Patent Application: US-201414902804-A

Abstract:
disclosed is a method of preparing pure or substantially pure d - myo - inositol - 3 - phosphate from glucose - 6 - phosphate and / or fructose - 6 - phosphate . the method may also be applied to protected and / or derivative forms of glucose - 6 - phosphate and / or fructose - 6 - phosphate so as to form protected / derivative forms of d - myo - inositol - 3 - phosphate , for use in further chemical reactions . the enzyme d - myo - inositol - 3 - phosphate synthase is contacted with the glucose - 6 - phosphate and / or fructose - 6 - phosphate to generate labeled or unlabeled , protected or unprotected d - myo - inositol - 3 - phosphate , which may be further reacted and / or purified .

Description:
the present invention will now be further described by way of example and with reference to the figures which show : fig2 : shows the expression and purification of recombinant tbino1 : ( a ) tbino1 was cloned into the expression vector pet 15b ( n - terminal hexa - his tag ) and transformed into rosetta competent cells , and grown , expressed and purified as described in experimental procedures . tbino1 protein samples from each purification step were separated on a 10 % sds - page gel and stained with coomassie brilliant blue . lane 1 , cleared lysate ; lane 2 , unbound to ni ′ column ; lane 3 , washed column ; lane 4 , pooled elutions from ni 2 + column ; lane 5 , pooled elution from hi - trap blue sepharose . ( b ) purified recombinant tbino1 was passed through a high - resolution gel - filtration sephacryl s - 300 column . absorbance at 280 nm was monitored against elution volume . the column was calibrated with globular protein standards ( biorad ). fig3 : shows the separation of reaction products from tbino1 assay by dionex hplc . panel a shows separation of reaction products from standard assay using glucose - 6 - phosphate as substrate ( i ), with elution at identical times as standards of inositol - 1 - phosphate ( ii ), glucose - 6 - phosphate ( iii ) and fructose - 6 - phosphate ( iv ). panel b shows separation of reaction products from assay mixture using fructose - 6 - phosphate as substrate at various time points . fig4 shows the expression and purification of surface immobilized recombinant tbino1 on ni sepharose : ( a ) tbino1 was cloned into the expression vector pet 15b ( n - terminal hexa - his tag ) and transformed into rosetta competent cells , and grown , expressed and purified as described in experimental procedures . tbino1 protein samples from each purification step were separated on a 10 % sds - page gel and stained with coomassie brilliant blue . protein gel from typical tbino1 affinity chromatography purification . w = whole cell , s = supernatant , ft = flow through , elution &# 39 ; s of 10 , 20 , 50 , 100 , 250 and 400 = concentration of imidazole in mm . fig5 shows a 400 mhz 1 h nmr spectra in d 2 o of d - myo - inositol - 3 - phosphate product obtained after a 12 hour period of 10 mm d - glucose 6 - phosphate , 2 mm nad + , 1 mm 2 - mercaptoethanol and 50 mm ammonium bicarbonate ( ph 8 . 5 ) solution passed through a 5 ml ni - sepharose tbino1 affinity column , prepared as described herein . the spectrum has been annotated to indicate which proton signals have been assigned to the corresponding adjacent c1 - c6 carbons , numbered as shown in the representation shown above the spectrum ). the tbino1 was pcr amplified from tbino1 - pbad ( described previously { martin , 2006 # 113 }) using the primers 5 ′- ctcgag atgccagccgtccgtacg - 3 ′ and 5 ′- ggatcc tcaacttcccacgccgcc - 3 ′ containing xhoi and bamhi restriction sites respectively ( underlined in primer sequences ). the purified pcr product was digested with xhoi and bamhi and ligated into pet15b ( invitrogen ) via the same restriction sites and sequenced . site directed mutagenesis was performed using a quickchange kit ( stratagene ) according to manufacturers &# 39 ; instructions with tbino1 - pet15b as the template and sequenced to confirm presence of desired mutation . the pair of complementary primers to mutate the selected amino acids of tbino1 to alanine are as follows ; the over expression construct of interested was freshly transformed into bl21 rosetta ( de3 ) cells ( invitrogen ). a single colony was used to inoculate lb - amp broth and the cells were grown at 37 ° c . with shaking until the od ( 600 nm ) was approximately 0 . 