Molecular chaperone for improving effective display efficiency of Fab on yeast surface and application of molecular chaperone

Abstract

The invention relates to the technical field of antibody engineering, in particular to a molecular chaperone PDI for improving the effective display efficiency of Fab on the surface of yeast and application of the molecular chaperone PDI, and the molecular chaperone PDI comprises an amino acid sequence as shown in SEQ ID NO: 1. The effective display efficiency and stability of Fab can be remarkably improved through co-expression of the molecular chaperone and Fab, Fab fragments with high affinity or specificity can be efficiently screened out through flow cytometry (FACS), and an effective strategy is provided for accelerating discovery of high-performance antibodies.

Description

Technical Field

[0001] This invention relates to the field of antibody engineering technology, specifically to a molecular chaperone P for improving the Fab display efficiency on yeast surfaces and its applications. Background Technology

[0002] Fab yeast display is a technique that displays antigen-binding fragments (Fab) of antibodies on the surface of yeast cells, combining the advantages of yeast display and antibody engineering. Specifically, it involves fusing antibody molecules with yeast membrane surface molecules for expression, displaying the antibody molecules on the yeast membrane surface for rapid screening. The yeast display system based on Saccharomyces cerevisiae Aga1-Aga2 is the most popular system. This method is based on the expression of exogenous proteins fused to the Aga2 protein. The Aga2 protein itself is anchored to the Aga1 protein on the cell membrane via two disulfide bonds, forming a covalent complex on the yeast surface. Exogenous proteins and peptides can be fused to the N-terminus or C-terminus of Aga2. Using this system, more than 3 × 10⁻⁶ antibodies can be displayed on the surface of a single yeast cell. 4 A heterologous protein molecule. EBY100, an engineered strain of *Saccharomyces cerevisiae*, can be used in the Aga1-Aga2 yeast display system. However, existing technologies for yeast cell surface Fab display have significant shortcomings: the effective assembly and folding of the VH-CH1 domain of the heavy chain and the light chain in the Fab molecular structure are usually limited, resulting in low effective display efficiency of Fab, which cannot be used for subsequent screening and thus cannot follow the technical path of screening for advantageous antibody molecules.

[0003] Molecular chaperones primarily function during protein expression to assist in the correct folding of nascent peptide chains, maintain protein stability, prevent aggregation, and participate in the repair or degradation of misfolded proteins. In mammalian cells, PDIs (protein disulfide isomerases) ensure the correct folding and disulfide bond formation of secreted proteins, especially monoclonal antibodies. Molecular chaperones are also a preferred and crucial tool for optimizing the expression of multi-structured proteins or membrane proteins. Summary of the Invention

[0004] To address the above-mentioned technical problems, the first aspect of the present invention provides a molecular chaperone PDI for improving the effective display efficiency of Fab, wherein the molecular chaperone PDI comprises an amino acid sequence as shown in SEQ ID NO: 1.

[0005] A second aspect of the present invention provides a recombinant vector comprising a nucleotide sequence encoding the molecular chaperone PDI.

[0006] Preferably, the recombinant vector comprises a GAL1,10 promoter and an S. cerevisiae GAL1 promoter, wherein the GAL1,10 promoter bidirectionally initiates transcription and subsequent translation of the Fab sequence, and the S. cerevisiae GAL1 promoter mediates transcription and subsequent translation of the molecular chaperone PDI.

[0007] Preferably, the recombinant vector further includes one or more expression elements such as enhancers and terminators.

[0008] Preferably, the recombinant vector comprises a nucleotide sequence as shown in SEQ ID No. 2.

[0009] The third aspect of this invention provides an application of the aforementioned molecular chaperone PDI for improving Fab display efficiency, which improves the effective display efficiency of Fab by introducing a strategy of co-expression of the molecular chaperone and its mutants.

[0010] The present invention has the following advantages and beneficial effects: The molecular chaperone co-expression of this invention aims to improve the inadequate efficient assembly and folding of the VH-CH1 and VL-CL domains of Fab within the endoplasmic reticulum, providing an effective approach to enhance the display efficiency of functional Fab fragments on the yeast cell surface. This also offers an optimized strategy for screening advantageous antibody sequences in antibody engineering.

