Paul.xu at 01:44, 2 April 2012

2012-04-02T01:44:39Z

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Procedure

Day 0

Start an overnight culture of the bacterial cells in LB broth.

This step has been done for you.

 

Day 1

Materials:

Sterile Luria Bertani (LB) broth containing 100mg/ml of neomycin (an antibiotic)

50mM IPTG (isopropyl-B-thiogalactopyranoside) in ethanol

plastic cuvettes and spectrophotometer

50mM Tris-HCl, pH7.4, 10% sucrose

10mM PMSF in methanol (phenylmethanesulfonyl fluoride) A common serine protease inhibitor PMSF is toxic and should always be handled with gloves)

Procedure:

1. Inoculate 50ml of sterile LB broth with 5ml of a saturated bacterial culture ("overnight culture" )

2. Allow culture to shake vigorously at 37ºC. Monitor bacterial growth by determining the absorbance of the culture.

a. Place 0.5ml of LB in a plastic cuvette and "blank" the spectrophotometer.

b. Remove 0.5ml of culture and place in plastic cuvette.

c. Place cuvette in spectrophotometer and determine the absorbance at 600nm.

3. When the absorbance of the culture reaches 0.3, add 0.5ml of 50mM IPTG (Final concentration = 0.5mM).

4. Allow the culture to continue shaking at 37ºC for 1-2 hours.

5. Remove flask from shaker and swirl in an ice/water bath for ~5min.

6. Transfer culture to centrifuge tube. Place tube in centrifuge and pellet the cells at 10,000rpm, for 20 minutes.

7. Discard supernatant, and resuspend the pellet in 0.2ml of 50mM Tris, pH 7.4, 10% sucrose. Transfer cells to a 1.5ml eppendorf tube and immediately place in an ethanol dry ice bath at -70ºC. Store at -80° C.

Day 2

Materials:

10mg/ml lysozyme in 0.25M Tris-HCl, pH7.4 (prepared immediately before use)

Metal affinity resin

Wash Buffer: 50mM Tris-HCl, pH8.0; 450 mM KCl, 10% glycerol, 2.5mM imidazole

Elution Buffer: 50mM Tris-HCl, pH 7.5; 350 mM KCl; 10% glycerol; 0.6M imidazole

10mM PMSF (Phenylmethanesulfonyl fluoride) WEAR GLOVES!

Procedure:

1. Thaw cell pellet between finger tips, and immediately place tube in ice.

2. Add 10ul of 10mM PMSF. PMSF is a serine protease inihibitor and will help stabilize all proteins in the crude extract from degradation.

3. Add 10ul of freshly prepared lysozyme solution (solution contains: 10mg/ml lysozyme in 0.25M Tris, pH 7.4) This will lyze the bacterial cells so the intracellular contents are in solution. Lysozyme is an endoglycosidase that breaks down the cell wall and outer membrane of bacterial cells.

4. Leave on ice 30 minutes, mix occasionally (every 5-10 minutes). During this incubation - the metal affinity resin must be equilibrated! Take 50 µl of metal resin (disperse resin first by pipetting up and down), add it to a tube with 10 volumes (500 µl) of Wash Buffer. Centrifuge at 700 g for 2 min. (~3 rpm) for 2 min to remove the storage buffer from the resin bed. Carefully remove and discard supernatant. Add 500 µl of Wash Buffer and mix briefly to pre-equilibrate the resin. Re centrifuge 700 g 2 min. discard supernatant, repeat wash 1 more time. Keep on ice.

5. Place eppendorf tube in a micro-centrifuge and spin at 15,000 rpm for 20 minutes. This spin will pellet the bacterial membranes and DNA.

6. Transfer supernatant, using P200 pipetman, to a fresh eppendorf tube. The supernatant will contain the INT protein as well as other cytosolic proteins. KEEP SUPERNATANT (= clarified CELL EXTRACT) ON ICE AT ALL TIMES

7. Transfer 20 ml of the cell extract to a fresh 1.5ml eppendorf tube. Label "Crude" and place in ethanol dry ice bath.

8. Add 50 ml of equilibrated metal affinity resin (disperse resin first by pipetting up and down) to the remaining cell extract.

