Paul.xu at 22:03, 2 May 2018

2018-05-02T22:03:28Z

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Lymphocytes were isolated from 44 donors with various ethnical backgrounds using the Lymphoprep Kit (Progen, Heidelberg) according to the manufacturers instructions. Total RNA was isolated using Trizol (Invitrogen, Karlsruhe) and mRNA (from all samples) was isolated using the QuickPrep micro mRNA Purification Kit (GE Healthcare, München) leading to 81 RNA preparations. Then cDNA was synthesised separately from each RNA sample using Superscript II (Invitrogen), random hexamer oligonucleotide primers and 50–250 ng mRNA or 2–20ug total RNA, respectively. To amplify the antibody gene fragments (kappa, lambda, VH) 27 individual first PCRs of each cDNA sample were performed separately for each cDNA preparation in a volume of 50uL using Red Taq (Sigma, Hamburg) and 0.4uMof each primer (Table 1) for 30 cycles (1 min 94 ◦C, 1min 55 ◦C, 2min 72 ◦C) followed by a 10 min final synthesis step. The PCR products were purified by agarose gel electrophoresis using the Nucleospin Extract 2 Kit (Macherey-Nagel, Düren). The VH, kappa or lambda PCR products were pooled separately. To add restriction sites for cloning, 100 ng of the purified and pooled VH, kappa or lambda PCR products of the first PCR for each of the 27 s PCR reactions were used in a volume of 100uL using Red Taq and 0.2uM of each primer (Table 1) for 20 cycles (1 min 94 ◦C, 1min 57 ◦C, 2min 72 ◦C) followed by a 10min final synthesis step at 72 ◦C. The PCR products were purified by agarose gel electrophoresis using the Nucleospin Extract 2 Kit and VH, kappa or lambda were pooled separately. In total 2187 first PCR and 2187 s PCR reactions were performed. For VL cloning 5ug vector pHAL14 and 2ug kappa or lambda PCR products were digested in a volume of 100uL using 30U MluI and NotI (NEB, Frankfurt) at 37 ◦C for 2 h. This step was performed multiple times to produce enough material for up to 20 transformations. For quality control each digestion was performed separately with each enzyme in parallel. The digestions were inactivated at 65 ◦C for 10 min, followed by adding 0.5U calf intestine phosphatase (CIP) (MBI Fermentas, St. Leon-Rot) and incubation at 37 ◦C for 30 min, this step was repeated once. The vector and the PCR products were purified using the Nucleospin Extract 2 Kit, removing short stuffer fragments containing multiple stop codons between MluI and NotI in pHAL14. Each ligation was performed with 1ug digested vector and 270 ng PCR product using 3U T4 ligase (Promega, Mannheim) in a volume of 100uL overnight at 16 ◦C. The ligations were inactivated at 65 ◦C for 10 min and precipitated with 10uL 3Msodium acetate pH 5.2 and 250uL ethanol for 2min at RT, followed by 5min centrifugation at 16,000×g and 4 ◦C. The pellet was washed two times with 500uL 70% (v/v) ethanol. The dried pellet was resuspended in 35uL dH2O and mixed on ice with 25uL electrocompetent XL1-Blue MRF'. The electroporation was performed with 0.1cm prechilled cuvettes using aMicroPulser electroporator (BIO-RAD, München) at 1.7 kV. Immediately, 1mL prewarmed 37 ◦C SOC medium pH 7.0 (2% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.05% (w/v) NaCl, 20mM Mg solution, 20mM glucose) was added and incubated for 1 h at 600rpm and 37 ◦C. The transformation was plated out on 25cm square Petri dishes with 2xYT-GAT agar (1.6% (w/v) tryptone, 1% (w/v) yeast extract, 0.5% (w/v) NaCl, 100mMglucose, 100ug/mL ampicillin, 20ug/mL tetracycline, 1.5% (w/v) agar–agar) and incubated overnight at 37 ◦C. In parallel 10−6 dilutions were plated out to determine the transformation rate. These plates were used also for quality control by colony PCR using the oligonucleotide primers MHLacZ-Pro f and MHgIII r (Table 1) to verify the insert size. The colonies were scraped from the 25cm plates using 40mL 2xYT medium (1.6% (w/v) tryptone, 1% (w/v) yeast extract, 0.5% (w/v) NaCl) and 5mL were directly used for midi plasmid preparation using the Nucleobond Plasmid Midi Kit (Macherey-Nagel).

