Expression and Purification of Enzymes

Solutions

Resuspension Buffer

• 400 mM NaCl
• 50 mM NaHPO4
• 2.5% (v/v) glycerol
• 15 mM Imidazole
• 0.05% Triton X-100
• pH 8.0

Taq Ligase Storage Buffer

• 10 mM Tris-HCl
• 50 mM KCl
• 1 mM DTT
• 0.1 mM EDTA
• 200 μg/ml BSA
• 50% v/v Glycerol
• pH 7.4 @ 25°C

T5 Exonuclease Storage Buffer

• 50 mM Tris-HCl
• 100 mM NaCl
• 1 mM DTT
• 0.1 mM EDTA
• 50% v/v Glycerol
• 0.1% Triton® X-100
• pH 7.5 @ 25°C

Methods

Transforming the Synthesized Plasmid

1. The genes for Taq Ligase and T5 exonuclease were designed synthesized in a factory plasmid by GeneArt
2. Prepare 100 mL stocks of BL21(DE3) competent cells (stored at -80°C)
3. Allow to unthaw, and add 2 uL DNA (using 2uL water as a control) to competent cell cultures.
4. Let sit on ice for ten minutes
5. Heat-shock cells for 45 seconds in 42°C water bath
6. Return cells to ice for two minutes
7. Allow cells to recover by adding 900 uL LB and putting into 37°C shaking incubator for 1 hour
8. Plate 100 uL on Kan(50) LB agarose plates and incubate for 16 hours at 37°C
9. Determine which colonies have successfully taken up plasmid by cPCR

Colony PCR Protocol

Each tube:

• 25uL PCR tube contents:
• 1uL of Forwards and backwards Primers at 10uM concentration
• 10.5uL Nuclease Free H20
• 12.5uL goTaq
• Cell Culture

Method:

1. Pick a colony from the cell culture using a sterile toothpick
2. Streak out on Kan(50) lysogeny broth (LB) agarose
3. Place in the 25uL Reaction Mix and spin toothpick for a few seconds

PCR Block Settings:

• 1. Initial Denaturation: 2.5 min @ 95°C
• 2. Denaturation: .5 min @ 95°C
• 3. Annealing: .5 min @ primer annealing temperature
• 4. Extension: 1 min @ 72°C
• Repeat steps 2-4 30 times
• 5. Final Extension: 3 min @ 72°C
• 6. Final Hold: indefinitely @ 4°C

Starting Overnights of Cultures

• A single colony selected from each cPCR Streak plate was used to inoculate a 5 mL Kan(50) LB culture.
• Grow for ~16 hours at 37°C (Ensure that the proper antibiotic (kanamycin in this case) is added to the cultures)
• Two cultures containing each plasmid will be grown out (T5 exonuclease, Taq Ligase, and the empty pET vector)

Culture Growth

1. Overnight cultures are to be diluted using LB to an OD600 of 1.0 in order to use 2mL of diluted culture to inoculate 200mL Terrific broth
2. This is then grown at 37°C while checking the OD600 of the culture every hour or so.
3. Upon reaching an OD600 of 0.700, the culture is to be induced with IPTG, to a final concentration of 500 uM.
4. The cultures are then to be shaken for 16 hours at 20C°.

Preparation of -80C Freezer Stocks

Combine 400uL of culture with 400uL of 40% v/v filter-sterilized glycerol in labelled (strain, date, initials) in a cryotube.

Store at -80C.

Preparation of Columns

1. Clean column using washes of EtOH and 2%SDS
2. 3 final rinses with Deionized water
3. Fill 15mL column with resuspension buffer, add 1mL Ni-NTA resin beads
4. Rock on ice for 20 min
5. Allow buffer to flow through column until it is mostly empty (Note: Never allow the Ni-NTA layer to be dry or disturbed)
6. Column is ready to bind protein

Purification of Enzymes

Weigh centrifuge tubes into which to place the cultures.

The bacteria are then to be pelleted by centrifugation at 4250 RCF for as long as necessary to pellet debris.

The supernatant is then removed and the pellet mass is determined.

The cells are then resuspended using 4mL of resuspension buffer for every g of wet pellet weight.

Resuspended cells are sonicated on ice for 2 min total in 2 sec pulses with 4 sec in between.

Centrifuge lysate at top centrifuge speed to remove debris

Add 125 uL of DTT

Apply Supernatant to column and allow to flow through

Wash 3 times with wash buffer

Elute with mL elution buffer

Bradford Assay

In a 96 well plate add to each well:

• 3uL of sample, or BSA standards
• 297uL of bradford reagent

• Incubate in the dark for 15 min
• Use plate reader to collect absorbance at 595 for each well

SDS-PAGE Gel Analysis

1. Prepare a 5mL page gel of the appropriate percentage acrilimide
2. Make fresh 5x loading dye which is :10% w/v SDS, 10 mM Beta-mercapto-ethanol, 20 % v/v Glycerol, 0.2 M Tris-HCl, pH 6.8, 0.05% w/v Bromophenolblue,
3. Mix sample 4:1 with loading dye
4. Boil mixture for 3 minutes
5. Load 20-30ug of soluble fraction, 1-4ug eluent and 20uL 1:1 diluted insoluble fraction into appropriate wells immediately

Buffer Exchange

Place purified protein solution in a >30 KD exclusionary protein concentration spin column

Spin for 30 min

Dump out flow through

Fill column with 2x appropriate protein storage buffer

Spin for 30 min

Dump flow through

Repeat 2 more times

Collect purified protein in storage buffer from the top of the column and store in 500uL aliquots at -20 C

Assays

T5 Exonuclease Assay

Day One

Start two overnights of DH5 alpha pETALM Taq Ligase (add kanamycin to concentration of 0.05M)

Day Two

1. Mini-prep and nanodrop the overnights to determine DNA concentration
2. Perform a PCR reaction using the mini-prepped DNA
3. We wanted a lot of DNA, so we set up 24 50 uL reactions in three strips of PCR tubes.
4. The pETALM Taq Ligase DNA was diluted to 30 ng/uL
5. PCR Reaction Per Tube
   • 2uL T7 Promoter Primer
   • 2uL T7 Terminator Primer
   • 1uL DNTPs
   • 5uL 10x PFU Buffer
   • 1uL PFU Enzyme
   • 2uL pETALM Taq Ligase DNA
   • 37uL Nuclease Free Water
6. After PCR Reaction PCR Purify products
7. DPN1 Digest for 1 hr at 37C using 1uL Dpn1, 4uL NEB Buffer 4, 5uL Water and 30uL DNA solution
8. Set up following reactions using enzyme dilutions:

   	Reaction 1	Reaction 2	Reaction 3	Reaction 4	Reaction 5	Reaction 6
   Linear DNA	1.3uL	1.3uL	1.3uL	1.3uL	1.3uL	1.3uL
   T5 Exonuclease	0uL	1uL 1:10 dilution Our enzyme	1uL 1:100 dilution Our enzyme	1uL 1:1000 dilution Our enzyme	1uL 1:10000 dilution Our enzyme	1uL Commercial Enzyme
   NEB Buffer 4	1uL	1uL	1uL	1uL	1uL	1uL
   Water	7.7uL	6.7uL	6.7uL	6.7uL	6.7uL	6.7uL