1.What is the Yeast Display Platform

Yeast display refers to the expression of foreign proteins/peptides on the cell surface by linking or anchoring to the yeast cell wall composition. To achieve this, the foreign gene is fused to the cell wall protein gene, of which Aga2p is most commonly used for antibody display. Aga2p belongs to the family of yeast lectin proteins responsible for the fusion event and is fixed to the cell surface through two disulfide bonds on Aga1p. One of the advantages of using Aga2p as a fusion protein is that it is far away from the cell wall, so the fused antibody is more flexible in space, avoiding the loss of activity due to steric hindrance. Another advantage is that Aga2p is expressed under the control of the GAL1 promoter after cell growth, protecting the yeast cells from potentially toxic antibodies, thereby ensuring the display of all antibodies in the library. Yeast displaying antibodies or other proteins are then screened with antigen-coated magnetic beads, followed by several rounds of FACS screening for antibodies/proteins with desired properties.

Figure 1: Schematic diagram of yeast library display

2. What is Phage Display

Bacteriophages are a type of virus that wraps DNA or RNA in a coat protein. They can infect bacteria as hosts, insert their genes into the bacterial genome, and produce coat proteins and genes to reconstitute phage particles in the cytoplasm, which are then secreted into the periplasm. Plasmids that can produce phages after transforming bacteria are called phagemids. When foreign genes are inserted into phagemids and transformed into bacteria, phages with foreign proteins/peptides displayed on their capsids are produced.

3. What is the Difference between Yeast Display and Phage Display?

Yeast display has advantages over phage display in some aspects, but it also has some disadvantages.

(1) In the screening/panning step, yeast display is suitable for FACS, which can accurately screen antibodies with the desired binding properties. However, in phage display, the affinity of antibodies to antigens can only be roughly stratified by adjusting the composition of the washing buffer.

(2) Due to its eukaryotic expression system, yeast-displayed antibodies can be correctly folded and modified, such as glycosylation, and are closer to their natural structures in mammals than antibodies expressed by E. coli in phage display.

(3) Due to the low transformation efficiency of yeast, the diversity of antibodies displayed by yeast may be lower than that of phage (10^7-10^9 for yeast and 10^11 for phage at most).

(4) Since the number of antibody copies on the surface of yeast (10^4-10^5) is much greater than that of phage, the yeast display system can be used to screen low-affinity antibodies based on antibody affinity.

4. Tek Biotech can Provide Customers with High-quality Phage Library Construction Services

The M13 single-chain filamentous phage display system is currently the most widely used library display system. By fusion expression of proteins, antibodies or peptides with phage pIII protein, they are involved in phage assembly and displayed on the phage surface to form a phage display library. TechBio uses M13 filamentous phage shell protein pIII and PVIII display technology to build natural or immune phage antibody display libraries of different species for customers. Through antibody library technology, multi-species monoclonal antibodies are prepared, such as monoclonal antibodies from human, mouse, rabbit, chicken, sheep, goose, pig, cattle, horse, donkey, camel, alpaca, shark and other species. Customized services can be provided according to customer needs, and the library capacity can reach 10^11.

5. Introduction to the Whole Process of Phage Library Construction Service Provided by Tek Biotech for Customers

Tek Biotech provides phage library construction service for customers, the process includes: total RNA extraction, reverse transcription to obtain cDNA, PCR amplification, vector and PCR product restriction ligation, bacterial library construction, phage library construction, phage library titer detection.

Figure 3: Phage display antibody library construction process

5.1 Total RNA Extraction

Peripheral blood lymphocytes were taken out of the refrigerator and packaged, chloroform was added, and the supernatant was added with isopropanol after centrifugation. The precipitate was retained after centrifugation, and 75% ethanol was added and the precipitate was retained after centrifugation. DEPC water was added after drying, and the RNA was incubated to ensure that the RNA was dissolved. The total RNA was obtained by mixing the tubes into one tube. 1μl of the total RNA was taken for electrophoresis, and 2μl was taken for measuring the concentration using a nucleic acid concentration meter.

