pBL166 IPTG-based rAB Expression

Expression and purification of rAB’s cloned into the pBL166 expression vector.

1.0 Introduction

This method is intended for the small (50-100ml) expression and purification of rABs that have been cloned into the pBL166 rAB expression vector.

2.0 Materials

Glassware/Plasticware

• 250 mL baffled flasks
• 50 mL Conical Tube (Bioexpress, LLC C-3394-4)
• Econo-pac 1.5x12cm Chromatography Column (Biorad 732-1010)
• 15ml Conical Tube (Bioexpress, LLC C-3394-1)
• 14ml Disposable culture tube (VWR 60818-703)

Reagents

Solutions

1X TB Expression Media

• 1X Sterile Terrific Broth made according to manufacturers instructions and add Glycerol (0.4% Final), Carbenicillin (50ug/ml Final), Chloramphenicol (50ug/ml Final)

Elution buffer

• 100mM Acetic Acid

Neutralization Buffer

• 1M Tris pH 11

3.0 Transformation

• 3.1 Transform <50ng sequence verified expression plasmid into C43 (DE3) chemically competent cells using standard laboratory transformation protocols and plate on LB Carb

4.0 Expression

• 4.1 Pick an isolated colony from step 3.2 above into 5ml LB+50ug/ml Carb and Chlor into 14ml culture tube and grow 16-18 hours in shaking incubator at 37C
• 4.2 Dispense 100ml 1X TB expression media into 250ml baffled flasks
• 4.3 Inoculate with 200ul overnight culture from step 4.1
• 4.4 Incubate culture at 37C in shaking incubator (200-250 rpm) to an OD₆₀₀ of 0.6-0.8
• 4.5 Add IPTG to final concentrations of 1mM then reduce incubator temperature to 25C, and incubate for an additional 16-18 hours
• 4.6 Transfer ½ the volume of culture to a 50 mL Conical tube and pellet cells at >4000xg 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 Resuspend and lyse pellet by the addition of 5 mL of B-per lysis buffer and add 5ul DNaseI
• 5.2 Incubate at room temperature for 10 minutes
• 5.3 Add 5ml PBS to lysed cells
• 5.4 Transfer lysed cells with tightly closed tube cap to 60C water bath and incubate for 20 minutes
• 5.5 Centrifuge at 30,000xg for 20 minutes to pellet cell debris
• 5.6 Meanwhile, prepare Protein A purification column by adding 1ml Protein A sepharose slurry to a 15ml GF column. Wash Protein A with 10ml ddH2O then equilibrate with 15ml PBS
• 5.7 Transfer clarified cell lysate to the equilibrated and capped Protein A column and allow binding to proceed for 30 min at 4^oC with occasional mixing of the resin with the supernatant
• 5.8 Drain the supernatant from the column retaining the flow-through and wash the resin bed with 40 ml 1X PBS
• 5.9 Add 1ml of 1M Tris pH 8 added to 15ml Falcon tube for collecting eluted protein
• 5.10 10ml Fab elution buffer (above) added to each column
• 5.11 Concentrate and buffer exchange by dialysis or spin concentration into PBS