BACKGROUND
the 2-oxoglutarate-dependent dioxygenases  of gibberellin  biosynthesis have a key role in plant hormone metabolism.
three principal classes of 2odds control the later stages of the pathway where bioactive ga is produced and inactivated .
activation and deactivation of the ga molecule are performed by ga 3-oxidase and ga 2-oxidase enzymes, respectively.
the genes encoding ga 2odds have been described from many species.
they belong to multigene families which show spatial and temporal patterns of expression  <cit> .
factors including hormone levels and light have been shown to influence ga 2odd gene expression  <cit> , suggesting that the enzymes are important in regulating the overall ga pathway.
the genetic manipulation of ga levels for agricultural purposes has often involved 2odd-catalysed steps.
the ga 2odds are well suited to in vitro studies because they retain good activity when expressed in escherichia coli and their natural substrates are mostly known and available in synthetic form.
thus, a reasonable amount of data relates to the activity of recombinant ga 2odds, e.g.
<cit> .
their catalysis generally appears to reflect that of the native enzymes.
unfortunately, the excellent literature on the activity of recombinant ga 2odds is accompanied by a lack of progress on their purification and structural characterisation.
in native form the enzymes displayed a lability that hindered their complete purification  <cit> .
the problems associated with this lability have ostensibly not been overcome through using recombinant expression methods.
the sole report on a pure ga 2odd relates to a native ga 20-oxidase from pumpkin endosperm  <cit> .
herein, we report the successful purification of a recombinant ga 2-oxidase from pisum sativum l.  and a ga 3-oxidase from arabidopsis thaliana , findings that represent a significant step for an important group of enzymes.
RESULTS
expression and purification of the ga 2-oxidase, psga2ox1
the pea ga 2-oxidase, psga2ox <dig>  accumulated intact and in soluble form in e. coli when expressed as a gst fusion in pgex.
the expression conditions employed were determined to be optimal with respect to incubation temperature and induction conditions.
trials with a gst fusion expression system driven by the stronger t <dig> promoter did not improve expression levels .
affinity chromatography of gst-psga2ox <dig> from the soluble cell fraction gave a high degree of purification .
subsequent removal of the gst tag yielded psga2ox <dig> with only two major contaminants .
gel filtration removed these contaminants without significant loss of psga2ox <dig> and increased its purity to > 95% as estimated from sds-page analysis .
pure psga2ox <dig> was obtained in milligram quantities and was concentrated to  <dig> mg/ml .
psga2ox <dig> was active at all stages of purification .
edta had an inhibitory effect on activity that was removed most effectively by dialysis rather than dilution.
however, when crude samples were dialysed, activity diminished, presumably because of enzyme inactivation or degradation.
in addition, endogenous components of e. coli lysates are suspected to inhibit ga 2odds  <cit> .
therefore, the activity measurements after the first two stages of purification  gave apparent values.
concentrated highly pure psga2ox <dig> retained activity after storage at -70°c.
kinetic analysis of psga2ox1
the activity of this pure psga2ox <dig> displayed a saturation curve of the michaelis menten type in response to increasing concentration of substrate ga <dig> .
the km was calculated to be  <dig>  μm and the vmax  <dig>  pkat/mg.
expression and purification of the ga 3-oxidase, atga3ox4
the arabidopsis ga 3-oxidase, atga3ox <dig>  accumulated intact and in soluble form when expressed as a poly-tagged trx fusion in pet <dig> to a level that was discernible by sds-page analysis of the soluble cell fraction .
during purification atga3ox <dig> tended to precipitate, although precipitation could be minimised during the first step by dilution of the soluble cell lysate and the use of a buffer of ph  <dig>  with  <dig> mm β-me.
trx-atga3ox <dig> was purified to a high degree by immobilised metal affinity chromatography .
cleavage of the trx tag from atga3ox <dig> was enabled by a thrombin site engineered into the expression construct – the factor xa site encoded by pet <dig> failed to cleave.
atga3ox <dig> was difficult to separate from trx, remaining strongly associated with it subsequent to thrombin cleavage.