Figure 4: VHH RNA extraction

5.2 Reverse Transcription to Obtain cDNA

Prepare cDNA according to the instructions of the commercial kit. Split the RNA obtained in the previous step into two parts and reverse transcribe them into cDNA. Oligo dTprimer and random 6-mers are used as reverse transcription primers.

5.3 PCR Amplification

5.3.1 First Round of PCR

Use cDNA as a template for the first round of PCR reaction, and use HS Ex Taq enzyme for PCR amplification. The PCR reaction system is:

The obtained PCR amplification products were subjected to 1% agarose gel electrophoresis, and the target band was found for PCR amplification under the above conditions. All PCR production areas were subjected to 1% agarose gel electrophoresis, and DNA was recovered from the cut gel strips using a universal DNA purification and recovery kit.

Figure 5: Results of the first round of PCR amplification

5.3.2 Second Round of PCR

The DNA fragments amplified and recovered in the previous step of PCR were used as templates to amplify specific antibody fragments again. The PCR reaction system was:

The obtained PCR amplification product was recovered using a DNA purification recovery kit according to the instructions.

Figure 6: Second round PCR amplification results

5.4 Enzyme Digestion and Ligation of Vector and PCR Product

The second round PCR product was ligated to the phage plasmid pADL-10b by enzyme digestion to construct a phage plasmid library containing the amplified fragment. The ligation product was recovered using a DNA purification recovery kit according to the instructions, and 2μl was taken to detect the concentration of the recovered product using a nucleic acid concentration meter.

5.5 Bacterial Library Construction

The ligation product was electroporated to construct an E. coli library containing the target antibody fragment.

Take SS320 E. coli competent cells, add the recovered ligation product, transfer the mixed competent cells and ligation product to a pre-cooled electroporation cup, and use the transformation program preset by the electroporator for electroporation. After electroporation, add 950μl SOC medium to the electroporation cup immediately, and perform at least 20 electroporations. After the cells are revived, spread them on agarose culture plates containing ampicillin resistance and grow overnight. The cells on the culture plate grown overnight in the previous step were washed and scraped with 2xYT medium and a coating rod, and 20% glycerol was added to measure the OD600nm value and then stored at -80°C, which is the bacterial library.

Figure 7: VHH positive rate: >90%

5.6 Phage Library Construction

5.6.1 Phage Library Amplification

The scraped bacteria from the previous step were mixed and transferred to 100 mL of 2x YT culture medium pre-added with tetracycline, and cultured at 37°C, 250rpm until OD600nm reached 0.5-0.55.

After adding helper phage at a ratio of 20:1, continue to culture at 37°C for 30 minutes. Add Kana with a final concentration of 50μg/mL and PTG with a final concentration of 0.2mMI, and culture overnight at 30°C in a shaking incubator.

5.6.2 Phage Library Preparation

Centrifuge the overnight cultured bacteria at 4°C 4000 rpm for 20 minutes, transfer the supernatant to a new centrifuge tube, add 1/4 volume of pre-cooled 20% PEG/2.5M NaCl, and incubate on ice for 30 minutes. Centrifuge at 4000 rpm for 20 minutes at 4℃, remove the supernatant, and add 1 mL PBS buffer to dissolve the precipitate. Add 1/4 volume of pre-cooled 20% PEG/2.5M NaCl again and incubate on ice for 10 minutes. Centrifuge at 12000 xg at 4℃ for 10 minutes, remove the supernatant and dissolve the precipitate in 1 mL PBS to obtain the phage library, which can be stored at -80℃ for a long time or at 4℃ for a short time (1-2 weeks).

5.7 Phage Library Titer Detection

After culturing the SS320 strain stored in a -80℃ refrigerator, pick a single colony from the single colony plate and culture it overnight in 5ml 2×YT medium. The OD600 of the overnight culture solution is 0.5-0.55. Take the prepared phage library and dilute it into 12 gradients. Add 90μl of SS320 bacterial solution to each gradient for culture. Take 5μl from each dilution tube and add it to 2×YT solid culture medium (Amp) for overnight culture. Then the titer can be calculated.