6 . protein expression was induced by the addition of 1 mm iptg , and the cells were grown further 16 hrs at room temperature . tbino1 has been over - expressed using the expression plasmid pet15b ( invitrogen ), which has increased the yield of recombinant protein from approximately 2 mg / l to 20 mg / l . tbino1 was highly purified via a three step purification protocol using ni - nta affinity , hitrap blue sepharose affinity and size exclusion chromatography . small - scale protein purification of scino1 and the various site directed mutated tbino1 were performed according to martin and smith unpublished . for larger scale purification of wild type tbino1 for biochemical characterisation , the cells were suspended in buffer a ( 20 mm tris ph 7 . 5 , 250 mm nacl , 50 mm imidazole , 3 mm 2 - mercarptoethanol ) and lysed using a one - shot cell disrupter ( constant systems ) at a pressure of 30 000 psi . insoluble protein and cell debris was collected by centrifugation ( 50 000 g , 30 min 4 ° c .). the resulting supernatant was loaded onto a hi - trap chelating sepharose hp column ( amersham ) which was pre - equilibrated with buffer a . protein was eluted with a linear gradient of 0 - 100 % buffer b ( 1 . 6 m imidazole ) with tbino1 elution with approximately 25 % buffer b ( 400 mm imidazole ). fractions were chosen based on purity determined by sds - page analysis and dialysed extensively against buffer c ( 20 mm tris ph 7 . 5 , 50 mm nacl and 3 mm 2 - mercaptoethanol ) using snakeskin dialysis tubing ( pierce ) with a mwco of 10 kda . the dialysed sample was then applied to a hi - trap blue sepharose column ( amersham ) which had been pre - equilibrated with buffer c . protein was eluted with a line gradient of 0 - 100 % buffer d ( 2m nacl ). one pool of tbino1 was found to elute from the column prior to the nacl gradient , a second pool of tbino1 eluted with approximately 1 . 25m nacl . each pool was dialysed separately against buffer e ( 20 mm tris ph7 . 5 , 50 mm nacl and 5 mm dtt ) prior to concentration to approximately 80 mg / ml using centrifugal concentrators ( mwco 30 kda , vivaspin ). the concentrated protein was then loaded onto a superdex 200 column ( amersham ) which had been pre - equilibrated with buffer e and eluted with the same buffer . fractions from this size elusion chromatography were chosen conservatively from the centre of the protein peak . the purity of tbino1 was confirmed firstly by sds - page ( fig1 a ) and secondly by maldi - tof / ms analysis ( fig1 b ), both methods showed the recombinant protein to have a molecular size of approximately 60 kda , which is in good agreement with the predicted size of 59 . 2 kda . a secondary peak is present on the maldi - tof / ms spectrum which corresponds to the doubly protonated species with a molecular weight of 30 . 4 kda . size elusion chromatography data showed that in its native state tbino1 predominantly exists as a tetramer ( fig1 c ), which is supported by data from analytical ultra centrifugation ( fig1 d ). various glucose analogues ( 10 - 45 mg ) were dissolved in 1 ml of tris . hcl ( 100 mm , ph 8 . 0 ) long with atp ( 100 mm ) and mgcl 2 ( 50 mm ), yeast hexokinase ( 100 units ) was added and incubated at 30 ° c . overnight . the reaction mixture is passed through a 5 × 1 cm column of dowex ( ag1 , borate form ) to bind excess phosphate and eluted with a gradient of borate , followed by a 3 × 1 cm column of ag50w × 8 ( h + ) to form the free acid . eluted products were freeze - dried and checked by es - ms prior to phosphate quantification by acid phosphatase treatment followed by malachite green analysis . the following compound were prepared galactose - 6 - phosphate , 2 - fluoro - glucose - 6 - phospahte , 3 - fluoro - glucose - 6 - phospahte , 4 - fluoro - glucose - 6 - phospahte , 3 - o - methyl - glucose - 6 - phospahte and glucose - 1 - o - methyl - 6 - phospahte . an assay to test the glucose - 6 - phosphate isomerase activity of tbino1 was performed using a coupled assay with inositol - 3 - phosphate monophosphatase ( impase , available from sigma ). glucose - 6 - phosphate is isomerised into d - inositol - 3 - phosphate by the action of tbino1 . the reaction is monitored by following the concentration of phosphate from the formed inositol - 3 - phosphate ( not glucose - 6 - pbosphate ) which can be cleaved by impase to leave myo - inositol