the single factor identified that most obviously mitigated this association was β-me, with low ph further aiding separation.
resolution of the two proteins was achieved by gel filtration in a buffer of ph of  <dig>  with  <dig> mm β-me.
highly pure atga3ox <dig> was thus obtained in microgram quantities .
once pure, atga3ox <dig> resisted concentration.
the problem was partially overcome by the addition of triton x- <dig>  however, the maximum concentration of atga3ox <dig> achieved was  <dig>  mg/ml and its activity did not tolerate freezing.
otherwise, atga3ox <dig> was active at all stages of purification, permitting a kinetic study on highly pure atga3ox <dig>
removal of the trx tag had no obvious affect on atga3ox <dig> activity levels.
triton x- <dig> had an inhibitory effect but this seemed to disappear with dilution.
the use of low ph buffers during purification did not obviously dampen atga3ox4's activity.
indeed, we found atga3ox <dig> displayed  <dig> % activity when assayed in the phosphate buffer of ph  <dig>  used for the first purification step, providing the reducing agent dtt was present .
however, the buffer of ph  <dig>  in the second step was not compatible with activity assays.
in light of the difficulties associated with obtaining atga3ox <dig> in high yield, further purification of the fusion, trx-atga3ox <dig>  was conducted.
milligrams of highly pure trx-atga3ox <dig> were obtained.
the protein was amenable to concentration  and could be frozen at -70°c without activity loss.
kinetic analysis of atga3ox4
the activity of highly pure atga3ox <dig> displayed a saturation curve of the michaelis menten type in response to increasing concentration of ga <dig> .
the km was calculated to be  <dig>  μm and the vmax  <dig>   <dig> pkat/mg.
discussion
high levels of activity are frequently obtained for ga 2odds in e. coli expression systems in the absence of accumulated soluble enzyme  <cit> .
the instability generally reported in native 2odds appears to be a feature of the recombinant enzymes.
in early trials of ga 2odds, the pea ga 2-oxidase, psga2ox <dig>  and the arabidopsis ga 3-oxidase, atga3ox <dig>  showed promise as candidates for purification .
in both cases, a n-terminal fusion tag was needed to obtain soluble intact protein.
the purification of the pea ga 2-oxidase, psga2ox <dig>  was straightforward.
no problems were encountered during chromatography, tag removal or enzyme concentration.
all steps of purification were performed in a buffer that preserved enzyme activity.
the ph was within physiological range when the chelator edta and the reducing agent dtt were present.
.
the milligram amounts of psga2ox <dig> that we obtained are unprecedented for a highly pure ga 2odd.
the gene encoding psga2ox <dig> is affected by the null mutation, sln, which causes the accumulation of ga <dig> within maturing pea seed  <cit> .
the mutation has a lesser effect on ga <dig> levels in the stem, presumably because other ga 2-oxidases compensate for its loss in this tissue.
mature seeds contain at least two ga 2-oxidases  <cit> .
the sln phenotype indicates that psga2ox <dig> is the main catalyst for the conversion ga <dig> to ga <dig> at latter stages of seed maturation, when 2-oxidation of ga <dig> takes place at high levels.
although the sln mutation is not deleterious to developing seeds, the mature seeds contain abnormally high amounts of ga <dig>  which, after germination, is converted to the bioactive ga <dig> resulting in the slender  phenotype of the seedling  <cit> .
the levels of ga20present in mature sln and sln seeds are  <dig> ng/g and  <dig>  ng/g, respectively  <cit> .
the difference can be solely attributed to the action of psga2ox <dig> in planta.
multiple ga 2-oxidases have been previously partially purified in low yield from pea seeds of unspecified maturity  <cit> .
the incomplete purification was attributed to a low abundance of the enzymes in the tissue, and instability during purification.
it can be assumed that native psga2ox <dig> was among these ga 2-oxidases because its gene is expressed throughout seed maturation  <cit> .