[0011] This invention improves the efficiency of Fab display by introducing a strategy of co-expression of molecular chaperones and their mutants. Attached Figure Description

[0012] Figure 1 The construction process of recombinant plasmid pDEST1-B6H12 in this embodiment of the invention is as follows; Figure 2 The construction process of recombinant plasmid pDEST2-B6H12 in this embodiment of the invention is as follows; Figure 3 The construction process of recombinant plasmid pDEST3-B6H12 in this embodiment of the invention; Figure 4 Flow cytometry was used to detect the differences in display efficiency of B6H12 Fab by different recombinant plasmids; Figure 5 Flow cytometry was used to detect the differences in binding efficiency of different recombinant plasmids to positive cells. Detailed Implementation

[0013] To better understand the present invention, the following embodiments are further illustrations of the present invention, but the content of the present invention is not limited to the following embodiments.

[0014] CD47 is a 42-52 kDa N-linked glycoprotein. The CD47 antigen, also known as integrin-associated protein (IAP), is expressed in all hematopoietic cells, including leukocytes, platelets, and erythrocytes. It is also expressed in epithelial cells, endothelial cells, fibroblasts, and many tumor cell lines. CD47 may play a signal transduction role in regulating cation flux across cell membranes and in the chemotactic and adhesion interactions between leukocytes and endothelial cells. Antibodies against CD47 (B6H12) can block the phagocytosis of microparticles by peripheral blood granulocytes. It has also been reported to induce the proliferation of CD3-activated T cells.

[0015] This invention utilizes the above-mentioned expression approach to perform Fab display efficiency and binding efficiency tests on CD47(B6H12) monoclonal antibody sequences.

[0016] Example 1

[0017] The technical solution of this invention, which utilizes molecular chaperones and their mutants to improve Fab display efficiency, is summarized as follows: A method for improving Fab display efficiency using molecular chaperones, wherein the Fab display expression vector has the following structure: pDEST1-B6H12 (without molecular chaperone elements): pDEST-GAL1--------Myc-CL-VL-GAL10-GAL1-VH-CH1-HA-Aga2p, pDEST2-B6H12 (wild-type PDI): pDEST-GAL1-PDI--------Myc-CL-VL-GAL10-GAL1-VH-CH1-HA-Aga2p, pDEST3-B6H12: pDEST-GAL1-PDI1--------Myc-CL-VL-GAL10-GAL1-VH-CH1-HA-Aga2p, wherein PDI (derived from a publicly available sequence at www.uniprot.org / , numbered C4R938) and PDI1 (SEQ ID No: 1 of this invention).

[0018] Except for B6H12 VH (SEQ ID NO: 3), B6H12 VL (SEQ ID NO: 4) and PDI1, all other elements in the recombinant vectors pDEST1-B6H12, pDEST2-B6H12, and pDEST3-B6H12 are disclosed in the prior art. Synthesizing the vector based on these elements is a conventional technique, and their sequences and synthesis methods will not be described in detail here.

[0019] The sequences of the GAL10-GAL1 promoter are also publicly available. The present invention uses the BioVector plasmid vector strain cell protein antibody gene preservation center, the promoter of the pYD1 vector, the sequence of pDEST1-B6H12 is shown in SEQ ID NO: 5, and the sequence of pDEST2-B6H12 is shown in SEQ ID NO: 6.

[0020] It should be noted that, except for the heavy chain variable region VH and the light chain variable region VL, the sequences of the recombinant vectors pDEST2-B6H12 and pDEST3-B6H12 are all synthesized by genes. The PDI and PDI1 elements are also synthesized by genes.

