Editing FLP recombinase Expression

Escherichia coli Strain HBlOl&nbsp; was used for constcuction and propagation of<br/>plasmids.

The construction of the substrate plasmids pBAl12, pLB112 and pCS38<br/>has been described previously (Andrew6 et al.. 1985; Beatty L SedOW6ki. 1988;<br/>described previously Isabineau. et al, 19851. SChWartz 6 Sadowski. 19901. Plasmids pov56. pDv64, cvI9 and pBAlO4 were<br/>Plasmid pLO3 contains the FLP codlng sequence joined to the<br/>promoter. This plasmid was constructed in three steps [see Figure 81. (a1<br/>ribosome binding site of the T7 gene 10 protein downstream from the Tl(10<br/>The entire FLP coding sequence contained on a 1.4 kb fragment was exclsed<br/>pRR3039 (from F.W. Studieri. This plasmid contains the T7t10 promoter. the<br/>from plasmid pDv56 with SrI and ligated into the =HI site of plarnld<br/>tranblation initiation site for the T7 gene 10 protein, a %HI cloning Site<br/>followed by the transcrlptian terminator for T7 RNA polymerase. Cloning of<br/>the FLP gene fragment from pOV56 gives a coding sequence consisting of the<br/>first 11 codons from the gene 10 protein, four extraneous codons followed by<br/>the entire FLP coding sequence. This plasmid was called pDC-1 (Figure EA!.<br/>Although this Construct produced a larqe amount of a protein that was the<br/>correct size to be the FLP fusion product inor shown!. the protein was<br/>apparently insoluble and inactive. Plasmid pDC-1 was therefore modified to<br/>remove the extraneous amino acid codons in front of the FLP coding region.<br/>lb) Plasmid pOY56 was linearized with *I, which cuts in the ATG initiation<br/>codon of the FLP gene. The 3' protruding ends were made blunt with T4 DNA<br/>polymerase and deoxytriphosphates and a double nonphosphorylated +I linker<br/>precipltation to remove excess linkers and heating to 80' for 5 minutes. the<br/>15'-CATATGCRTATG-3'1 was ligated to the blunted termini. After spermine<br/>plasmid termini were allowed to anneal and the resultant plasmids were<br/>transformed into HB101. This pcocedure resulted in the replacement Of the<br/>*I site in the ATG of the FLP gene with an -1 site. The resulting<br/>plasmid was called pLB4 (Figure 801. (c1 A 1.1 kb fragment from pLB4 was<br/>excised with *I and transferred to plasmid pDC-1 Chat had been Cut with<br/>+I. The resulting plasmid. pLD3. has therefore lost the 15 extra codons<br/>that were originally present in pDC-1 (Figure 8Cl. The DNA sequence of the<br/>junction ceglon was verified by DNA sequencing. The plarrold was then<br/>Brookhaven ~ational Laboratory!. This Strain contains a lambda prophage with<br/>transferred into strain BL2 (*DE31 pLy6 5 [obtained fcom F.W. Studier,<br/>the gene for Tl RNA polymerase under control of the & VV 5 pcomoter as well<br/>as a plasmid (p~yssl that constitutively expresses the qene encoding T7<br/>polymerase activity that ib made from the & UV5 promoter (Studier et al..<br/>lysozyme. This enzyme binds to and inhibits any basal amount of T7<br/>19901.&nbsp;

Assays of PLP Activity<br/>which Contains the A form of the 2 micron plasmid. One unit of activity is<br/>ILP activity in crude extracts was assayed using the inversion plasmid cv19.<br/>B form of the 2 micron plasmid (Bablneau et al., 19851. The activity of FLP<br/>the amount Of protein required to invert 50 per cent Of the Substcate to the<br/>was followed during the pur~flcdtion procedure using the llnear excision<br/>Substrate, plasmid pBRlO4. A unlt Of activity is the amount of FLP required<br/>to excise 50 per cent of the substrate (Babineau et al., 19851. The<br/>composition of vac~ous fractions was assessed by SDS polyacrylamide gels 115<br/>per cent separatlng el. cast in Bio-Rad mini-spparaturl, followed by<br/>staining with coomassie blue. Protein concentrations were detemlned by the<br/>Bradford colorlnetric method [Bradford, 19761.