Anecdotally pATH11 gives the highest protein yields. You can choose appropriate
cDNAs; that end at a 5' synthetic Eco R1 site, or engineer one in by site directed
mutagenesis, or use a naturally occurring site. Otherwise there are many other restriction
sites in the polylinkers to choose from. Don't worry about the C-terminus, as all three
frames stop soon after the polylinker. All of these vector DNAs grow very poorly, so be
prepared to put all of your miniprep plasmid DNA in one track to see it on a gel.
Including tryptophan in the growth medium may help avoid selecting non-expressing
mutants. Once you have your subclone in E. coli HB101; do the following:
1) Grow a 5ml seed culture in LB + 50 µg/ml ampicillin; shaken at 37šC to saturation
(overnight).
2) Inoculate 250 ml of M9CA; + 50 µg/ml ampicillin in a 2 liter flask with 2.5 ml of the
seed culture and grow shaken at 37šC to A600 = 0.2 (about two hours).
3) Add 250 µl of 10 mg/ml 3 -indole-acrylic acid; (Sigma No.I-1625) in propan-1-ol and continue to incubate shaken at 37šC for at least four hours (I usually go overnight).
4) Make up 50 ml of TEN+ and store on ice.
5) Spin down the cells and resuspend in 25 ml TEN+ and transfer to a 50 ml disposable polypropylene tube, add a dash of lysozyme; (Sigma No. L-6876) and incubate on ice for 25 minutes.
6) Add 2.5 ml of a 10% solution of triton X-100; (Sigma No. T-6878) in water, vortex and incubate on ice for 10 minutes.
7) Add 1.25 ml of a 10% solution of Zwitergent; (Calbiochem No. 693017) in water, vortex and incubate on ice for 10 minutes.
8) Sonicate at maximum microtip energy in repeated 10 second bursts with cooling on ice for a total of 60 seconds, or until the viscosity drops significantly. This step breaks E.coli DNA, which otherwise reduces the effects of the subsequent wash steps and can interfere with the running of preparative gels.
9) Keep 50 µl of the total extract and load the rest carefully (so as to avoid mixing) onto a 10 ml sucrose cushion (40% sucrose in TEN) in a 50 ml polycarbonate tube. Mark the position of the interface.
10) Spin for 30 minutes at 4šC at 12,000 rpm in an HB-4 rotor; (or similar).
11) Take off the supernatant above the interface, keep a 50 µl aliquot handy and store the rest frozen at -20šC. Then remove and discard the sucrose.
12) Resuspend the pellet in 5 ml TEN+ and transfer to a 15 ml corex tube on ice. To break up the pellet you will need to thoroughly beat up on it, vortex it, draw it through a pipette tip or use any other violent means.
13) Spin at 9,000 rpm at 4šC for 5 minutes, and remove and discard the supernatant.
14) Repeat steps 12 and 13 until the supernatant becomes clear or you think you will run out of TEN+, or you get bored. I usually do three such washes.
15) Resuspend the pellet in 2 ml TEN+, keep a 50 µl aliquot handy and store the rest at -20šC.
16) Pour a 9% acrylamide SDS gel; at pH 8.8 with a pH 6.8 stacker. For my size apparatus these are as below, but can be scaled to fit:
9.6 ml 30:0.8 acrylamide:bis; 1.66 ml 30:0.8 acrylamide:bis
12.0 ml 1 M Tris ;pH 8.8 (from base) 1.25 ml 1 M Tris pH 6.8 (from base)
10.0 ml distilled water 6.92 ml distilled water
320 µl 10% SDS; 100 µl 10% SDS
160 µl fresh 10% Ammonium persulfate; 50 µl fresh 10% Ammonium persulfate
30 µl TEMED; 12 µl TEMED
17) Load 15 µl each of the total and supernatant and 5 µl of the pellet in equal volumes of 2X Lämmli buffer;. Also run appropriate size standards; (such as Sigma No. SDS-6H). It is a good idea to do a parallel prep on induced vector alone so that you can see the 37 kD TrpE protein.
18) Run the gel as fast as you can while keeping it cool in 1X PGB and then stain for one hour in coomassie; stain.
19) Destain repeatedly until satisfied with the result (overnight works well), and dry the gel down onto 3MM paper.
20) Comparison between the vector and insert preps should immediately reveal your fusion protein when the gel is stained, hopefully at the correct molecular weight. If you can't see anything quit now and get a steady job. If you have what you want, scale up the gel and run the pellet out on a prep gel.
21) Stain the prep gel for only two minutes in aqueous coomassie, and destain extensively in water.
22) Put the gel in a pyrex dish and view it on a white light box. Cut out the band and electroelute in your favorite apparatus in 1XPGB in a cold room. You can follow the passage of the coomassie blue.
23) Dialyse extensively (at least four changes over 24 hours) against 1X PBS + 2mM d ‘mercapto ethanolQ (140 µl per 1 liter) + 1.74 µg/ml PMSF.
24) Run an aliquot adjacent to a dilution series of a known protein (eg BSA) and assess its concentration.
25) Store in aliquots at -20šC. This is your antigen.
M9CA - see buffers/media section
TEN+ (50 ml)
0.5 ml 1 M Tris pH 8.0
0.2 ml 250 mM EDTA
2.0 ml 5 M NaCl
7 µl d -mercapto ethanol
50 µl 1.74 mg/ml Phenylmethane-sulfonyl, in propanol ;(Boehringer Manheim)
50 µl 1 M Benzamidine; in water (Sigma B-6506 stored at -20šC in 1 ml aliquots)
50 µl 2 mg/ml aprotinin; in water (Boehringer Manheim stored at -20šC in 1 ml aliquots)
50 µl 1 mg/ml leupeptin; in water (Boehringer Manheim stored at -20šC in 1 ml aliquots)
50 µl 1 mg/ml pepstatin; in methanol (Boehringer Manheim stored at -20šC in 1 ml aliquots)
distilled water to 50 ml
10% triton X-100; (Sigma No. T-6878) in water
10% Zwitergent ;(Calbiochem No. 693017) in water
40% sucrose ;(in TEN)
1.0 ml 1 M Tris pH 8.0
0.4 ml 250 mM EDTA
4.0 ml 5 M NaCl
40 g sucrose
distilled water to 100 ml, filter sterilise and store at 4šC.
2X Lämmli buffer ;
20 % glycerol
4 % SDS
250 mM Tris pH 6.8
10 % -mercapto ethanol
0.05% bromophenol blue
5X PGB; (Protein Gel Buffer)
30.27 g Tris base
144.1 g glysine
10 g SDS
water to 2 liters, store at room temperature
stain - 50 ml glacial acetic acid; (to 10%)
125 ml Ethanol; (to 25%)
0.25 g coomassie brilliant blue; (Sigma No. B-1131)
distilled water to 500 ml, store at room temperature (reusable)
destain - Same as stain less the coomassie
aqueous coomassie
0.25 g coomassie brilliant blue (Sigma No. B-1131)
distilled water to 500 ml, store at room temperature (reusable)
1X PBS
0.1 M sodium phosphate buffer pH 7.2
0.7 % NaCl