[0021] Specifically, the first step is to design and construct the basic plasmid framework for Fab expression. The specific approach is as follows: pDEST1-B6H12: pDEST1-GAL1--------Myc-CL-VL-GAL10-GAL1-VH-CH1-HA-Aga2p, as shown Figure 1 As shown, GAL1-GAL10 in the element is a commonly used bidirectional promoter for yeast Fab display. The GAL1-GAL10 promoter expresses the light chain and Myc tag of the Fab antibody, as well as the variable and constant regions of the heavy chain CH1, HA tag, and Aga2p; pDEST2-B6H12: pDEST2-GAL1-PDI--------Myc-CL-VL-GAL10-GAL1-VH-CH1-HA-Aga2p, as shown. Figure 1 As shown, GAL1-GAL10 in the element are bidirectional promoters commonly used in yeast Fab display. The GAL1-GAL10 promoter expresses the light chain and Myc tag of the Fab antibody, as well as the variable and constant regions of the heavy chain CH1, HA tag, and Aga2p. The GAL1 promoter expresses the wild-type molecular chaperone element PDI; pDEST3-B6H12: pDEST3-GAL1-PDI1--------Myc-CL-VL-GAL10-GAL1-VH-CH1-HA-Aga2p, as shown. Figure 1 As shown, GAL1-GAL10 in the element are bidirectional promoters commonly used for yeast Fab display. The GAL1-GAL10 promoter expresses the light chain and Myc tag of the Fab antibody, as well as the variable and constant regions of the heavy chain CH1, HA tag, and Aga2p. The GAL1 promoter expresses the molecular chaperone element PDI1, and PDI1 is a preferred mutant. The antibody heavy chain variable region VH and light chain variable region VL gene fragments of B6H12 were obtained by PCR cloning. PCR reaction system: 10×PCR buffer, 5 μl; dNTP (2.5 mM), 4 μl; primer F (10 μM), 1 μl; primer R (10 μM), 1 μl; Pfu polymerase, 2 μl; template, 1 μl; add ddH2O to 50 μl. PCR amplification system: 95℃, 5 min; 95℃, 30 s, 55℃, 30 s, 72℃, 1 min, 30 cycles; 72℃, 5 min; 4℃, 10 min. Relevant amplification primers are shown in Table 1. The obtained PCR products were recovered using 0.8% agarose gel. The recovery method followed the instructions of the Kangwei Century Gel Extraction Kit Agarose Gel DNA Recovery Kit Cat. No. CW2302. Table 1

[0022] The pDEST1 empty vector was first digested with restriction endonucleases KpnI and PaeI, and then recovered via agarose gel electrophoresis. It was then homologously recombinated with the recovered B6H12-VL product. The product was directly transformed into *E. coli* Top10 cells. After colony PCR verification and sequencing confirmation, an intermediate plasmid containing the light chain was obtained. This intermediate plasmid was then digested with SalI and NheI, recovered via agarose gel electrophoresis, and homologously recombinated with the recovered B6H12-VH product. The enzyme ligation product was again transformed into *E. coli* Top10 cells. After colony PCR verification and sequencing confirmation, the final plasmid pDEST1-B6H12 was obtained. The construction process is detailed in the appendix. Figure 1 The vectors and restriction enzyme sites involved in the construction are shown in Table 2, and the restriction enzyme system is shown in Table 3. Table 2

[0023] Table 3

[0024] 4. Endotoxin-free extraction of pDEST1-B6H12, pDEST2-B6H12, and pDEST3-B6H12 plasmids was performed, following the instructions of Kangwei Century EndoFree Plasmid Midi Kit Cat. No. CW2105S. Plasmid electroporation: The constructed pDEST1-B6H12, pDEST2-B6H12, and pDEST3-B6H12 plasmids were incubated with 400 μL of competent cells of Saccharomyces cerevisiae EBY100 (URA+,leu-,trp-) for 10 min, then transferred to electroporation cuvettes. The electroporation was performed at 2500 V and 0.2 cm, with a time of 3.0–4.5 ms. The cells were incubated at 30 °C and 180 rpm for 1 hour, and then plated on SD-Trp agar plates. Culture and induction: The above monoclonal yeast transformants were inoculated into SD-Trp (formula reference: A Streamlined Approach for the Construction of Large Yeast Surface Display Fab Antibody Libraries). Incubate in liquid medium at 30°C and 180 rpm until OD 600 The yeast was centrifuged at 3000g and the supernatant was discarded. The yeast was then transferred to SG-Trp liquid medium (formula referenced in A Streamlined Approach for the Construction of Large Yeast Surface Display Fab Antibody Libraries) and cultured at 20℃ and 180 rpm for 48 h to induce expression of the antibody Fab fragment. Two copies of each of the pDEST1-B6H12, pDEST2-B6H12, and pDEST3-B6H12 expression samples were taken: one copy was used for Fab display efficiency detection of B6H12, and the other copy was used for activity detection.