<br/>Pucification of FLP Protein<br/>Induction of PLP expression from Tl promoter We previously induced FLP<br/>expression from the promoter by the addition of IPTG to the medium<br/>pcarnoter using the same protocol. we were unable to detect any activity at<br/>IBebineau et al., 19851. when we attempted to produce FLP from the T7<br/>levels Of protein where FLP activity was readily detectable fcm the<br/>construct. It was possible that the FLP protein was being highly expressed<br/>from the Tl promoter, but that the protein vas inactive in the 5. cell<br/>amount of a band that coaigrated with authentic FLP protein when cells<br/>[Marston, 19861. This hypothesis was confirmed by the finding of a large<br/>containing the plasmid pL03 were lnduced with IPTG, and analyzed by SDS-PAGE<br/>fallwed by coomasrie blue staining Inat shown). This band was present one<br/>hour after induction and its intensity increased markedly after three hour6<br/>of induction. It therefore seemed that a large amount of inactive FLP<br/>protein was being expressed from the Tl promoter. We therefore sought<br/>methods which might facilitate the synthehls of enzymatically competent<br/>protein and found that inductlan of FLP expression at low tenrperature led to<br/>the synthesis of substantla1 amounts of active protein. When we induced FLP<br/>expression at 2S0c we obtalned about forty-fold more FLP activity in crude<br/>cell extracts than we previously detected in cell extracts where FLP was<br/>expcessed from the % promoter at 31'.<br/>Growth of cells A 200 1 volume of LB Brath 110 9 Tryptone. 5 g yeast<br/>extract. 5 g NaCl, 0.1 ml. of 10 N NaUH per I! was inoculated with a 2 liter<br/>overnight culture of strain PSl31 IBL2IACE31, pLysS, pLO31 that had been<br/>grown at 31.. Both cultures canrained 20114 per ml of ampicillin and 30ug per<br/>ml of chloramphenicol. The ZOO 1 culture was grown at 31' with an air flow<br/>of 4 cubLC feet per minute, and an agltation speed of 200 cpm in a New<br/>cooled to 22' and solid lPTG was added to a concentration of 5OppI. Growth<br/>Brunswick fermentor, After the reached 0.73. the temperature was<br/>was continued far six hours at 22O: the cells ".ere collected by<br/>Centrlfugation and stored at -70'.&nbsp;<br/>Preparation of crude extras 1\11 the procedures were carried out at 0.4'~<br/>weight1 were thawed with gentle stirring on ice in 450 m1 of sonication<br/>unle55 otherwise indicated. One hundred and fifty grams Of frozen cells (wet<br/>mixture was sonicated for 3 minutes to suspend the cells and then for twenty<br/>buffer I50mM Tris-C1, pH 1.4, 50mM INH412SD4, 1nM EUTR, 0.5mM OTTI. The<br/>periods of one minute. interspersed wrth 45 seconds of coaling in an ice-salt<br/>bath. The sonicated extract I520 ml 18 ag/ml Of protein] was centrifuged in a<br/>Beckman Type 70 Ti rotor at 40,000 rpm for 40 nin. The supernatant fraction<br/>1500ml. 14 mq/ml Of protein) was loaded directly onto a Bio-Rex 10 column.<br/>- Bio-Rei 10 chromatography Bia-Rex 70 cesln 1100-200 mesh1 vas packed into d<br/>radial flow column (8.6 cm diameter. 11.5 cm high, superflo-500 column.<br/>Sepragen Corpocat~onl. The column was equlllbrsted with BiO-Rex buffer 150mn<br/>NaP04 buffer, pH 6.5. laH EDTA. 0.5~" DTT, 158 glycerol). The crude extract<br/>was applied dlrectly to the column and the pa%s-throUgh fraction 1500 nll was<br/>Cole-Parmer pump. The column was then washed with 1000 m1 of Bio-Rex buffer.<br/>collected. The flow rate was maintained at 25-35 ml/nrin with the aid of a<br/>with I000 a1 of Bio-lex buffer containing 700 an NaCl. The two 700 mM NaC1<br/>and eluted with 1500 ml of Bio-Rex buffer containing 300 nM NaCl and finally<br/>fractions (500 ml each1 contained >90% of the FLP activity. They were pooled<br/>and the protein was concentrated with ammonium Sulfate.<br/>Ammonium sulfate precipitation The pooled 700 all NaCl fractions from the<br/>010-Rex column (1000 mlI were brought to 60% Saturation by the addition of<br/>390 grams of sol~d ammaniun sulfate Iultrapure, Schwarz/Mann Biotech.] which<br/>was dissolved by stirring at 4'c for 30 mln.. The protein was pelleted by<br/>precipitate vas dlssalved in 25 a1 of Blo-Rex buffer IRS fraction, pcotein<br/>centrifugation at 16.000 rpm for 20 m~n. in the Sarvall SS34 rotor. The<br/>concentration - 19 ng/mli and the fraction was frozen immediately at - 10'C