and free phosphate . the concentration of phosphate released can then detected colormetrically by biomol ( malachite ) green by measuring absorbance at 620 nm . therefore the amount of inositol - 3 - phosphate formed from glucose - 6 - phosphate can be monitored . a portion of the reaction ( 10 ul ) was removed and the reaction stopped by heating to 100 ° c . for ten minutes . the impase assay was then performed on this small aliquot to ensure all the glucose - 6 - phosphate had been isomerised into inositol - 3 - phosphate , i . e . gone to completition . the impase part of the coupled assay was tested first in order to verify the commercially bought impase activity and obtain a standard curve for phosphate detection . the assay was run in a clear 96 well plate with 50 μl of reaction mixture in each well with the following constituents with either inositol - phosphate or free phosphate as the substrate : the reaction was started by adding impase to each well last and once added the plate was incubated for 1 hour at 37 ° c . the reaction was stopped by heating to 100 ° c . for 10 minutes , followed by the addition of biomol ( malachite ) green ( 100 μl ). the plate was incubated at room temperature for 20 minutes and then the absorbance at 620 nm was read using a spectrophotometer . a control with impase replaced with water was used to account for any free phosphate contained in the reaction mixture . the phosphate standard curve was formulated using free phosphate as the substrate and this standard curve was used to calculate the amount of inositol - 1 - phosphate cleaved by the impase . the final product ( inositol - 3 - phosphate ) was obtained from the reaction mixture by first removing the protein from the reaction mixture by centrifugation with a protein membrane ( 10 kda molecular weight cut off ) and then an overnight freeze - drying to remove the water and the ammonium acetate buffer ( which evolves as ammonia , carbon dioxide and water ), yielding essentially pure inositol - 3 - phosphate . an alternative reaction mixture consisted of 2 mm tris - ac ( ph 9 ), 2 mm glucose - 6 - phosphate , 1 mm nad + , 1 mm dtt , 2 mm nh 4 ac and 5 μg protein in a final volume of 20 μl . the reaction was incubated at 37 ° c . for 1 hr and terminated by heating at 100 ° c . for 10 min and the addition of 804 of 10 mm naoh . for inhibitor studies the standard reaction mixture was prepared without glucose - 6 - phosphate , the inhibitor was added to the reaction and incubated on ice for 10 min prior to the addition of glucose - 6 - phosphate and subsequent incubation of 37 ° c . for 1 hr . after quenching the reaction mix was analysed by high performance anion - exchange chromatography using a dionex hplc system with a carbopacpa - 1 column and pa - 1 guard column ( dionex ). the column was pre - equilibrated with 95 % buffer a ( 1 mm sodium hydroxide ) and 5 % buffer b ( 1m sodium acetate + 1 mm sodium hydroxide ). an aliquot ( 204 ) of the quenched reaction mixture was injected onto the column and eluted a linear gradient of 5 - 40 % buffer a , detected with pulsed amperometric detection and analysed with chromeleon software ( dionex ). the substrate glucose - 6 - phosphate and the product inositol - 3 - phosphate are clearly separated by this method . previously preliminary biochemical characterisation of tbino1 was performed using a coupled assay with a commercial inositol - 3 - phosphate monophosphatase ( impase , as described above ) and detection of the release of free phosphate by colorimetric analysis ( martin and smith 2006 ). however , to overcome the disadvantages of using this assay system , in particular the potential inhibition of the impase and the high levels of free phosphate in some inhibitors to be tested , it was decided to employ high performance ion - exchange chromatography to quantify enzyme activity ( described above ). using this method glucose - 6 - phosphate and inositol - 3 - phosphate were successfully separated from each other , as well as from other components of the reaction mixture ( fig2 a ). an additional peak was observed in the traces from enzyme assays , which was not present in the control reactions ( enzyme and substrate blanks ) ( fig2 a ), suggesting that the unidentified peak was a result of catalysis by tbino1 . this unidentified peak had an identical elution time as fructose - 6 - phosphate , suggesting that in