[0025] Yeast display efficiency testing procedure: Sample preparation: Take 1×10⁻⁶ samples after induction. 8 50 μL of yeast was centrifuged to remove the supernatant, washed twice with PBS, and blocked with 100 μL of 1×PBS-5% BSA at room temperature for 30 min. After blocking, the sample was centrifuged, washed twice with PBS, and the antibody was diluted 1:500. 100 μL of Elab Fluor® 647-conjugated Myc-Tag Monoclonal Antibody (catalog number: AN00300M) diluted with 1×PBS-5% BSA was added, and the sample was incubated at 4 °C for 30 min. After washing three times with pre-chilled PBS, the sample was resuspended in 1 ml of pre-chilled PBS and placed on ice. After centrifugation, wash twice with PBS, resuspend yeast cells in 200 μL PBS, and analyze by flow cytometry. The fluorescence intensity of yeast cells was detected by flow cytometry using a 488nm (Elab Fluor® 647) laser excitation. Yeast binding efficiency testing procedure: Test sample: Take 1×10 after induction 850 μL of yeast was centrifuged to remove the supernatant, washed twice with PBS, and blocked with 100 μL of 5% 0.1% BSA-1×PBS at room temperature for 30 min. After blocking, the sample was centrifuged, washed twice with PBS, and the antibody was diluted 1:500. 100 μL of Elab Fluor® 647-conjugated Myc-Tag Monoclonal Antibody (catalog number: AN00300M) diluted with 1×PBS-5% BSA was added, and the sample was incubated at 4 °C for 30 min. After washing three times with pre-chilled PBS, the sample was resuspended in 1 ml of pre-chilled PBS and placed on ice for later use. Preparation of positive cells: After centrifuging the cultured Jurkat, Clone E6-1 (human T-lymphocytic leukemia cells) (catalog number CL-0129), wash once with 1×PBS, and then resuspend the cells in 1×PBS to a density of 1x10⁻⁶ cells / mL. 7 / mL; Labeled stock solution: Dissolve 1 mg CFDA-SE in 0.1794 mL DMSO to obtain 10 mM CFDA-SE (fluorescence signal can be detected under excitation light at 488 nm); Labeling working solution: Dilute the labeling stock solution with 1×PBS solution at a volume ratio of 1:1000; Cell labeling: Centrifuge at 1000 g for 3-5 minutes at 4°C, discard the supernatant, wash twice with PBS for 5 minutes each time, add 1 mL of CFDA-SE working solution, incubate at room temperature for 30 minutes, centrifuge at 400 g for 3-4 minutes at 4°C, wash twice with PBS for 5 minutes each time, and finally resuspend the cells with 1×PBS-5%BSA. Take 5x10 5 The labeled cells were mixed with the sample to be tested and inverted at 4°C for 1 hour before fluorescence signal detection. like Figure 4As shown, flow cytometry results indicate that the pDEST1-B6H12, pDEST2-B6H12, and pDEST3-B6H12 plasmid systems can all enable Fab... The B6H12 antibody was displayed in a yeast display system. In the diagram, P1 in "ad" refers to the yeast cell gate (debris removal). "a" is the negative control, showing that the Myc-647 antibody does not specifically bind to yeast cells. "b" represents yeast transformed with the pDEST1-B6H12 plasmid, with a Myc tag positivity rate of 57.14%, indicating successful induced expression of the Fab fragment. "c" represents yeast transformed with the pDEST2-B6H12 plasmid, with a Myc tag positivity rate of 45.65%, indicating successful induced expression of the Fab fragment. "d" represents yeast transformed with the pDEST3-B6H12 plasmid, with a Myc tag positivity rate of 33.12%, indicating successful induced expression of the Fab fragment. The display efficiency corresponding to the Myc-647 tag signal, from highest to lowest, is: pDEST1-B6H12 > pDEST2-B6H12 > pDEST3-B6H12. The introduction of the molecular chaperone reduced the display efficiency to some extent. like Figure 5As shown, the flow cytometry results indicate that: a) excludes non-specific binding between yeast and Jurkat cells; b) shows the binding of yeast transformed with pDEST1-B6H12 plasmid to fluorescently labeled Jurkat cells, with double-positive cells (Myc tag and fluorescently labeled cells) accounting for 1.26% of the total cells; c) shows the binding of yeast transformed with pDEST1-B6H12 plasmid to fluorescently labeled Jurkat cells, with double-positive cells accounting for 16.25% of the total Jurkat cells. This indicates that the Fab displayed on the yeast surface can bind to Jurkat cells, but the proportion is small, suggesting that the pDEST1-B6H12 plasmid system... The Fab fragments displayed on the yeast surface lacked proper folding; d shows the binding of yeast cells in fluorescently labeled Jurkat cells after induction with pDEST2-B6H12 plasmid, with Myc-labeled and fluorescently labeled cells accounting for 2.93% of the total cells; e shows the binding of yeast cells in fluorescently labeled Jurkat cells after induction with pDEST2-B6H12 plasmid, with double-positive cells accounting for 36.25% of the total Jurkat cells. This indicates that although the pDEST2-B6H12 plasmid system has a lower display efficiency for Fab fragments on the yeast surface compared to pDEST1-B6H12, it is still comparable to J... The significantly increased binding rate of Jurkat cells indicates an improved probability of correct Fab folding and effective display rate, which is attributed to the introduction of wild-type PDI chaperone elements into the pDEST2-B6H12 plasmid system. f shows the binding of yeast cells infused with pDEST3-B6H12 plasmid to fluorescently labeled Jurkat cells; the proportion of Myc-tagged and fluorescently labeled cells was 8.15% of the total cells. g shows the binding of yeast cells infused with pDEST3-B6H12 plasmid to fluorescently labeled Jurkat cells; the proportion of dual-positive cells (Myc tag and fluorescently labeled cells) was 63.72% of the total Jurkat cells, indicating that the mutation... PDI improves the normal folding of Fab on the yeast surface to the greatest extent; P2 in figures c, e, and g refers to the fluorescently labeled Jurkat cell gate: removing cell debris; the above shows that the three sets of yeast display plasmids pDEST1-B6H12, pDEST2-B6H12, and pDEST3-B6H12 can all display Fab-type antibodies and can all bind to positive cells, but there are great differences in the effective display rate of the three groups of yeast cells. Among them, the cell binding efficiency (effective display process) is pDEST3-B6H12 > pDEST2-B6H12 > pDEST1-B6H12. This demonstrates that the molecular chaperone PDI mutant of the present invention can efficiently display functional Fab on the surface of yeast. The binding efficiency of the molecular chaperone PDI mutant of the present invention is better than that of the selected wild-type molecular chaperone and better than the scheme without molecular chaperone. Moreover, there are few literature reports on the binding efficiency of yeast display Fab to cells, and the limited data are all lower than those of the present invention.