recombinant expression methods have now enabled us to complete the successful purification of psga2ox <dig>
at  <dig>  μm, the km of recombinant psga2ox <dig> toward ga <dig> was much lower than that obtained for the native pea seed ga 2-oxidases   <cit> .
the comparison of km results is complicated, however, because the native preparation contained multiple activities.
certainly, a high substrate affinity of psga2ox <dig> is consistent with its ability to clear the maturing seed of ga <dig>
the gene encoding psga2ox <dig> is expressed relatively strongly throughout the latter weeks of pea seed maturation  <cit> .
that the enzyme is abundant in mature seeds was supported by a study of ga 2-oxidases from this tissue in the related species phaseolus vulgaris  <cit> .
if present in abundance, it is plausible that psga2ox <dig>  could account for the difference in ga <dig> levels between sln and sln seeds.
the vmax of the partially pure native ga 2-oxidases toward ga <dig> was  <dig>  pmol/h mg   <cit> .
the higher figure of the recombinant enzyme suggests that its purity and/or retention of activity was enhanced compared with the native preparation.
interestingly, the vmax of psga2ox <dig> was several orders of magnitude lower than the vmax of atga3ox <dig> toward ga <dig> .
however, this difference is difficult to interpret considering the two enzymes are from different species and are likely to be expressed differentially within the plant alongside isoenzymes.
with their role in ga activation, the ga 3-oxidases are particularly interesting to study, but in native form the enzymes are difficult to purify.
it required  <dig>  immature seeds for kwak et al.
<cit>  to obtain  <dig>  mg of enzyme described as partially pure.
recombinant methods enabled us to partially overcome the problems associated with purification of a recombinant ga 3-oxidase.
our experience provides valuable insight for future work.
the first step we used in the purification of the arabidopsis ga 3-oxidase, atga3ox <dig>  was imac chromatography.
this method does not permit the inclusion of ddt or edta in buffers and therefore has been considered unsuitable for purifying 2odds  <cit> .
nonetheless, atga3ox <dig> with high specific activity was obtained using this method.
evidently,  <dig> mm β-me was able to protect the enzyme active site from metal-catalysed oxidation during chromatography.
the result suggests that this widely used and highly efficient purification strategy does not necessarily compromise the activity of a 2odd.
atga3ox <dig> showed a tendency to precipitate, one that became more pronounced when it was cleaved, but not physically separated, from trx.
this diminished the yield of the protein, particularly in the final step.
a beneficial effect from low ph and β-me implied that an exposed cysteine was causing the precipitation.
however, a combination of factors were probably to blame because the aforementioned conditions did not fully overcome the problem.
furthermore, replacement by site-directed mutagenesis of the cysteine predicted as most likely to be exposed  brought no improvement.
certainly, atga3ox <dig> displayed hydrophobic behaviour when pure, sticking to tubes unless triton x- <dig> was present.
hydrophobicity was also a feature of the ga 3-oxidase of bean  <cit> .
good scope exists for further optimisation of the atga3ox <dig> purification method.
efficient separation of atga3ox <dig> and trx will lead to a greatly increased yield.
hydrophobic interaction chromatography is a possible alternative to gel filtration in separating the two proteins.
atga3ox <dig> was amenable to concentration and freezing in the presence of trx, whether or not it was chemically attached to it.
the identification of buffer ingredients that replace trx's stabilising role, such as alternative detergents, salts or organic solvents may improve the behaviour of purified atga3ox <dig>
atga3ox <dig> is encoded by a member of a family of four ga 3-oxidase genes in arabidopsis.
generally the non-13-hydroxylated ga pathway predominates in arabidopsis, however recent studies found comparable levels of non-13-hydroxylated and 13-hydroxylated gas in developing seeds  <cit> .
this tissue is the main site of expression of the gene encoding atga3ox <dig> , therefore ga <dig> is likely to be a natural substrate of atga3ox <dig>  the high vmax gained here shows that atga3ox <dig> can efficiently metabolise ga <dig>  the value of  <dig>  pkat/mg is higher than the vmax of  <dig> nmol/min mg  reported for a purified gst fusion of another arabidopsis ga 3-oxidase, atga3ox <dig>  <cit> .
atga3ox4's km of  <dig>  μm toward ga <dig> was lower than that of atga3ox <dig>   <cit> , yet higher than the value for the bean seed ga 3-oxidase   <cit> .
accumulated kinetic information with respect to both 13-hydroxylated and non-13-hydroxylated gas on each of the ga 3-oxidases of arabidopsis will give valuable insight into the control of active hormone levels in this model species.
