pSFV4-Avi IPTG-based Biotinylated rAB Expression

Expression and purification of biotinylated rAB’s cloned into the pSFV4 expression vector with a Heavy Chain Avi Biotinylation tag.

1.0 Introduction

This method is intended for the small (50-100ml) expression and purification of biotinylated rABs that have been cloned into the pSFV4-Avi rAB expression vector. Please be aware that if transformed directly into C43(DE3) cells the protein will be minimally biotinylated and to obtain more robust biotinylation the C43(DE3) cells should be first transformed with a BirA expression plasmid, then made competent prior to transformation with the rAb plasmid.

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 BirA expression plasmid into chemically competent C43 (DE3) and select for cells containing BirA plasmid on Kanamycin plates. Make cells chemically competent by standard laboratory methods.
• 3.2 Transform <50ng sequence verified expression plasmid into competent cells generated in 3.1 using standard laboratory transformation protocols and plate on LB Carb, Chlor, and Kan plates. and incubate 16-18 hours at 37C

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 Biotin and IPTG to final concentrations of 5uM and 1mM, respectively.  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