9. Rotate tube at 4ºC for 1 hour.

10. Spin tube for 30sec. Remove the supernatant being careful to ensure that the resin remains at the bottom of the tube. Transfer this supernatant to a fresh 0.5ml eppendorf tube. Label "Unbound" and place in ethanol dry ice bath.

11. Add 250 ml of "wash buffer" to the pelleted resin. Mix gently, and rotate tube at 4ºC for 15minutes.

12. Spin tube for 30 seconds at the lowest speed setting of a microfuge. Remove the supernatant being careful to ensure that the resin remains at the bottom of the tube. Transfer buffer to a fresh 1.5ml eppendorf tube. Label "Wash"and place in ethanol dry ice bath.

13. Add 50 ml of "elution buffer" to the pelleted resin. Mix gently, and place on ice. Continue mixing gently every 30 seconds for 5 minutes.

14. Spin tube for 30 seconds. Remove the supernatant and transfer to a fresh 1.5ml eppendorf tube. Label "Pure" and place in ethanol dry ice bath.

 

Day 3

Electrophoretic Mobility Shift Assay (EMSA)

Materials:

Rec Mix: 50mM Tris-HCl, pH 8.0; 12.5 mM Spermidine; 1 mg/ml BSA,

1:100 IHF (from Segall’s stock) IHF is needed by INT to bind to DNA.

32P-end labeled attDNA (DNA that contains att sites, attachment sites for INT) ALWAYS wear gloves when handling radioactivity!

5% native polyacrylamide gel (Native means that this is a non-denaturing gel)0.5X TBE Procedure:1. Remove the four tubes from the freezer and immediately place on ice.2. Label five 1.5ml eppendorf tubes, 1-5.Assemble the following on ice.3. Add 8 ml of "Rec. Mix" to each of the five tubes.

4. Add 2 ml of each of the protein preparation to each tube:

tube Protein preparation

1 crude

2 unbound

3 wash

4 pure

5 distilled water (negative control)

Add 1 ml IHF (from 1/50 dilution)

6. Add 7 ml 40% glycerol.

7. Add 2ul of radioactively labeled attDNA to each tube. Final volume is 20 ml total. Instructor will demonstrate the necessary precautions for working with radioactive material.

 

8. Make sure that all reagents are mixed gently in all tubes and let tubes sit at room temperature for 10 minutes.

9. While reactions are incubating, begin pre-running vertical, 5% native acrylamide gel, 0.5XTBE, at 200Volts.

10. Turn off power supply, and load 15ml of the reaction into individual wells in the gel.

11. Electrophorese the samples at 200 volts for 1 hour.

12. Disassemble gel apparatus, and place gel onto gel blotting paper. Instructor will dry the gel and expose to autoradiography film.

 

Purification of His-tagged Int. E. coli BL21 (λDE3 pLysS) cells (250 ml) carrying the Int gene cloned into the pET28a vector (Novagen) were grown in Luria broth supplemented with neomycin sulfate (100 μg/ml; Sigma) at 37°C to mid-log phase. Int expression was induced with 0.5 mM IPTG (isopropyl-β-d-thiogalactopyranoside; Bachem) for 3 to 4 h; during induction, cultures were shaken at room temperature. Cell pellets were resuspended in extraction buffer (50 mM TrisCl [pH 8], 0.8 M KCl, 10% sucrose) after one freeze-thaw cycle. Extracts were made by homogenization on ice in the presence of phenylmethylsulfonyl fluoride, pepstatin A, leupeptin, and soybean trypsin inhibitor (concentrations of protease inhibitors were as for the previous cell extracts). The supernatant was frozen at −80°C, and the pellet was reextracted in 400 μl of extraction buffer. The supernatants were combined and mixed with 200 to 300 μl of Talon metal-affinity resin (Clontech) which had been washed in binding buffer (50 mM Tris-Cl [pH 8], 300 mM KCl, 10% glycerol, 10 mM β-mercaptoethanol). The beads were rotated with the protein extracts for 2 h at 4°C and then washed five times (30 min each) with 500 μl of extraction buffer. Two sequential elution steps were performed, each with 150 μl of elution buffer (50 mM Tris-Cl [pH 8], 600 mM KCl, 10% glycerol, 0.6 M imidazole or histidine, 10 mM β-mercaptoethanol). The resin was stripped with elution buffer containing 1 M imidazole. Fractions showing the greatest activity were quantitated by Western blot analysis.