CONCLUSIONS
the previous lack of reports on the complete purification of ga 2-oxidases and ga 3-oxidases is probably due to a combination of their lability and their low abundance in most plant tissues.
one might speculate that a crucial role in the control of active hormone levels requires a short enzyme half-life.
this study on psga2ox <dig> and atga3ox <dig> shows that ga 2-oxidases and ga 3-oxidases are not necessarily inherently unstable and can be purified active from e. coli expression systems under standard conditions.
indeed, the ga 2-oxidase, psga2ox <dig>  was exceptionally stable throughout purification.
problems were encountered with the arabidopsis ga 3-oxidase, atga3ox <dig>  due to its hydrophobicity and the presence of hydrostatic charges, rather than metal-catalysed autocleavage or proteolytic breakdown.
indeed, plant 2odds from secondary metabolic pathways are also problematic during purification  <cit> .
yet in some cases, they have been purified in high yield permitting detailed structural and mechanistic studies  <cit> .
we note the recent elucidation of the structure of the labile 2odd-related enzyme, acc-oxidase, of ethylene biosynthesis  <cit> .
our work opens the door to similar studies on the ga 2odds.

 the 2-oxoglutarate-dependent dioxygenases of gibberellin biosynthesis have a key role in plant hormone metabolism. three principal classes of 2odds control the later stages of the pathway where bioactive ga is produced and inactivated. activation and deactivation of the ga molecule are performed by ga 3-oxidase and ga 2-oxidase enzymes, respectively. the genes encoding ga 2odds have been described from many species. they belong to multigene families which show spatial and temporal patterns of expression. factors including hormone levels and light have been shown to influence ga 2odd gene expression, suggesting that the enzymes are important in regulating the overall ga pathway. the genetic manipulation of ga levels for agricultural purposes has often involved 2odd-catalysed steps. the ga 2odds are well suited to in vitro studies because they retain good activity when expressed in escherichia coli and their natural substrates are mostly known and available in synthetic form. thus, a reasonable amount of data relates to the activity of recombinant ga 2odds, e.g.. their catalysis generally appears to reflect that of the native enzymes. unfortunately, the excellent literature on the activity of recombinant ga 2odds is accompanied by a lack of progress on their purification and structural characterisation. in native form the enzymes displayed a lability that hindered their complete purification. the problems associated with this lability have ostensibly not been overcome through using recombinant expression methods. the sole report on a pure ga 2odd relates to a native ga 20-oxidase from pumpkin endosperm. herein, we report the successful purification of a recombinant ga 2-oxidase from pisum sativum l. and a ga 3-oxidase from arabidopsis thaliana, findings that represent a significant step for an important group of enzymes.  expression and purification of the ga 2-oxidase, psga2ox1 the pea ga 2-oxidase, psga2ox accumulated intact and in soluble form in e. coli when expressed as a gst fusion in pgex. the expression conditions employed were determined to be optimal with respect to incubation temperature and induction conditions. trials with a gst fusion expression system driven by the stronger t promoter did not improve expression levels. affinity chromatography of gst-psga2ox from the soluble cell fraction gave a high degree of purification. subsequent removal of the gst tag yielded psga2ox with only two major contaminants. gel filtration removed these contaminants without significant loss of psga2ox and increased its purity to > 95% as estimated from sds-page analysis. pure psga2ox was obtained in milligram quantities and was concentrated to mg/ml. psga2ox was active at all stages of purification. edta had an inhibitory effect on activity that was removed most effectively by dialysis rather than dilution. however, when crude samples were dialysed, activity diminished, presumably because of enzyme inactivation or degradation. in addition, endogenous components of e. coli lysates are suspected to inhibit ga 2odds. therefore, the activity measurements after the first two stages of purification gave apparent values. concentrated highly pure psga2ox retained activity after storage at -70°c. kinetic analysis of