the standard reaction mixture with glucose - 6 - phosphate as the substrate , tbino1 was able to form fructose - 6 - phosphate in addition to inositol - 3 - phosphate . fructose - 6 - phoshate was tested as a substrate in the standard reaction mixture , peaks with identical elution times to fructose - 6 - phosphate , glucose - 6 - phosphate and inositol - 3 - phosphate standards respectively , were observed ( fig2 b ). when the reaction was allowed to proceed for longer time periods only inositol - 3 - phosphate was observed ( fig2 b ), showing the quantitative conversion of fructose - 6 - phosphate to inositol - 3 - phosphate with an intermediate conversion to glucose - 6 - phosphate . the production of myo - inositol - 3 - phosphate from fructose - 6 - phospahte was confirmed two - fold , firstly the presence of myo - inositol was confirmed by gc - ms analysis after acid hydrolysis , and secondly this was shown to be inositol - 3 - phosphate , as it was used as a substrate for bovine impase ( data not shown ). the production of glucose - 6 - phosphate from fructose - 6 - phosphate was established using a traditional assay for glucose - 6 - phosphate isomerase activity , via a coupled assay with glucose dehydrogenase . glucose - 6 - phosphate isomerase activity was measured using the coupled assay described previously [ gracy and tilley 1975 ]. the standard assay mixture contained 0 . 1 mm tris - hcl ( ph 9 ), 2 mm edta , 0 . 5 mm β - nadp + , 1 mm fructose - 6 - phosphate , 1u glucose - 6 - phosphate dehydrogenase ( type xv from bakers yeast , sigma ) and 100 μg recombinant protein . enzyme activity was measured as an increase in absorbance at 340 nm . one unit of activity is defined as 1 μmoles of p - nadp + reduced per minute under the assay conditions . using the crystal structure of the archaeolglobus fulgidus ( at ) ino1 and associated site directed mutagenesis data , as well as the crystal structure of the saccharomyces cerevisiae ino1 , a series of tbino1 mutants were generated , over - expressed in e . coli and purified using ni - nta beads , expression levels of all the mutant proteins was approximately the same as wild type tbino1 . after purification the mutated recombinant protein was tested for activity using the standard reaction mixture using either glucose - 6 - phosphate or fructose - 6 - phosphate as the substrate and the reaction products separated and quantified by hplc as described previously . with the exception of the tbk353a mutant , all the tbino1 mutations resulted in the complete abolishment of the ino1 activity while retaining normal glucose - 6 - phosphate isomerase activity . the k353a mutant resulted in a decrease of ino1 activity to approximately 10 %, whilst retaining all glucose - 6 - phosphate isomerase activity . it was not surprisingly that mutant k353a lost ino1 activity whilst retaining glucose - 6 - phosphate activity as the corresponding afino1 mutant was unable to bind nad + which is essential for ino1 activity but not required for glucose - 6 - phosphate isomerase activity . however , the ino1 activity was not completely abolished in tbk353a suggesting that there may be weak binding of the nad + still occurring . afd332 was suggested to be a ligand for the second metal ion required for catalysis and afk306 is thought to work in with afd332 aiding in this interaction , and therefore the mutation of this residues resulted in the loss of activity as afino1 has a requirement for metal ions for activity . however , tbino1 does not require this metal ion for catalysis , but surprisingly mutation of the corresponding amino acids k421a and k395a in tbino1 resulted in the total loss of ino1 activity , suggesting that in tbino1 these residues has another essential roles for ino1 activity . however , mutation of these residues had no effect on the pgi activity , suggesting that they do not play crucial roles in pgi activity . mutant afk367a displayed no glucose - 6 - phosphate binding and therefore was inactive . likewise the corresponding mutant in tbino1 , k477a was inactive . however , this mutant still displayed glucose - 6 - phosphate isomerase activity . suggesting either that there is a secondary active site on the protein for glucose - 6 - phosphate isomerase activity , or that glucose - 6 - phosphate is still binding to the protein , but the mutation is disrupting something else that is essential to the ino1 