4. METHODS
4.2 Yeast surface display library construction and establishment of randomized sublibraries for affinity maturation
4.2.1 CDR3-randomized library construction
Blood samples of three individuals of the bamboo shark (C. plagiosum) were harvested from the caudal vein and subsequently transferred into Tri® Reagent BD (Sigma Aldrich). Total RNA from three individuals were used as template for cDNA synthesis using the Omniscript® Reverse Transcription Kit (Qiagen) and OdT-Oligonucleotides. From each individual three reactions were processed in parallel, each containing approximately 2 µg of RNA. From each reaction 5 µl were used for the follow-up gene-specific amplification as mentioned above. PCR products were pooled and purified using the Wizard® SV Gel and PCR Clean-Up System (Promega) according to the manufacturer’s protocol. The PCR amplified vNAR products were used as starting material for the semi-synthetic library construction.
The initial library was established in three consecutive PCR-steps (Fig. 14). For all reactions the conditions were: 94 °C for 2 min, 35 cycles of 30 s at 94 °C, 30 s at 55 °C and 40 s at 72 °C, followed by 72 °C for 7 min. Primer sequences are listed in section 3.4. In the first reaction PCR amplified vNAR product from the natural repertoire was used as template with the primer combinations FR1/CDR1/Tyr_up and FR3_lo. The forward primer replaced Cys by Tyr and incorporated a marginal diversity within CDR1. The PCR product was purified via Wizard® SV Gel and PCR Clean-up System (Promega). Subsequently, the second PCR was performed to fully randomize CDR3 using the primer-combination FR1_up and CDR3/rand/FR4_lo. After purification by Wizard® SV Gel and PCR Clean-up System, the PCR product was used as a template for the third reaction using primers with overlaps up- and downstream of the Nhe I and Bam HI restriction sites of the pCT-plasmid, respectively (GR_up and GR_lo) and purified by Wizard® SV Gel and PCR Clean-up System.
The pCT vector (3.2.1) was digested with Nhe I and Bam HI. The reaction was performed in a volume of 100 µl. For this, approximately 20 µg plasmid were used as well as 100 U restriction enzyme, respectively, and incubated overnight at 37 °C. The reaction mixture was purified via Wizard® SV Gel and PCR Clean-up System.
Library was established via an improved yeast transformation method according to Benatuil et al. in a homologous recombination-based process referred to as plasmid gap repair.[181] For electroporation 1-2 µg of the digested plasmid and 6-8 µg of insert were used. Settings on the Gene Pulser® were as followed: 2.5 kV and 25 µF. Time constants ranged from 3.0 to 4.5 ms. Library size was calculated by dilution plating after 2 days. Yeast cells (EBY 100) were transferred into SD-CAA medium. Stocks were stored at -80 °C in yeast freezing solution. For yeast surface display cells were grown overnight at 30 °C in SD-CAA medium, transferred into SG-CAA medium and incubated for 1-2 days at 20 °C.
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4.2.2 Establishment of randomized sublibraries for affinity maturation
For affinity maturation of the particular enriched antigen binding population, plasmid-DNA was isolated from yeast cells after sorting the cells via FACS and incubation at 30 °C for two days. To this end, 2 ml of yeast cell suspension were centrifuged (16100 g, room temperature, 1 min) and resuspended in 200 µl yeast lysis buffer. Chloroform and phenol were added in a volume of 100 µl, respectively, and cells were incubated at room temperature and 1000 rpm for approximately 10 min. After centrifugation (16100 g, 4 °C, 15 min) the supernatant was used as template for library establishment in a volume of 3 µl per PCR.
Sublibraries with totally randomized CDR1 were constructed in a 3-step PCR with the following conditions: 94 °C for 2 min, 35 cycles of 30 s at 94 °C, 30 s at 55 °C and 40 s at 72 °C, followed by 72 °C for 7 min. Akin to randomization of CDR3, in the first PCR reaction, the primer pair CDR1rand_up/GR_lo was used to randomize CDR1 followed by two consecutive PCR reactions with primer pairs FR1_up/GR_lo and GR_up/GR_lo, respectively. After each reaction step, the PCR product was purified via Wizard® SV Gel and PCR Clean-up System. Gap repair cloning and transformations were executed as described above (4.2.1).
4.2.3 Construction of a HV2-diversified library for the isolation of bi-specific IgNAR V domains
For the construction of a HV2-randomized yeast surface display library based on EpCAM-binding, affinity optimized single clone α-EpCAM-vNAR 5005, total DNA was isolated and used as starting material. For this, 2 ml of yeast cell suspension were centrifuged and plasmid DNA was isolated as aforementioned (4.2.3).
This particular library was established in a consecutive two-step splicing by overlap extension PCR. For the first PCR step, two reactions were carried out in parallel, each containing 3 µl isolated plasmid DNA as template. In one reaction, primer pair HV2_SOE_rand_up/pCT_Seq_lo was used. In the other reaction, primer pair pCT_Seq_up/HV2_SOE_lo was used. PCR conditions were as followed: 94 °C for 2 min, 35 cycles of 30 s at 94 °C, 30 s at 55 °C and 40 s at 72 °C, followed by 72 °C for 7 min. The respective PCR products were purified using Wizard® SV Gel and PCR Clean-up System. For the subsequent PCR, 1 µl of PCR product was used as template, respectively. After 6 cycles, primer pair pCT_Seq_up/pCT_Seq_lo was added. The resulting PCR product was purified via Wizard® SV Gel and PCR Clean-up System. Gap repair cloning and transformations were executed as described above.
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