psga2ox1 the activity of this pure psga2ox displayed a saturation curve of the michaelis menten type in response to increasing concentration of substrate ga. the km was calculated to be μm and the vmax pkat/mg. expression and purification of the ga 3-oxidase, atga3ox4 the arabidopsis ga 3-oxidase, atga3ox accumulated intact and in soluble form when expressed as a poly-tagged trx fusion in pet to a level that was discernible by sds-page analysis of the soluble cell fraction. during purification atga3ox tended to precipitate, although precipitation could be minimised during the first step by dilution of the soluble cell lysate and the use of a buffer of ph with mm β-me. trx-atga3ox was purified to a high degree by immobilised metal affinity chromatography. cleavage of the trx tag from atga3ox was enabled by a thrombin site engineered into the expression construct – the factor xa site encoded by pet failed to cleave. atga3ox was difficult to separate from trx, remaining strongly associated with it subsequent to thrombin cleavage. the single factor identified that most obviously mitigated this association was β-me, with low ph further aiding separation. resolution of the two proteins was achieved by gel filtration in a buffer of ph of with mm β-me. highly pure atga3ox was thus obtained in microgram quantities. once pure, atga3ox resisted concentration. the problem was partially overcome by the addition of triton x- however, the maximum concentration of atga3ox achieved was mg/ml and its activity did not tolerate freezing. otherwise, atga3ox was active at all stages of purification, permitting a kinetic study on highly pure atga3ox removal of the trx tag had no obvious affect on atga3ox activity levels. triton x- had an inhibitory effect but this seemed to disappear with dilution. the use of low ph buffers during purification did not obviously dampen atga3ox4's activity. indeed, we found atga3ox displayed % activity when assayed in the phosphate buffer of ph used for the first purification step, providing the reducing agent dtt was present. however, the buffer of ph in the second step was not compatible with activity assays. in light of the difficulties associated with obtaining atga3ox in high yield, further purification of the fusion, trx-atga3ox was conducted. milligrams of highly pure trx-atga3ox were obtained. the protein was amenable to concentration and could be frozen at -70°c without activity loss. kinetic analysis of atga3ox4 the activity of highly pure atga3ox displayed a saturation curve of the michaelis menten type in response to increasing concentration of ga. the km was calculated to be μm and the vmax pkat/mg. discussion high levels of activity are frequently obtained for ga 2odds in e. coli expression systems in the absence of accumulated soluble enzyme. the instability generally reported in native 2odds appears to be a feature of the recombinant enzymes. in early trials of ga 2odds, the pea ga 2-oxidase, psga2ox and the arabidopsis ga 3-oxidase, atga3ox showed promise as candidates for purification. in both cases, a n-terminal fusion tag was needed to obtain soluble intact protein. the purification of the pea ga 2-oxidase, psga2ox was straightforward. no problems were encountered during chromatography, tag removal or enzyme concentration. all steps of purification were performed in a buffer that preserved enzyme activity. the ph was within physiological range when the chelator edta and the reducing agent dtt were present.. the milligram amounts of psga2ox that we obtained are unprecedented for a highly pure ga 2odd. the gene encoding psga2ox is affected by the null mutation, sln, which causes the accumulation of ga within maturing pea seed. the mutation has a lesser effect on ga levels in the stem, presumably because other ga 2-oxidases compensate for its loss in this tissue. mature seeds contain at least two ga 2-oxidases. the sln phenotype indicates that psga2ox is the main catalyst for the conversion ga to ga at latter stages of seed maturation, when 2-oxidation of ga takes place at high levels. although the sln mutation is not deleterious to developing seeds, the mature seeds contain abnormally high amounts of ga which, after germination, is converted to the bioactive ga resulting in the slender phenotype of the seedling. the levels of ga20present in mature sln and sln seeds are ng/g and ng/g, respectively. the difference can be solely attributed to the action of psga2ox in planta. multiple ga 2-oxidases have