Purification of non-His-tagged Int. E. coli BL21 (λDE3 pLysS) or HN1463 (IHF−HU−) cells (500 ml) carrying the Int gene cloned into a pT7 vector (41) or in a pLex 5B vector (5) were grown in Luria broth supplemented with ampicillin (100 μg/ml; Sigma) at 37°C to mid-log phase. Int expression was induced with 0.5 mM IPTG (Bachem), and cultures were shaken at room temperature for 4 h. Cell extracts were prepared as described above for His-tagged Int purification. Int was isolated from these extracts by mixing 200 μl of phosphocellulose equilibrated in buffer X (50 mM Tris-Cl [pH 8], 400 mM KCl, 1 mM EDTA, 10% glycerol, 10 mM β-mercaptoethanol) with 800 μl of cell extract. After being rotated for 2 h at 4°C, the resin was pelleted gently, the supernatant containing unbound proteins was removed, and the resin was washed with 400 μl of buffer X for 30 min. Two sequential elution steps were performed with 100 μl of buffer X. The first elution step was performed with buffer containing 600 mM KCl, while the second elution step was performed with buffer containing 1 M KCl.

Gel mobility shift and in vitro recombination assays.Linear substrates containing the attL or attL tenP′1(27) sequence were end labeled using T4 polynucleotide kinase (NEB) and [γ-32P]ATP (NEN). All recombination and binding reactions were performed in 20-μl total volumes. Recombination reaction mixtures contained 1 nM labeled attsite (179 bp long) and 4 nM unlabeled att site (496 bp long), while binding reaction mixtures generally contained 1 to 4 nM labeled att site, as specified in figure legends. Each reaction mixture contained 0.1 to 1 μg of sonicated salmon sperm DNA, 44 mM Tris-Cl (pH 8), 60 mM KCl, 0.05 mg of bovine serum albumin per ml, 11 mM Tris-borate (pH 8.9), 1 mM EDTA, 13.6% (vol/vol) glycerol, and 5 mM spermidine. Int and integration host factor (IHF) were present at 50 and 35 nM, respectively. 

 

Integrase acts to recombine the attP site on bacteriophage lambda (about 240 bp) with the attB site on the E. coli genome (about 25 bp) (Weisberg, R. A. and Landy, A. in Lambda II, p. 211 (1983), Cold Spring Harbor Laboratory)), to produce the integrated lambda genome flanked by attL (about 100 bp) and attR (about 160 bp) sites. In the absence of Xis (see below), this reaction is essentially irreversible. The integration reaction mediated by integrase and IHF works in vitro, with simple buffer containing spermidine. Integrase can be obtained as described by Nash, H. A., Methods of Enzymology 100:210-216 (1983). IHF can be obtained as described by Filutowicz, M., et al., Gene 147:149-150 (1994).

In the presence of the λ protein Xis (excise) integrase catalyzes the reaction of attR and attL to form attP and attB, i.e., it promotes the reverse of the reaction described above. This reaction can also be applied in the present invention.

One exemplary buffer for lambda integrase is comprised of 50 mM Tris-HCl, at pH 7.5-7.8, 70 mM KCl, 5 mM spermidine, 0.5 mM EDTA, and 0.25 mg/ml bovine serum albumin, and optionally, 10% glycerol.

One preferred buffer for P1 Cre recombinase is comprised of 50 mM Tris-HCl at pH 7.5, 33 mM NaCl, 5 mM spermidine, and 0.5 mg/ml bovine serum albumin.

