Expression, Purification and Isolation of the MinE protein By Arsalan Wasim and Nicholas Wong

Incubation of bacteria containing the MinE gene Using the PET system which is a strain of E coli bacteria that has the ability to clone proteins at a very quick rate The specific gene sequence that is desired is inserted into this E coli plasmid which is under strong T7 RNA polymerase control T7 RNA polymerase control can allow the cell to spend almost all of the cell resources on making the desired protein until the cell itself is over 50% filled with the desired product

Incubation of bacteria containing the MinE gene The PET vector is under lacUV5 control and replication of the MinE protein can be induced by IPTG or lactose or an autoinduction medium IPTG acts as a clone to allolactose which triggers the transcription of the lac operon

Incubation of bacteria containing the MinE gene Cells are plated on agar mediums and allowed to grow for 48 hours Bacterial colonies are then cultivated using a sterile toothpick and incubated in 3mL of Luria-Bertani broth at 37 o C for 12 hours in a shaking incubator –shaking allows good exchange of oxygen and carbon dioxide while making sure all bacteria in the broth is suspended instead of sedentary

Luria-Bertani Broth Liquid medium high in nutrients used for the incubation of bacteria Generally composed of –10g tryptone (short peptide used as a protein source) –5g yeast extract –10g NaCl (variable depending on the bacteria) –1L deionized water Autoclave at 121 o C and keep sterile

Isolation of the MinE Protein Resuspend the bacteria in LB broth by vortexing the tube Transfer 1mL of the broth mixture to a 1.5mL microcentrifuge tube Centrifuge at 13000rpm for 30 seconds to pellet bacteria

Isolation of the MinE Protein Pipette off supernatant leaving the pellet Repeat until all 3mL of broth has been pelleted Resuspend pellet in 150ul lysis buffer

Lysis Buffer The lysis buffer is composed of –10mM tris-HCl –5mM EDTA –200mM NaCl –0.2% Sodium Dodecyl Sulfate (SDS)

Isolation of the MinE Protein Lyse cells in mixture through sonication –The sonicator transfers electrical power into mechanical energy at the tip of the titanium rod which causes micro-bubbles to form and implode causing strong shockwaves lysing cells

Isolation of the MinE Protein Centrifuge cells at 13000rpm and discard the supernatant

Isolation of the MinE protein Resuspend the pellet by adding 150ul resuspension buffer and vortex contents

Resuspension buffer The resuspension buffer is composed of –25mM Tris-HCl –10mM EDTA –50mM glucose

Isolation of the MinE Protein Pour the mixture into the His-Bind Nickel column –The His-Bind Nickel column has a solid stationary phase which binds to Histidine through chelation with the nickel ions on the column –Other contaminants that do not bind through chelation would elute out of the column

Isolation of the MinE Protein Imidazole is naturally found on histidine which allows the binding of the histidine to the nickel column

Purification of the MinE protein Elute non specifically bound proteins and contaminants using 300ul wash buffer Repeat 3 times

Wash Buffer The wash buffer is composed of –50mM sodium phosphate –300mM NaCl –5mM imidazole

Purification of the MinE protein Elute the now purified MinE protein using 300ul of elution buffer Repeat 3 times

Elution Buffer Elution buffer is composed of –50mM sodium phosphate –300mM NaCl –150mM imidazole

Purification of the MinE protein The high concentration of imidazole will displace the his-bound proteins and elute the protein

Western Blot Bradford Assay used to determine the amount of protein present - Prepare standards using stock solution of bovine serum albumin (BSA) - Serial Dilutions - Add Bradford Reagent (900µl) - Read Absorbance at 595nm

Western Blot SDS-Polyacrylamide Gel Electrophoresis - discontinous system is used - Heat all samples for 5min at 100°C before loading - load rainbow markers - Run the gel for 30 min at 200V

Western Blot Staining The Gel - Rinse the gel 3 X 5min in 100ml of deionized water - Add 20ml of GelCode Blue Stain Reagent - Continue gentle shaking until bands develop, max 1 hr - Rinse with 2 X 5min in 100 ml of deionized water

Western Blot Electrophoretic Protein Transfer And Protein Staining - BioRad Transblot semi-dry transfer unit - Transferred on PVDF membrane soaked in transfer buffer - 14 V applied for 30 min - Place membrane in TBS+Tween 20

Western Blot TBS+Tween 20 is composed of: –Trizma base – 61g –NaCl – 90g –Distilled water – 1000ml –Tween 20 – 5ml

Western Blot Blocking Non-Specific Binding Sites & Incubation with Primary Antibody –Place membrane in dilute solution of BSA –Protein attaches to the membrane in places where the target protein has not been attached –Add antibody, attaches on sites specific to the target protein

Western Blot Blocking Non-Specific Binding Sites & Incubation with Primary Antibody –Wash membrane in 100ml blocking buffer (nonfat dry milk ) for 1 hr –Wash with TBS+Tween 20, 1X15min, 2X5min –Place membrane in antibody for 1hr –Wash with TBS+Tween 20, 1X15min, 2X5min

Western Blot Detection Of The Antigen-Antibody Complex –Place the membrane in 2° antibody, horseradish peroxidase for 30 min –Wash 1X15 min and 4X5min with TBS+Tween 20 –Wash with water 2x10min –Incubate in 5 ml Western Blue Stabilized Substrate for Alkaline Phosphatase –Wash with water 2X5min to stop reaction

Summary 1.Incubate the MinE protein using the PET vector in LB broth induced with IPTG 2.Resuspend and pellet bacteria discard supernatant 3.Resuspend bacteria and lyse using lysis buffer 4.Sonicate bacterial cells to lyse contents 5.Centrifuge contents and discard supernatant 6.Resuspend pellet in resuspension buffer 7.Purify using a his-affinity nickel column 8.Wash with wash buffer 9.Elute with elution buffer

Summary 1.Bradford Assay, prepare standards from stock solution of BSA 2.Perform Serial dilutions and read abs at 595nm 3.SDS-Polyacrylamide Gel Electrophoresis 4.Staining The Gel with 20ml of GelCode Blue Stain Reagent 5.Electrophoretic Protein Transfer And Protein Staining using the BioRad Transblot semi-dry transfer unit 6.Blocking Non-Specific Binding Sites & Incubation with Primary Antibody 7.Detection Of The Antigen-Antibody Complex