been previously partially purified in low yield from pea seeds of unspecified maturity. the incomplete purification was attributed to a low abundance of the enzymes in the tissue, and instability during purification. it can be assumed that native psga2ox was among these ga 2-oxidases because its gene is expressed throughout seed maturation. recombinant expression methods have now enabled us to complete the successful purification of psga2ox at μm, the km of recombinant psga2ox toward ga was much lower than that obtained for the native pea seed ga 2-oxidases. the comparison of km results is complicated, however, because the native preparation contained multiple activities. certainly, a high substrate affinity of psga2ox is consistent with its ability to clear the maturing seed of ga the gene encoding psga2ox is expressed relatively strongly throughout the latter weeks of pea seed maturation. that the enzyme is abundant in mature seeds was supported by a study of ga 2-oxidases from this tissue in the related species phaseolus vulgaris. if present in abundance, it is plausible that psga2ox could account for the difference in ga levels between sln and sln seeds. the vmax of the partially pure native ga 2-oxidases toward ga was pmol/h mg. the higher figure of the recombinant enzyme suggests that its purity and/or retention of activity was enhanced compared with the native preparation. interestingly, the vmax of psga2ox was several orders of magnitude lower than the vmax of atga3ox toward ga. however, this difference is difficult to interpret considering the two enzymes are from different species and are likely to be expressed differentially within the plant alongside isoenzymes. with their role in ga activation, the ga 3-oxidases are particularly interesting to study, but in native form the enzymes are difficult to purify. it required immature seeds for kwak et al. to obtain mg of enzyme described as partially pure. recombinant methods enabled us to partially overcome the problems associated with purification of a recombinant ga 3-oxidase. our experience provides valuable insight for future work. the first step we used in the purification of the arabidopsis ga 3-oxidase, atga3ox was imac chromatography. this method does not permit the inclusion of ddt or edta in buffers and therefore has been considered unsuitable for purifying 2odds. nonetheless, atga3ox with high specific activity was obtained using this method. evidently, mm β-me was able to protect the enzyme active site from metal-catalysed oxidation during chromatography. the result suggests that this widely used and highly efficient purification strategy does not necessarily compromise the activity of a 2odd. atga3ox showed a tendency to precipitate, one that became more pronounced when it was cleaved, but not physically separated, from trx. this diminished the yield of the protein, particularly in the final step. a beneficial effect from low ph and β-me implied that an exposed cysteine was causing the precipitation. however, a combination of factors were probably to blame because the aforementioned conditions did not fully overcome the problem. furthermore, replacement by site-directed mutagenesis of the cysteine predicted as most likely to be exposed brought no improvement. certainly, atga3ox displayed hydrophobic behaviour when pure, sticking to tubes unless triton x- was present. hydrophobicity was also a feature of the ga 3-oxidase of bean. good scope exists for further optimisation of the atga3ox purification method. efficient separation of atga3ox and trx will lead to a greatly increased yield. hydrophobic interaction chromatography is a possible alternative to gel filtration in separating the two proteins. atga3ox was amenable to concentration and freezing in the presence of trx, whether or not it was chemically attached to it. the identification of buffer ingredients that replace trx's stabilising role, such as alternative detergents, salts or organic solvents may improve the behaviour of purified atga3ox atga3ox is encoded by a member of a family of four ga 3-oxidase genes in arabidopsis. generally the non-13-hydroxylated ga pathway predominates in arabidopsis, however recent studies found comparable levels of non-13-hydroxylated and 13-hydroxylated gas in developing seeds. this tissue is the main site of expression of the gene encoding atga3ox, therefore ga is likely to be a natural substrate of atga3ox the high vmax gained here shows that atga3ox can efficiently metabolise ga the value of pkat/mg is higher than the vmax of nmol/min mg reported for a purified gst fusion of another arabidopsis ga 3-oxidase, atga3ox. atga3ox4's km of μm toward ga was lower than that of atga3ox, yet higher than the value for the bean seed ga 3-oxidase. accumulated kinetic information with respect to both 13-hydroxylated and non-13-hydroxylated gas on each of the ga 3-oxidases of arabidopsis will give valuable insight into the control of active hormone levels in this model species.  