Editing Construction of the human antibody gene libraries

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

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

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

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