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

Terrific Broth

Add the following to 800ml distilled H2O:
• 12g Tryptone
• 24g Yeast extract
• 4ml Glycerol
• Adjust to 900ml with distilled H2O
• Sterilize by autoclaving
• Allow to cool to room temperature
• Adjust volume to 1000ml with 100ml of a filter sterilized solution of 0.17M KH2PO4 and 0.72M K2HPO4

Wash Buffer

• PBS with 25 mM imidazole
• pH 7.4

Elution Buffer

• PBS with 250 mM imidazole
• pH 7.4

PFU Resuspension Buffer

• 50 mM Tris-Cl pH8
• 150 mM NaCl
• 1 mM EDTA
• 1 mM PMSF
• 10 ug/ml E-64
• 80 mg lysozyme

1x PFU Storage Buffer (2x lacks glycerol)

• 50 mM Tris pH 8.3
• 50% Glycerol
• 0.1 mM EDTA
• 1.0 mM DTT
• 0.1% IGEPAL (aka NP-40)
• 0.1% Tween

10x PFU Reaction Buffer

• 200 mM Tris pH 8.8
• 100 mM KCl
• 100 mM (NH4)2SO4
• 20 mM MgSO4
• 1.0% Triton X-100
• 1.0 mg/ml BSA

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

Ethidium Bromide Staining Solution

• 20 uL ethidium bromide
• 200 uL milliQ water

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
5. Repeat steps 2-4 30 times
6. Final Extension: 3 min @ 72°C
7. 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(or Amp(100) for PFU culture).
• Grow for ~16 hours at 37°C (Ensure that the proper antibiotic (kanamycin in this case, or Ampicillin for PFU) 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 For Purification

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 250 uM.
4. The cultures are then to be shaken for 16 hours at 20C°.

Preparation of -80°C Freezer Stocks

1. Combine 400uL of culture with 400uL of 40% v/v filter-sterilized glycerol in labelled (strain, date, initials) in a cryotube.
2. Store at -80C.

Preparation of Columns(Not Necessary for PFU)

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 Taq Ligase and T5 Exonuclease

1. Weigh centrifuge tubes into which to place the cultures.
2. The bacteria are then to be pelleted by centrifugation at 4250 RCF for as long as necessary to pellet debris.
3. The supernatant is then removed and the pellet mass is determined.
4. The cells are then resuspended using 4mL of resuspension buffer for every g of wet pellet weight.
5. Resuspended cells are sonicated on ice for 2 min total in 2 sec pulses with 4 sec in between.
6. Centrifuge lysate at top centrifuge speed to remove debris
7. Add 125 uL of DTT
8. Apply Supernatant to column and allow to flow through
9. Wash 3 times with wash buffer
10. Elute with mL elution buffer

Purification of PFU

1. Remove the cultures from the shaking incubator. Transfer culture to conical tubes and spin down for ten minutes at 25°C (Note: these pellets can be stored at -20°C or long-term at -80°C)
2. Pour cell culture into 50 mL conical tubes and spin down. Pour off supernatant and resuspend in lysis buffer:
3. Made 40 mL (20 mL for uninduced and induced)
4. Added 12 mL NaCl, 400 uL PMSF, 400 uL E-64, 160 mg lysozyme, 80 uL EDTA, and 4 mL Tris-Cl (stock solution pH=8.5)
5. Let the resuspended pellets sit on ice for 30 minutes
6. Sonicate the cell solution four times for one minute each in an ice bath
7. Use 20% Amplification, with the pulse on for two seconds and off for 5 seconds
8. Transfer the cell solution from a conical tube to 1 mL eppendorf tubes. Centrifuge the cell samples (35,000g for 30 minutes at 4°C) using a tabletop centrifuge.
9. Transfer supernatant to new tube

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 acrylamide
2. Make fresh 5x loading dye which is :10% w/v SDS, 10 mM Beta-mercaptoethanol, 20 % v/v Glycerol, 0.2 M Tris-HCl, pH 6.8, 0.05% w/v Bromophenol blue,
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

Gibson Reaction Protocol

5x Isothermal Reaction Buffer

• 1 M Tris-HCl pH 7.5
• 3 mL 2.5 M MgCl2
• 120 μL 10 mM dNTP 600 μL
• PEG-8000 1.5 g
• NAD 19.9 mg
• 1 M DTT 300 μL
• H2O 1.98 mL

Gibson Master Mix Components

• 5x isothermal reaction buffer 320 uL
• T5 exonuclease (10 U/uL) 0.64 uL
• Taq ligase (40 U/uL) 160 uL
• Pfu polymerase 20 uL
• Water 700 uL
• *Can be stored at -20C.uL

Reaction Protocol

1. Synthesize forward and reverse primers to amplify the chosen insert and vector. Ensure that a minimum of 20 base pair homologous region overhang is included.
2. PCR amplify the insert and vector. PCR purify the DNA, and perform and DpnI digest.
3. Add Gibson Master Mix accompanied by the vector and insert in appropriate amounts:
   • Gibson Master Mix 7.5 μL Plasmid
   • 50 ng Insert
   • 2-3 fold plasmid amount
4. Incubate at 50C for one hour and run out on agarose gel.
5. Add 2 μL of reaction mix to competent cells (chemically competent DH5α), and transform according to standard protocol.

Transformation Protocol

1. Add 2.5 uL of DNA
2. Prepare 100uL aliquots of DH5α
3. Add 2 uL of desired DNA to each aliquot
4. Allow to sit on ice for 10min
5. Heat shock in 42ºC water bath for 45 seconds
6. Plunge into ice for 2 min
7. Add 900uL of media to each tube and incubate for 1 hour
8. Plate 100uL of culture onto LB Agarose

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 37°C 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

9. Incubate above reaction 20min at 37°C
10. Run reaction products out in 1% Agarose gel without ethidium bromide
11. Stain the gel in ethidium bromide solution (20 uL EtBr, 200 uL MilliQ water) for 15 minutes (gentle agitation), followed by a 15 minute water wash (gentle agitation).
12. To determine the proper concentration of T5 exonuclease to use, select the enzyme concentration that most closely matches the commercial enzyme based on the gel.

Taq Ligase Assay

1. Assay was performed to test the concentration of Taq Ligase necessary to mimic the activity of the commercially available Taq Ligase. To do this, several concentrations of Taq Ligase were added and compared to the commercial Taq Ligase. A master mix for the reaction was made containing 294 uL nuclease-free water, 35 uL Taq DNA ligase reaction buffer, and 14 uL lambda DNA. Pipette 49 uL of the reaction mixture into six tubes (use a PCR strip tube).
2. Incubate at  45°C for 15 minutes. Terminate the reaction is by adding 10 uL (6X) stop dye (50% glycerol, 50 mM EDTA and a pinch of bromophenol blue). Stop dye acts as loading dye
3. Heat at 70°C for 10 minutes and then load onto a 1% agarose gel. Run for 30 minutes. Taq  on 0.7% gel. Stain with ethidium bromide, and wash with water.
4. To determine which concentration of your enzyme to use, select the concentration that most closely matches the commercial Taq Ligase lane based on the gel.