1. What is the yeast display platform?

Yeast display (or yeast surface display) is a powerful protein engineering technology that uses the expression of recombinant proteins integrated into the yeast cell wall to isolate and engineer antibodies. Since its first publication in 2000, yeast surface display has been widely used to design various proteins to improve affinity, specificity, expression, stability and catalytic activity.

2. Yeast display principle

Yeast antibody display technology (Yeast Display Technology) refers to the expression of the variable region of the antibody sequence fused with the lectin Aga2p. The Aga2p protein subunit binds to the Aga1p protein subunit fixed on the yeast cell wall through two disulfide bonds. Combined with flow sorting technology, the specific antibodies targeting the antigen are screened out.

Figure 1: Yeast library display diagram

3. Nanobody

Nanobody (Nbs) is a single-chain antibody produced by the peripheral serum of camelids. These antibodies are composed only of heavy chains and lack light chains and functional CH1 and CH4 domains. The VHH crystal is 2.5nm, 4nm long, and has a molecular weight of only 15KDa. Compared with traditional antibodies, nanobodies are easier to penetrate the blood-brain barrier due to their small molecular weight; they have high expression levels in prokaryotic or eukaryotic systems; they have strong specificity and high affinity; they have weak immunogenicity to humans, etc., so they have great value in the fields of anti-tumor drug development, cardiovascular and cerebrovascular diseases, and immune diseases.

Figure 2: 3D structure of nanoantibodies

4. Advantages and disadvantages of nanoantibody yeast display

4.1 Advantages

(1) As a eukaryotic expression system, yeast can be post-translationally modified to express and fold complex eukaryotic proteins;

(2) Compared with phage display library technology, yeast display technology has higher antibody activity after trace antibody expression. Based on this, the affinity of antibodies can be distinguished during flow screening;

(3) Insertion of heterologous proteins will not destroy the structure of surface proteins, nor will it affect the efficiency of surface display.

4.2 Disadvantages

(1) Compared with phage display technology, yeast display library capacity is relatively low, so it is mainly used in antibody discovery projects where customers do not have high requirements for project library capacity, PBMC cells are sorted after secondary sorting after animal immunization, and disulfide bond formation is avoided;

(2) Due to the presence of multiple copies of proteins on the yeast surface, undesirable multivalent binding with oligomeric protein targets may occur.

5. Application of yeast antibody display

With the continuous improvement of this technology, the application of yeast display now includes enzyme engineering; protein epitope mapping, identification of protein-protein interactions, immunobiocatalysis and biosensors, and display of proteins and enzymes on yeast cells for biotechnology and biomedical applications.

6. Techbio can provide yeast antibody display technology services

Techbio can provide customers with high-quality nanoantibody yeast display services. The main service processes are as follows: antigen preparation, alpaca immunization, PBMC isolation, RNA extraction, cDNA preparation, library construction, library screening, antibody expression.

6.1 Antigen preparation

6.1.1 Codon optimization + gene template synthesis + expression vector construction;

6.1.2 Plasmid extraction + transfection (mammalian expression system) target protein detection (WB, SDS-PAGE);

6.1.3 Protein expression amplification + protein affinity purification

6.2 Animal immunization

6.2.1 Animals were immunized 4 times, with one booster shot, for a total of 5 shots;

6.2.2 Negative serum was collected before immunization, and the 4th shot of blood was used for ELISA to test the serum titer;

6.2.3 If the 4th shot of serum antibody titer (protein antigen>10^5; polypeptide antigen>10^4) meets the requirements, another booster shot will be given 7 days before blood collection. If it does not meet the requirements, conventional immunization will continue;

6.2.4 If the titer is qualified, blood (>100ml) will be collected to isolate mononuclear cells.

6.3 cDNA preparation

6.3.1 PBMC total RNA extraction (refer to RNA extraction kit);

6.3.2 High-fidelity RT-PCR preparation of cDNA (refer to reverse transcription kit).

6.4 Library preparation

6.4.1 Using cDNA as template, two rounds of PCR amplification of VHH gene;

6.4.2 Yeast expression plasmid construction and transformation: VHH gene splicing yeast display vector, electroporation transformation of yeast host bacteria, construction of antibody library, the number of electroporation is not less than 10 times;

6.4.3 Identification: Randomly select 24 clones, PCR identification positive rate + PCR identification diversity + insertion rate.

Figure 3: VHH positive rate: >90%

6.5 Library screening

6.5.1 Magnetic bead sorting;

6.5.2 FACS sorting (immune double labeling sorting);

6.5.3 Plating + positive clone bacterial culture + antibody gene sequencing; (>20, up to 96).

6.6 Antibody expression

6.6.1 Construct a suitable expression vector (mammalian expression) with the obtained antibody sequence for expression + affinity purification + antibody protein quantification;

Figure 4: Fc-VHH nano antibody expression results

6.6.2 Elisa verifies antibody binding to antigen and performs downstream verification (delivery of EC50 data);

6.6.3 BLI method verifies antibody affinity;

6.6.4 Cell function verification: flow cytometry verification;

6.6.5 Verify cell line (antigen receptor overexpression cell line).

7. Advantages of yeast display service

(1) Wide adaptability to species: Monoclonal antibody development of human, mouse, rabbit, sheep, alpaca, camel, fish and other species is compatible

(2) Mature technology platform: The library capacity of the provided level 1 immune library is 10^6-10^7; the insertion rate is >95%, and the affinity of the screened antibodies is generally at the nM-pM level

(3) Short development cycle: After obtaining PBMC, it takes 3-4 weeks from library construction to screening to obtain antibody sequences

(4) High product delivery standards: For immune libraries, pre- and post-immunization sera, antibody display libraries, yeast expression strains, and antibody sequences are delivered

With the continuous development of antibody library technology, phage display antibody libraries have produced a large number of highly active antibodies. The widespread application of phage display technology has prompted scientists to continuously explore yeast library technology. As a eukaryotic expression system, yeast display technology has its unique advantages compared to phage display systems, and therefore also occupies an important position in the field of human treatment and diagnosis.