the previous lack of reports on the complete purification of ga 2-oxidases and ga 3-oxidases is probably due to a combination of their lability and their low abundance in most plant tissues. one might speculate that a crucial role in the control of active hormone levels requires a short enzyme half-life. this study on psga2ox and atga3ox shows that ga 2-oxidases and ga 3-oxidases are not necessarily inherently unstable and can be purified active from e. coli expression systems under standard conditions. indeed, the ga 2-oxidase, psga2ox was exceptionally stable throughout purification. problems were encountered with the arabidopsis ga 3-oxidase, atga3ox due to its hydrophobicity and the presence of hydrostatic charges, rather than metal-catalysed autocleavage or proteolytic breakdown. indeed, plant 2odds from secondary metabolic pathways are also problematic during purification. yet in some cases, they have been purified in high yield permitting detailed structural and mechanistic studies. we note the recent elucidation of the structure of the labile 2odd-related enzyme, acc-oxidase, of ethylene biosynthesis. our work opens the door to similar studies on the ga 2odds. 
the 2-oxoglutarate-dependent dioxygenases of gibberellin biosynthesis have a key role in the metabolism of a major plant hormone. the ga 2-oxidase from pisum sativum l., psga2ox was expressed as a glutathione s-transferase fusion. the activity of recombinant ga 2odds from many species has been characterised in detail, however little information relates to enzyme purification. native ga 2odds displayed lability during purification. two ga 2odds were expressed in escherichia coli and purified to homogeneity. the successful purification of milligram quantities of psga2ox enables mechanistic and structural studies not previously possible on ga 2odds. it displayed a km of μm and a vmax of pkat/mg toward ga the ga 3-oxidase from arabidopsis thaliana, atga3ox was expressed as a poly-tagged thioredoxin fusion. it was purified in the three steps of affinity chromatography, gst removal and gel filtration. it was purified by immobilised metal affinity chromatography followed by gel filtration. psga2ox1's action in planta as deduced from the effect of the null mutation sln on ga levels in seeds is in agreement with the kinetic parameters of the recombinant enzyme.
 the 2-oxoglutarate-dependent dioxygenases of gibberellin biosynthesis have a key role in the metabolism of a major plant hormone. the activity of recombinant ga 2odds from many species has been characterised in detail, however little information relates to enzyme purification. native ga 2odds displayed lability during purification.  two ga 2odds were expressed in escherichia coli and purified to homogeneity. the ga 2-oxidase from pisum sativum l., psga2ox was expressed as a glutathione s-transferase fusion. it was purified in the three steps of affinity chromatography, gst removal and gel filtration. highly pure psga2ox was obtained at a yield of mg/g of cells. it displayed a km of μm and a vmax of pkat/mg toward ga the ga 3-oxidase from arabidopsis thaliana, atga3ox was expressed as a poly-tagged thioredoxin fusion. it was purified by immobilised metal affinity chromatography followed by gel filtration. cleavage of the fusion took place between the two purification steps. highly pure atga3ox was obtained at a yield of mg/g of cells. it displayed a km of μm and vmax of pkat/mg toward ga  fusion tags were required to stabilise and solubilise psga2ox and atga3ox during e. coli expression. the successful purification of milligram quantities of psga2ox enables mechanistic and structural studies not previously possible on ga 2odds. a moderate yield of pure atga3ox requires the further optimisation of the latter stages of the enzyme purification schedule. psga2ox1's action in planta as deduced from the effect of the null mutation sln on ga levels in seeds is in agreement with the kinetic parameters of the recombinant enzyme. 