Paul.xu at 02:18, 30 August 2014

2014-08-30T02:18:06Z

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== Cloning, Growing and Induction ==

Transform wt SM2 in <strike>pT7A3 vector (2254 pT7A3-wtSM2)</strike>

2301 pR-wtSM2-NucA

into E coli K12 CAG597 or JM101. Grow cells in LB/amp or Kan, at 30 oC, induction at 30 oC o/n.

=== Ecoli strains for expression ===

'''NEB CAG597'''

F- lacZ(am) pho(am) tyrT[supC(ts)] trp(am) rpsL(StrR) rpoH(am)165 zhg::Tn10 mal(am) Strain Properties

dam/dcm Methylation: dam+ , dcm+

Protease-deficient: Yes

Laclq: No Drug Resistance: tet, str

 

'''Strain W3110 '''http://cgsc2.biology.yale.edu/Strain.php?ID=5446 CGSC#: 4474 Strain Designation Designation Source Preference W3110 Lederberg J. 1

Sex(Hfr,F+,F-,or F'): F- 3 Mutations: Mutation Location Certainty Display ''LAM- 17.40 1 λ- rph-1 82.20 1 rph-1''

 

'''HB101: ''' http://cgsc2.biology.yale.edu/Strain.php?ID=130465 ''F- mcrB mrr hsdS20(rB- mB-) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20(SmR) glnV44 λ-'' Please note that different sources have different genotypes so treat this information with caution. From a GIBCO BRL list of competent cells. Hybrid of E. coli K12 and E. coli B (but 98% K strain AB266 according to Smith et al.) Host for pBR322 and many plasmids Sigma lists the deletion Δ(gpt,proA). Check this. Promega does not list F-, mcrB, or mrr Streptomycin resistant

CGSC#: 12554 Strain Designation Designation Source Preference HB101 Boyer H. 1

Sex(Hfr,F+,F-,or F'): F- 13 Mutations: Mutation Location Certainty Display araC14 1.52 1 araC14 leuB6(Am) 1.74 1 leuB6(Am) DE(gpt-proA)62 5.51 1 Δ(gpt-proA)62 lacY1 7.79 1 lacY1 glnX44(AS) 14.99 1 glnX44(AS) galK2(Oc) 16.99 1 galK2(Oc) LAM- 17.40 1 λ- recA13 60.80 1 recA13 rpsL20(strR) 74.84 1 rpsL20(strR) xylA5 80.34 1 xylA5 mtl-1 81.26 1 mtl-1 thiE1 90.34 1 thiE1 hsdS20([]) 98.67 1 [hsdS20]

 

'''JM101: '''http://cgsc2.biology.yale.edu/Strain.php?ID=121248 Genotype F´ traD36 proA+B+ lacIq Δ(lacZ)M15/ Δ(lac-proAB) glnV thi

CGSC#: 12137 Strain Designation Designation Source Preference JM101 1

Sex(Hfr,F+,F-,or F'): F' 3 Mutations: Mutation Location Certainty Display DE(gpt-lac)0 5.51 1 Δ(gpt-lac)0 glnX44(AS) 14.99 1 glnX44(AS) thiE1 90.34 1 thiE1 

== Wild-Type Secreted Benzonase ==

 

SM2 nuclease in periplasm can be released from spheroplast using a protocol of Randall and Hardy (1986) 

1. Cells are harvested by centrifugation at 10,000 x g for 4 minutes.

2. The pellet is resuspended in a volume equal to the cell pellet of 10 mM Tris hydrochloride (Tris-HCl, pH 8.0) buffer 

3. The cell suspension is centrifuged again at the same settings. Discard the supernatant.

4. The cell pellet is suspended in an equal volume of spheroplasting buffer, containing 20% sucrose, 30 mM Tris-HCI (pH 8.0)

5. The cells are then converted to spheroplasts by incubation at 0°C for 30 min in the presence of 1/10 the cell pellet volume of 1mg/ml lysozyme freshly dissolved in 0.1 M EDTA (pH 7.5).

6. Spheroplast and periplasm are separated by centrifugation at 10,000 x g for 10 min.

7. The supernatant contains proteins that are secreted into the periplasm.  

 

Culture medium 1. Concentrate culture medium using ultrafiltration membrane with a cut off at a molecular weight of 10,000 (Millipore type PTGC0005). 

LB medium: 10 g NaCl/L--> 0.17 M

2. Add solid(NH4)2SO4 to cleared lysate to conc. of 80% (saturation) 3. Collect precipitate by spinning for 30 min at 14,000xg. 4. Dissolve pellets in l0 mM Tris-HCl pH 8.3. 5. Dialyse against the same buffer overnight. 6. Load to a <strike>DE-52 (Whatman) column (5 cmx5 cm),</strike>  30 ml of Q FF equilibrated with the same buffer.  7. Elute the adsorbed protein with the buffer containing a linear gradient of NaCl from 0 to 0.3 M. 8. Pool the nuclease active fractions. 9. Adjust to pH 5.6 with acetic acid. 10.Load to a <strike>CM-Sepharose CL-6B column (2.6 cmx l0 cm)</strike> 15~ 20 ml of SP-FF equilibrated with 20 mM sodium-acetate pH 5.6. (benzonase's pI: 6.23)

11. Elute the adsorbed nuclease with the buffer containing a linear gradient of NaCl from 0 to 0.5 M. 12. Pool the active fractions. 13. Dialyze against distilled water 14. Lyophilize 

 

Alternatively:

or P11 phosphate cellulose (cation exchange) 2.5 X 20 cm column, equilibrated with 50 mM NaAC, pH 5.2 buffer, wash with 200 ml the same buffer, elute with 500 ml linear gradient from pH 5.2 to pH 7.5, NaCl from 0 to 3 M, then dialyse pooled protein vs 10 mM Tris-HCl, pH8.2, 20 mM NaCl, load to DE52 (anion exchange, or QFF) column, Wash with 200 ml 10 mM Tris-HCl, pH 8.2, elute with 10 mM Tris-HCl, pH 8.2, 500 mM NaCl.

DE52: can be replaced by QFF

== His Tag Benzonase ==

[[Category:Protocols]]

Purification of Benzonase, SM2 Nuclease - Revision history

Paul.xu at 02:18, 30 August 2014