activity . all compounds listed in tables 2 and 3 were tested as potential substrates for tbino1 in the standard reaction mixture and the reaction products separated by hplc . however , no reaction products were observed for any of the compounds tested showing that tbino1 has a strict substrate specificity for glucose - 6 - phosphate and fructose - 6 - phosphate . these compounds were then tested for their ability to inhibit the tbino1 pgi or ino1 activity . the tbino1 was pre - incubated with potential inhibitor prior to the addition of glucose - 6 - phosphate or fructose - 6 - phosphate and quantification of inositol - 3 - phosphate and fructose - 6 - phosphate by hplc . a number of glucose - 6 - phosphate analogues were generated , and these in addition to commercially available phosphorylated sugars were tested and the results shown in table 2 . substitution of the 2 - position of glucose - 6 - phosphate resulted in an inhibitor of both ino1 and pgi activity , interestingly substitution with fluoro - was more potent than the deoxy - derivative . there was no inhibition observed with a substitution in the 2 - position of a non - phosphorylated glucose , indicating that phosphorylation is important for inhibition . substitution in the 3 and 1 - positions also resulted in inhibition of both ino1 and pgi activities . no inhibition of ino1 or pgi activites were observed in the presence of glucose - 1 - phosphate , glycerophosphate , 6 - phosphogluconic acid , acetylglucosamine - 1 - phosphate , acetylglucosamine - 6 - phosphate , mannose - 6 - phosphate and galactose - 6 - phosphate . a number of metabolic intermediates were also tested as potential inhibitors against ino1 and pgi activity , the results are shown in table 3 . inhibition was observed by gap , oaa , dhap against both ino1 and glucose - 6 - phosphate isomerase activity . interestingly no inhibition was observed due the presence of the non - phosphorylated counterparts of gap and dhap , glyceraldehyde and dha respectively , suggesting that the phosphate group is essential for efficient binding . no inhibition was observed in the presence of pep , pyruvate or vpa . an affinity column of immobilized tbino1 was prepared as follows . lysed e . coli was filtered and loaded onto a ni 2 + sepharose column , generally as described above ( in the section entitled , “ protein over expression and purification ”). loading was in the amount of 40 mg proteinaceous material per ml of resin beads . the column was then washed with a buffer solution of ( i ) 10 mm imidazole ( ii ) 50 mm ammonium bicarbonate ( at ph 8 . 5 ) ( iii ) 250 mm sodium chloride . subsequently a further wash with 50 mm ammonium bicarbonate solution was conducted . the purity of the immobilized tbino1 was confirmed by sds - page ( fig4 ). referring now to scheme 1 , below , columns of capacity 1 ml and 5 ml were prepared . 4 mm , 10 mm and 20 mm d - glucose 6 - phospate solutions ( in an nad + , 2 - mercaptoethanol , ammonium carbonate buffer solution ; ( ph 8 . 5 ) were prepared , as described above . the 4 mm d - glucose 6 - phospage solution was passed through the 1 ml column at a flow rate of 2 . 5 ml / hr for 12 hours . the 10 mm and 20 mm solution were each passed through the 5 ml column at the same flow rate and for the same period . the temperature of each column was maintained at 37 ° c . throughout . following purification by freeze - drying ( to remove the ammonium carbonate ), 42 mg of inositol - 3 - phosphate ( ino3p ) product was obtained from the 4 mm glc - 6p solution passed through the 1 ml column . the highest yield (˜ 100 %) from the 5 ml column , of 210 mg ino3p , was obtained was obtained using the 20 mm glc - 6p solution . nmr spectra of the purified ino 3p product were obtained and assigned and are consistent with high percentage conversion of the glc - 6p reagent into an enantiometrically pure product . an example spectrum ( obtained from the 10 mm glc - 6p solution ) is shown in fig5 . these initial results demonstrate the utility of the method for preparing large amounts of enantiomerically pure d - ino3p ( i . e . l - ino1p ) using a continuous flow process . in addition , the high yield from the 20 mm glc - 6p solution demonstrates how the use of an affinity column facilitates re - use of the immobilized ino1 enzyme . 1 . potter , b ., and lampe , d . 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