http://www.jbc.org/content/266/17/11347.long<br/>""til application to a gel filtration column.<br/>s?@hcryl 5-300 gel filtrat_lo-n chromatography A Sephacryl 5-100 IPharmacial<br/>column was packed at room temperature 15 Crn x 90 cm: 1,640 ml bed voluneI and<br/>equilibrated I" the cold wlth 5-100 elutlon buffer I50aN NIP04 buffer, pH<br/>6.7. ImH EDTA. 1mM DTT, 20% glycerol and 150aM NaC11. Ten a1 of the AS<br/>fraction were loaded onto the column and elution was continued with 5-100<br/>buffer at a flowrate of 2.5 al/min. The first 300 ml were collected as a<br/>single void fractlon and then 12 ml fractlons Yece collected with a fraction<br/>collector. Prote~n cancentrat~an was monitored by Bradford assays. The FLP<br/>actlvity eluted after about 0.6 column volume and about 15% of the starting<br/>activity was present in 6 fractions Of specific activity greater than IO5<br/>units per mg of protexn [Fig. 91\ lanes 2-11. These fractions were pooled and<br/>stored at -7O'C.<br/>FPLC ion exchange chromatography &n FPLC cation exchange column Iflono S HR<br/>5/5. 5x50 mnl was equllibrated in buffer A 150aM NaP04 buffer, pH 6.1, 0.2mN<br/>EDTA. ImH UTT. 25% glycerol) on a FPLC system (Phacmacia). Nine nl 14.1 mg<br/>protein) of the pooled fractions from Sephacryl s-300 column was directly<br/>applied to the column at a fiawrate of 0.5 al/ain at room temperature. The<br/>A. protein concentration was monitored by uv absorbance at 280 m and 0.5 ml<br/>&nbsp;ample vas eluted with a 32.6 ml salt gradient 10.00-700 ml NaCll in buffer<br/>fractions wece collected. FPLC fcactions were analyzed for purity by SDS-PAGE<br/>and staining with Coomassie Blue and for FLP activity. The pure fractions of<br/>specific actlvlty greater than 4.8~10~ units per mg Of protein were pooled<br/>and stared at -loot.<br/>CoYente on the purification The purification scheme is surnmarired in Table<br/>a fast flowrate. FurtheCmOre, the resin can be regenerated several tines with<br/>1. The initial Bio-Rex chromatography step used a radial flow column that has<br/>alkali and the column can be repacked. This step yields a fraction in which<br/>FLP in about 108 pure and the major contaminants are Of low molecular weight<br/>(Fig. 9 C and 0. lane 21. The Sephacryl column also flows rapidly, remove6<br/>meny Of these contaminants and results in a few fold purification with 61<br/>percent recovery [Fig. 9 C and D, lane 3. Table 11. In the purification we<br/>described here. we used 40 percent Of the annonivln Sulfate fraction. but<br/>double the volun~e can be applied to the Sephacryl column with little<br/>deterioration in purification. We have chromatographed less than 5 mg of the<br/>5-300 fraction to the Nono s column and recover 1.5-2 mg of homogeneous FLP<br/>per run. TO amass milligcamb of homogeneous FLP fcom 150 gcamb of frozen<br/>cells would require up to twenty FPLC runs, although several 1""s can be done<br/>in one day. This procedure is capable Of generating 30 mg of homogenous FLP<br/>Stability Of the purified ILP The aanonlum Sulfate fraction has been stable<br/>from 150 grams of cell6 with excellent yields.<br/>for one year at -70°C. The Sephacryl fractlons are stable far up to 6 months<br/>at -1O'C and will withstand three cycles of freezing and thawing. The FPLC<br/>several months.<br/>fraction can withstand freezing and thawing several times and is stable for<br/>when stained with Comna~sie Blue (Fig.90; 9C lane 41. Since the gel lane<br/>Purity Of the purified FLP FLP in FPLC fraction6 YEIS apparently homogenous<br/>contained 8 pg of FLP protein (Fig. 4C lane 41 and since 100 ng of protein is<br/>readily visible as a Coornambie slue-stained band in such a gel. we ebtinarc<br/>estimate of the degree of contamination, we stained the same gel with sllver<br/>that the pceparation is greater than 98% puce. To obtain a nore sensitive<br/>and found scarcely any contaminating bands. The Oo280/oo260 ratio was 1.99,<br/>indicating that the preparation 16 free of nucleic acid. The was<br/>11.61 using the Bradford method and bovine plesma albumin as the standard.&nbsp;<br/>The FLP protein war purified from an E.m transfarmant carry~ng plasmid<br/>pLD3 as described above. R Sephacryl S-300 fraction of greater than 85%<br/>purlty was used. ~estriction enzymes were from Boehringer-mnnhein, Pharaacia<br/>and BRL. AMY reverse transcriptase Yao fr
[[Category:Protein Expression]]