Lymphocytes were isolated from 54 donors with various ethnical backgrounds using the Lymphoprep Kit (Progen, Heidelberg) according to the manufacturers instructions. Total RNA was isolated using Trizol (Invitrogen, Karlsruhe) and mRNA was isolated using the Oligotex mRNA Minikit (Qiagen, Hilden). Then cDNA was synthesized using Superscript III (Invitrogen), random hexamer oligonucleotide primers and 50–250 ng mRNA. To amplify the antibody gene fragments (kappa, lambda, VH) 27 individual first PCRs of each cDNA sample were performed separately for each cDNA preparation in a volume of 50 μL using GoTaq (Promega, Mannheim) and 0.4 μM of each primer [40] for 30 cycles (1 min 95°C, 1 min 55°C, 2 min 72°C) followed by a 10 min final synthesis step. The PCR products were purified by agarose gel electrophoresis using the NucleoSpin Gel and PCR Clean-up Kit (Macherey- Nagel, Düren). VH, kappa or lambda PCR products were pooled separately. To add restriction sites for cloning, 100 ng of the purified and pooled VH, kappa or lambda PCR products of the first PCR for each of the 27 second PCR reactions were used in a volume of 100 μL using GoTaq and 0.2 μM of each primer for 20 cycles (1 min 95°C, 1 min 57°C, 2 min 72°C) followed by a 10 min final synthesis step at 72°C. PCR products were purified by agarose gel electrophoresis using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Düren). VH, kappa or lambda PCR products were pooled separately. In total 1458 first PCR and 1458 second PCR reactions were performed.