The reaction is reversible in the presence of the protein Xis (excisionase); recombination between attL, and attR precisely excise the λ genome from its integrated state, regenerating the circular λ genome containing attp and the linear E. coli genome containing attB.

 One hundred ng of plasmids were mixed in buffer (equal volumes of 50 mM Tris HCl pH 7.5, 25 mM Tris HCl pH 8.0, 70 mM KCl, 5 mM spermidine, 0.5 mM EDTA, 250 μg/ml BSA, 10% glycerol) containing Int (43.5 ng), Xis (4.3 ng) and IHF (8.1 ng) in a final volume of 10 μl. Reactions were incubated for 45 minutes at 25° C., 10 minutes at 65 ° C., and 1 μl was transformed into E. coli DH5α. After expression, aliquots were spread on agar plates containing 200 1μg/ml kanamycin and incubated at 37° C.

 Reactions (8 μl final volume) contained about 40 ng of each DNA, 1×Int buffer (25 mM Tris HCl pH 7.5, 25 mM Tris HCl pH 8.0, 70 mM KCl, 5 mM spermidine HCl, 0.5 mM EDTA, and 0.25 mg/ml BSA), 12.5% glycerol, 8 ng IHF, and 43 ng lambda integrase. Reactions were incubated at 25° C. for 45 minutes, then at 70° C. for 5 minutes, and then cooled.

 Use of Topoisomerase to Stimulate Recombination

The stimulation of the recombination reaction by making one or the parental plasmids linear was not expected. If the stimulation resulted from relief of some conformation constraint arising during the two recombination reactions (formation of the Cointegrate and resolution to the two daughter molecules), then unwinding of the plasmids with a topoisomerase might also be stimulatory when one or both parental plasmids were circular.

The Insert Donor was pEZC2901 (FIG. 7A), and the Vector Donor was pECZ3101 (FIG. 7B). A portion of pEZC3101 was linearized with Mlu I. 20 ng of pEZC2901 and/or pECZ3101 were used in each 10 μl reaction (29 ng Int, 2.9 ng Xis, 5.4 ng IHF in 50 mM Tris HCl pH about 7.8, 16.5 mM NaCl, 35 mM KCl, 5 mM spermidine, 0.375 mg/ml BSA, 0.25 mM EDTA, 2% glycerol). Topoesomerse I (from calf Thymus; Life Technologies, Inc.) was diluted from 15 units/μl to the concentrations indicated in Table 15 in 1×EZC buffer.

 

BP: Catalyzed by the bacteriophage lambda Integrase (Int) and E.coli Integration Host Factor (IHF) proteins -> BP Clonase II enzyme mix) LR: Catalyzed by the bacteriophage lambda Int and Excisionase (Xis) proteins, and E.coli Integration Host Factor (IHF) protein --> LR Clonase II enzyme mix, - The LR reaction facilitates recombination of an attL substrate (entry clone) with an attR substrate (destination vector) to create an attB-containing expression clone

Integration of the phage genome into the bacterial chromosome occurs between DNA sequences named attP and attB (the phage and host DNA sites, respectively) and requires the integration host factor (IHF) protein. Excision of the phage genome occurs between attL and attR sites (generated as a result of integration) and requires both IHF and the phage-encoded excisionase (Xis) protein.

 from Korean site http://www.elpisbio.com/: Lambda Integrase/Excisionase mediate the recombination reaction of lambda into the E. coli chromosome specific site. Lambda Integrase/excisionase perform a recombination reaction between an attL-containing DNA vector and an attR-containing vector to generate an expression clone. Lambda Integrase/excisionase is purified from a recombinant E.coli strain. 5x Lambda Integrase Reaction Buffer 200mM Tris-HCl, pH7.4, 10mM DTT, 10mM EDTA (pH 8), 25mM Spermidine, 500ug/ml BSA 1. Prepare Integrase/excisionase reaction mixture as follows plasmid DNA (attL contain) 1-5 ㎕ ( supercoiled 100-300ng ) vector DNA (attR contain) 1 ㎕ ( supercoiled 150ng/ul) 5x Reaction Buffer : 2 ㎕ Lambda Integrase/excisionase 2 ㎕ Adjust volume to 10 ㎕ with TE Buffer (pH8.0) 2. Incubate at 25°C for 1 hour . 3. Add 1 ㎕ of 2 ㎍/㎕ Proteinase K and incubate at 37°C for 10 min. 4. Transform E.coli. competent cells and select for the appropriate antibiotic-resistant clones. 