[0026] This invention adopts a two-step strategy. The first step is to select molecular chaperone elements and synthesize genes targeting the dominant mutants of the active site. The second step is to select the antibody sequence to be studied in the design scheme obtained in the previous step and perform Fab fragment display and binding efficiency verification of the B6H12 antibody.

[0027] The above description is merely a preferred embodiment of the present invention, and should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A molecular chaperone PDI for improving the effective display efficiency of Fab on yeast surfaces, characterized in that: The molecular chaperone PDI contains the amino acid sequence shown in SEQ ID NO:

1.

2. A recombinant vector, characterized in that: It contains a nucleotide sequence encoding the molecular chaperone PDI of claim 1.

3. The recombinant vector according to claim 2, characterized in that: The recombinant vector contains a GAL1,10 promoter and an S. cerevisiae GAL1 promoter. The GAL1,10 promoter bidirectionally initiates transcription and subsequent translation of the Fab sequence, while the S. cerevisiae GAL1 promoter mediates transcription and subsequent translation of the molecular chaperone PDI.

4. The recombinant vector as described in claim 2, characterized in that: The recombinant vector also includes one or more expression elements such as enhancers and terminators.

5. The recombinant vector according to claim 2, characterized in that: It contains the nucleotide sequence shown in SEQ ID No. 2.

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