For VL cloning 5 μg vector pHAL30 and 2 μg kappa or lambda PCR products were digested in a volume of 100 μL using 30 U MluI and NotI (NEB, Frankfurt) at 37°C for 3 h. This step was performed multiple times to produce enough material for up to 50 transformations. For quality control each digestion was performed separately with each enzyme in parallel. The digestions were inactivated at 80°C for 20 min, followed by adding 0.5 U calf intestine phosphatase (CIP) (MBI Fermentas, St. Leon-Rot) and incubation at 37°C for 30 min, this step was repeated once. Vector and PCR products were purified using the NucleoSpin Gel and PCR Clean-up Kit, removing short stuffer fragments between MluI and NotI in pHAL30. Each ligation was performed with 1 μg digested vector and 270 ng PCR product using 3 U T4 ligase (Promega, Mannheim) in a volume of 100 μL overnight at 16°C. Ligations were inactivated at 70°C for 10 min and precipitated with 10 μL 3 M sodium acetate pH 5.2 and 250 μL ethanol for 2 min at RT, followed by 5 min centrifugation at 16000 × g and 4°C. Pellet was washed two times with 500 μL 70% (v/v) ethanol. The dried pellet was resuspended in 35 μL dH2O and mixed on ice with 25 μL electrocompetent E. coli XL1-Blue MRF’. Electroporation was performed with 0.1 cm prechilled cuvettes using a MicroPulser electroporator (BioRad, München) at 1.7 kV. Immediately, 1 mL prewarmed 37°C SOC medium pH 7.0 (2% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.05% (w/v) NaCl, 20 mM Mg solution, 20 mM glucose) was added and incubated for 1 h at 600 rpm and 37°C. Transformation was plated out on 25 cm square Petri dishes with 2xYT-GAT agar (1,6% (w/v) Tryptone, 1% (w/v) yeast extract, 0,5% (w/v) NaCl, 100 mM glucose, 100 μg/mL ampiciline, 20 μg/mL tetracycline, 1.5% (w/v) agar-agar) and incubated overnight at 37°C. In parallel 10−6 dilutions of each transformation reaction were plated out to determine the transformation rate. These plates were used also for quality control by colony PCR using the oligonucleotide primers MHLacZ-Pro_f and MHgIII_r to verify the insert size. The colonies were scraped from the 25 cm plates using 40 mL 2xYT medium (1,6% (w/v) Tryptone, 1% (w/v) yeast extract, 0,5% (w/v) NaCl) and 5 mL were directly used for midi plasmid preparation using the Nucleobond Plasmid Midi Kit (Macherey-Nagel). For VH cloning material from 44 donors of the previous described HAL7/8 libraries [31] was used in addition to material of 54 new donors. In a first step 5 μg pHAL30-VL sublibraries and 2 μg VH PCR products were digested in a volume of 100 μL using 30 U NcoIHF and 30 U HindIII-HF (NEB) at 37°C for 3 h. This step was performed several times to produce enough material for 150–200 transformations. The digestions, ligations and transformations were performed as described for VL cloning with following modifications. Ligation was performed with 250 ng VH PCR product. After transformation of XL1-Blue MRF’, overnight incubation on 25 cm 2 × YT-GAT plates and resuspension in 25 mL 2 × YT medium, glycerol stocks of each library were made using 800 μL bacteria solution (~1010 bacteria) and 200 μL glycerol (Roth, Karlsruhe) and stored at −80°C. For the final library one glycerol stock of each sublibrary was thawed, mixed together, aliquoted (1 mL) and stored at −80°C. The antibody gene libraries HAL9/10 were packaged separately inoculating 400 mL 2 × YT-GA (2 × YT containing 100 ug/mL ampicillin and 100 mM glucose) with a library glycerol stock. The bacteria were grown to an optical density at 600 nm (OD600) of 0.4–0.5 at 37°C and 250 rpm. 25 mL bacterial culture (~1.25 × 1010 cells) were infected with 2.5 × 1011 Hyperphage particles [38], incubated at 37°C for 30 min without shaking followed by 30 min at 250 rpm. Infected cells were harvested by centrifugation for 10 min at 3220xg. The pellet was resuspended in 400 mL 2 × YT-AK (2 × YT containing 100 ug/mL ampicillin and 50 mg/mL kanamycin). Phage were produced at 30°C and 250 rpm over night. The bacteria were centrifuged for 20 min at 10,000 × g. Phage particles in the supernatant were precipitated with 1/5 volume of 20% (w/v) polyethylene glycol (PEG)/ 2.5 M NaCl solution for one hour on ice with gentle shaking and pelleted by centrifugation for one hour at 10,000 × g at 4°C. The precipitated phage were resuspended in 10 mL phage dilution buffer (10 mM TrisHCl pH 7.5, 20 mM NaCl, 2 mM EDTA), 1/5 volume of PEG solution was added and incubated on ice for 20 min, followed by centrifugation for 30 min at 10,000 × g and 4°C. The pellet was resupended in 1 mL phage dilution buffer. Residual bacteria and cell debris were removed by additional centrifugation for 5 min at 16000 × g at 20°C. Supernatants containing antibody phage were stored at 4°C. Phage titration was done as described before [41]. The scFv display rates of the packaged libraries were analyzed by 10% SDS-PAGE, Western blot and anti-pIII immunostain (mouse anti-pIII 1:2000, goat anti-mouse IgG AP conjugate 1:10000). Wildtype pIII has a calculated molecular mass of 42.5 kDa, but it runs at an apparent molecular mass of 65 kDa in SDSPAGE [42]. Accordingly, the scFv::pIII fusion protein runs at about 95 kDa.

Journal of Biotechnology, A human scFv antibody generation pipeline for proteome research, Michael Hust∗,
[[Category:Antibodies]]

Construction of the human antibody gene libraries - Revision history

Paul.xu at 22:03, 2 May 2018