 

Two-milliliter cultures of Escherichia coli BL21 (DE3) cells carrying the various pet22 integrase vectors were grown for 16 h at 37°C in Luria–Bertani (LB) broth supplemented with Ampicillin (100 µg/ml; Sigma). One-hundred milliters of fresh LB broth was then inoculated with 1 ml of these cultures and grown for 2–3 h to an A600 of ∼0.5. Integrase expression was induced with 0.1 mM IPTG (Invitrogen) for 3 h; cultures were shaken at 30°C during induction. The cells were pelleted, resuspended in binding buffer [10 mM Tris–HCl (pH 8), 150 mM NaCl, 10 mM imidazole and protease inhibitor cocktail tablet; complete mini EDTA free (Roche)] and lysed by sonication. Cell debris was removed by centrifugation at 13 000 r.p.m. for 45 min at 4°C. Cleared lysates containing the 6 × His-tagged integrase proteins were mixed with 1.5 ml of nickel-charged resin (Ni–NTA Agarose; QIAGEN) which had been pre-equilibrated in binding buffer. The beads were subsequently rotated with the protein extracts for 3 h at 4°C. The Ni–NTA resins were then washed six times with chilled wash buffer (PBS + 80 mM imidazole). Two sequential elution steps were performed, each with 120 µl of elution buffer [250 mM imidazole, 50 mM Tris–HCl (pH 8), 1 mM DTT, 150 mM NaCl and 10 mM EDTA]. The amount of protein released was determined using the Quick Start™ Bradford Reagent (Bio-Rad Laboratories) as per manufacturer’s instructions. The purity of all proteins was determined by Coomassie Blue staining of polyacrylamide gels (NuPAGE® Novex 4–12% Bis–Tris Gel; Invitrogen). In vitro recombination assay for purified proteins

One microgram of purified recombinant integrase protein was incubated with 200 ng of linear attH substrate and 100 ng of linear attPH substrate for 30 min at 37°C in recombination buffer (100 mM Tris pH 7.5, 500 mM NaCl, 25 mM DTT, 10 mM EDTA, 5 mg/ml bovine serum albumin). The reaction volume was 25 µl. One microliter of this reaction was subsequently used for real-time PCR quantification of recombination efficiency as described above. 

Int and C-domain proteins were produced from expression plasmids under the control of a T7 promoter in E.coli BL21 (DE3, pLysS) as described previously (Tirumalai et al., 1997, 1998). To overexpress the proteins, E.coli cells carrying the appropriate expression plasmids were grown at 37°C to an A600 of 0.4±0.6, at which point protein expression was induced by the addition of isopropyl-b-D-thiogalactopyranoside to a ®nal concentration of 1 mM and the cells were grown for a further 3 h. The cells were pelleted by centrifugation at 500 g for 10 min, washed with 50 mM Tris±HCl pH 8.0, 10% (w/v) sucrose, 100 mM sodium chloride, 10% (v/v) glycerol, 1 mM dithiothreitol (DTT), 1 mM EDTA (lysis buffer). The cell suspension was frozen as droplets directly in liquid nitrogen and the resulting `popcorn' was stored at ±80°C. Proteins were puri®ed to near homogeneity by minor modi®cations of the protocol described by Kikuchi and Nash (1978) as detailed earlier (Tirumalai et al., 1998). The puri®cation of the N-domain will be described elsewhere (D.Sarkar and A.Landy, in preparation). 

[[Category:Protocols]]

Integrase Purification - Revision history

Paul.xu at 01:44, 2 April 2012