the nanoparticle carrying the Lpp-OmpT membrane anchoring domain and the encoding sequence of the anti-eGFP nanobody uses pSC101 as the replicon and chloramphenicol as the resistance marker; the pMut mutant plasmid is shown in figure a. Figure 2 As shown in b, the sequences carrying TadA-8e 8e, PmCDA1-UGI ​​base editing-related sequences and the Mmp1 element, with ColE1 as the replicon and kanamycin as the resistance marker, are used.

[0024] 2. Transformation of two plasmids into competent states pDisplay plasmid and mutagenic plasmid were transformed into E. coli XL1-Blue competent cells, plated on LB agar plates containing 25 μg / mL chloramphenicol and 50 μg / mL kanamycin, and incubated overnight at 37°C.

[0025] 3. Colony PCR Identification Six single colonies were randomly selected and inoculated onto double-antibiotic LB liquid medium. After incubation, PCR identification was performed. The results showed... Figure 3 All colonies amplified approximately 745 bp of the pMut plasmid and approximately 400 bp of the pDisplay plasmid. The bands were single and consistent with the expected size, confirming successful co-transformation of the two plasmids and obtaining a dual-plasmid engineered strain that can be used for subsequent mutation and screening.

[0026] 1. Optimization of induction temperature The engineered bacteria with dual plasmids were inoculated into double-antibody LB liquid medium and cultured at 37°C until OD600≈0.6. Nanobody expression was then induced at 30°C, 37°C, and 42°C, respectively.

[0027] 2. Whole-cell antigen binding test After induction, bacterial cells were collected, washed with PBS and resuspended. The total protein concentration of the whole bacteria was determined using the BCA method, and a uniform loading volume was applied. eGFP antigen was added for incubation, and washing was performed to remove unbound antigen. Western blotting was performed using anti-eGFP monoclonal antibody as the primary antibody and HRP-labeled goat anti-mouse antibody as the secondary antibody. Results showed ( Figure 4 Specific bands appeared at 35-40 kDa, and the signal strength of different groups was observed to obtain the optimal conditions.

[0028] The engineered bacteria with dual plasmids were inoculated into double-antibody LB medium to induce nanobody display. An inducer was added to induce deaminase system expression, and the nanobody encoding gene was targeted and mutated. Continuous passage and mutant library construction: After multiple rounds of continuous passage and induction, the nanobody gene was amplified by PCR and high-throughput sequencing was performed. Sequencing results are shown below. Figure 5 The mutations were mainly C→T conversions, and the mutation sites were evenly distributed in the FR1, FR2, FR3, CDR1, and CDR2 regions. CDR1 and CDR2 showed obvious mutation hotspots, which are consistent with the characteristics of deaminase activity. An in vivo mutant library was successfully constructed.

[0029] 1. Preparation of antigen magnetic beads Streptavidin magnetic beads were incubated with biotinylated eGFP antigen to prepare antigen-immobilized functional magnetic beads, while empty magnetic beads conjugated only with biotin were set up as a control.

[0030] Whole-cell incubation and enrichment: The induced mutant library of bacteria was mixed with antigen magnetic beads and incubated to allow the bacteria displaying functional nanobodies on their surface to specifically bind to the magnetic beads.

[0031] 2. Washing and elution Magnetic field separation was performed, followed by rigorous washing to remove non-specifically bound bacteria. Positive clones bound to the magnetic beads were eluted and plated onto double-antibody LB agar plates. Six positive single colonies were obtained from the experimental group plates, while no colonies grew in the control group, indicating that the screening system has high specificity and extremely low background.

[0032] 3. Colony PCR Verification Randomly select single colonies for PCR identification, such as... Figure 6 As shown, the strain retained positive clones with stable coexistence of the two plasmids and carrying complete nanobody genes for subsequent iterations.

[0033] Using the positive clones obtained in the first round of screening as the starting bacteria, the in vivo mutation steps of Example 3 and the magnetic bead enrichment steps of Example 4 were repeated to complete three rounds of "in vivo mutation-magnetic bead enrichment" cyclic evolution. Single clones after each round of screening were sequenced, and the number of mutation sites and the proportion of mutations in the CDR region were statistically analyzed. The average number of mutation sites per single clone increased from 11 in the initial mutation library to 17 in the third round; the proportion of mutations in the CDR region increased from 27.27% to 35.29%. Mutations accumulated towards key antigen-binding regions under screening pressure, and the screening system effectively accumulated functional mutations.

[0034] 1. Three-dimensional structure prediction AlphaFold2 was used to predict the structure of the original nanobody and the mutant obtained through iteration. All models had pTM ≥ 0.87, ranking score ≥ 0.89, global RMSD < 0.2, and the skeletal structure remained stable.

[0035] 2. Molecular docking analysis Molecular docking of the mutant with eGFP was performed using HDOCK. The results showed that the docking score of the original antibody was -284.21, while the docking score of the preferred mutant was -293.21.

[0036] 3. Interaction analysis: Hydrogen bond interaction analysis was performed using PyMOL. The results showed that the original antibody formed 13 hydrogen bonds, while the mutant formed 18 hydrogen bonds. The mutation greatly optimized the binding interface between the antibody and the antigen.

Claims

1. A method for directed evolution of nanobodies based on intracellular mutagenesis and surface display in *Escherichia coli*, characterized in that: A display plasmid carrying a nanobody gene and a mutagenic plasmid carrying a base editor gene were constructed; the display plasmid and the mutagenic plasmid were co-transformed into Escherichia coli to obtain engineered bacteria; After induction, the nanobody gene undergoes random mutations within the cell, and the mutant nanobody is displayed on the bacterial surface. E. coli displaying mutant nanobody on the bacterial surface is co-incubated with pre-fixed antigen, and positive cells that specifically bind are enriched by washing and separation. The enriched positive cells are then subjected to multiple rounds of iterative induction mutation and enrichment screening. The mutant strains obtained from the final screening were sequenced, and the binding activity of the displayed antibodies was verified to obtain nanobodies with enhanced affinity.

2. The method according to claim 1, characterized in that: The proteins that display the nanobody are Lpp-OmpT (SEQ1) or Lpp-OmpA (SEQ2).

3. The method according to claim 1, characterized in that: The mutant enzyme carried by the plasmid is a dideminase such as TadA-8e (SEQ3) / PmCDA1 (SEQ4).

4. The method according to claim 1, characterized in that: The directed mutation of nanobodies depends on the MmP1 promoter (SEQ5), which encodes the protein LAVFIILENDV. The promoter is located between the nanobody and the display protein Lpp-OmpT or Lpp-OmpA via the amber codon UAT, or at the 3' end of the nanobody gene. A matching MmP1 phage RNA polymerase (SEQ6) is used to fuse a base editor to introduce mutations bidirectionally or unidirectionally during transcription.

5. The method according to claim 1, characterized in that: After screening, the mutant engineered bacteria can be directly cultured to complete multiple rounds of continuous cyclic screening.

6. The method according to claim 1, characterized in that: Nanobodies are small-molecule antibodies or antigen analogs that can specifically bind to other proteins.

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