RH2.2 IPTG-based rAB Expression

Expression and purification of rAB’s cloned into the RH2.2 expression vector.

1.0 Introduction

This method is for the mid-scale expression and purification of Fabs from the RH2.2 pTAC-based IPTGinducible vector.

2.0 Materials

Glassware/Plasticware

• 250 mL baffled flasks (Fisher Scientific, 10-140-6A)
• 50 mL Conical Tube (Bioexpress, LLC C-3394-4)
• 15 mL Biorad protein purification columns (EconoPac, 732-1010)
• 1.7 mL Eppendorf tubes (Axygen, MCT-150-C)

Reagents

Solutions

1X KCM

• KCl (500 mM)
• CaCL2 (150 mM)
• MgCl2 (250 mM)

2YT media

• 1% yeast extract (10 g)
• 1.6% bio-tryptone (16 g)
• 86 mM NaCl (5 g)
• Make up volume to 1L and adjust pH to 7.0

20X salt stock

• 500 mM Na2HPO4 (70.98 g)
• 500 mM KH2PO4 (68.05 g)
• 1M NH4Cl (53.4 g)
• 100 mM Na2SO4 (14.20 g)
• Mix with heating and sterile filter or autoclave until dissolved and make up to 1L. Store at RT

NZY media

• 1% N-Z-Amine (10g)
• 0.5% yeast extract (5 g)
• Mix in 500 mL and stir until dissolved
• Add 50 mL of 20X salt
• Add 2 mL of 1M MgSO4
• Make up volume to 980 mL and autoclave

Lysis buffer:

• 50mM Tris
• 150mM NaCl
• 1%Triton X-100
• 1mg/ml lysozyme
• 2mM MgCl2
• 10U benzonase

Elution buffer:

• 50mM NaH2PO4
• 140mM NaCl
• 100mM H3PO4
• pH 2.5

3.0 Transformation

• 3.1 Thaw 25 μL of chemically competent BL21-T1^r cells on ice
• 3.2 Add 50 ng of sequence-verified pTAC-based RH2.2 expression plasmid to a mixture of 4 μL of 5X KCM in 16 μL of MilliQ H₂O on ice
• 3.3 Chill mixture on ice for 10 minutes
• 3.4 Add 20 mL of chemically competent BL21-T1^r cells to the mixture of KCM and DNA
• 3.5 Incubate 20 min on ice, transfer to the benchtop, incubate 10 min at RT, return to ice and incubate 2 min on ice

4.0 Expression

• 4.1 Following the last stage of transformation on ice, transfer the entirety of the cell/DNA mixture in to 25 mL of 2YT supplemented with 50 μg/mL carb in a 50 mL Falcon tube, leaving the lid loose but taped secure to allow gas transfer
• 4.2 Inoculate 1/40^th volume (2.5 mL) of overnight culture into NZY media + 50 μg/mL carb (100 mL) supplemented with 1/20^th volume of 20X salt mixture
• 4.3 Grow 2-3 hr at 37^oC, 200rpm until OD 0.8-1.0 achieved
• 4.4 Induce culture with 1/1000^th volume of 1M IPTG (i.e. 100 μL in to 100 mL of culture)
• 4.5 Continue growing for 6-8 hrs at 30^oC and shaking at 200 rpm
• 4.6 Transfer ½ the volume of culture to a 50 mL Falcon tube and pellet cells at 8000 rpms for 20 min.
• 4.7 Discard supernatant and repeat to obtain a single pellet.
• 4.8 Discard remaining supernatant. At this point the pellet can be frozen for purification at a later date of lysed for purification as per the following step.

5.0 Purification

• 5.1 Lyse pellet by the addition of 10 mL of lysis buffer and nutation for 1.5-4 hrs at 4^oC
• 5.2 To ensure complete lysis, mixture can be sonicated for 1 min at 40% intensity using a 1 min program of 5 seconds on and 5 seconds off.
• 5.3 Centrifuge the pellet lysate at 9000rpm for 20min.
• 5.4 Add 250 μl of a protein A sepharose slurry to a 15 ml GF column.
• 5.5 Equilibrate resin with 25 mL of 1X PBS, drain and cap.
• 5.6 Transfer the lysate supernatant to the equilibrated protein A column and allow binding to proceed for 30 min at 4^oC with occasional mixing of the resin with the supernatant.
• 5.7 Drain the supernatant from the column retaining the flow-through and wash the resin bed with 40 ml of 1X PBS.
• 5.8 Add 60 μl of 1M Tris pH 8 added to Eppendorf collection tubes to neutralize the elution buffer.
• 5.9 Add 300 μl Fab elution buffer (above) to each capped column and incubate 5 min before draining in to an Eppendorf containing neutralization buffer.
• 5.10 Elution step can be repeated for a total elution volume of 600 μl neutralized with 1200 μl Tris.
• 5.11 Additional polishing steps can be carried out as necessary.
• 5.12 Columns can be regenerated with 10mL of 100mM H₃PO₄, washed with 20ml TBS, stored in 2ml 20% EtOH at 4^oC.

Please send corrections, modifications and suggestions to smiersch@recombinant-antibodies.org