of this key enzyme from Taxus baccata cell cul- tures are presented.
2. Methods
2
.
1
. Materials [1-
14
C] Isopentenyl diphosphate IPP with a specific activity of 1.96 Gbq mmol
− 1
was pur- chased from Amersham Pharmacia Biotech. Non-
labelled IPP, farnesyl diphosphate FPP and dimethyl allyl diphosphate DMAPP were synthe-
sised according to Davisson et al. [9]. Leupeptin hemisulfate was from ICN and PVPP from Sigma.
PD-10 columns, Phenyl Sepharose 6 Fast Flow, Mono Q 1010, Mono P 520 ready-made columns
were purchased from Amersham Pharmacia Bio- tech, while TSK-GEL G3000 SW 0.0075 × 0.6 m
was from Tosohaas. A Waters HPLC system con- sisting of a 616 pump, a 486 tuneable UV detector,
a 600 S system controller and a Rheodyne 7215 injection valve was used for the gel filtration
room temperature. All other purification steps were performed at 4°C with a FPLC system Phar-
macia consisting of a LCC-500 Liquid Chro- matography Controller, two P-500 pumps and a
MV-8 Motor Valve. Protein in the effluent was monitored by measuring the absorption at 280 nm
while the salt concentration was monitored by a conductivity meter. Gel electrophoresis was per-
formed on a PhastSystem Amersham Pharmacia Biotech using microgels and protein bands were
visualised by silver staining [10]. Ultrafiltration concentrator, YK-30 30 kDa cut-off point filters
and the Microcon-30 concentrators were from Amicon.
All the chemicals were of the highest purity commercially available.
2
.
2
. Plant material, extraction, enzyme assay The explants were derived from the T. baccata
621 tree from Pinetum Blijdestein in Hilversum, Holland, in June 1995. Cell suspension line T.
baccata C TBC was established in a B5M11 medium B5 salts supplemented with 100 mgl
meso-inositol, 10 mgl thiamine dihydrochloride, 1 mgl pyridoxine hydrochloride, 1.86 mgl NAA
and 20 gl sucrose from leaves-derived callus that grew on the same medium. Cultures were main-
tained under continuous light of 1000 Lux Philips TL 40W33 RS on a gyratory shaker at 110 rpm
and 25°C in 2 l flasks containing 500 ml of medium and they were harvested 2 days after
inocculation, at the time when enzyme activity was the highest [7]. Cells were filtered over a sintered
glass under vacuum and then washed with water. 800 g of cells were frozen in liquid N
2
and stored at − 80°C until required for protein extraction.
Fig. 1. Biosynthetic scheme of paclitaxel. The point of action of methyl jasmonate a paclitaxel production inducer is shown.
The frozen cells were ground in a liquid N
2
chilled Waring blender for 2 × 30 s at maximum speed
and a fine powder was obtained. Immediately 0.15 g of polyvinylpolypyrrolidone PVPP and 1 ml of
extraction buffer per gram of fresh weight, were added. The extraction buffer contained 50 mM
Tris – HCl pH 7.6, 1 Brij-35, 2 mM DTT, 10 mM leupeptin, 4 mM MnCl
2
, 2 mM MgCl
2
and 20 glycerol. The homogenate was mixed in a 30°C
waterbath until the temperature reached 4°C and was then passed through Miracloth and cen-
trifuged at 20 000 × g for 60 min and the superna- tant was used for the subsequent purification
steps.
The assay is based on the acid lability of the allylic diphosphates [11] and is described in detail
in [7,8]. GGPP synthase activity was measured using the following incubation mixture: 50 ml of
desalted protein extract, 50 mM Tris – HCl pH 7.2, 10 mM leupeptin, 4 mM MnCl
2
, 2 mM MgCl
2
, 20 glycerol and 25 mM KF in a total volume of
200 ml. Before addition of the substrates the mix- ture was preincubated for 10 min in the presence
of 10 mM iodoacetamide in order to inhibit the activity of IPP isomerase. The reaction was started
by addition of [1-
14
C]-IPP 4.86 mM final concen- tration, 55 mCimmol and FPP 43.5 mM final
concentration. After incubation for 30 min at 30°C the enzyme reaction was stopped by addition
of 500 ml EtOH:HCl 1:1. The hydrolysis of the allylic diphosphates was allowed to proceed for 20
min and then 2 ml of toluene were added in order to extract the allylic alcohols. 1 ml of the toluene
layer was removed and mixed with 10 ml of Opti- Fluor Packard and the radioactivity incorpora-
tion
was determined
by liquid
scintillation counting Tri-Carb 4530, Packard. Blanks were
performed with addition solely of [1-
14
C]-IPP, protein extract and omitting acidic hydrolysis as
well as incubation with [1-
14
C]-IPP and no protein extract,
showed negligible
incorporation of
radioactivity. Protein concentration was determined by using
the DC Protein Assay kit from Bio-Rad.
2
.
3
. Purification of GGPP synthase Ammonium sulphate was added in the 20 000 ×
g supernatant at 4°C and the activity precipitated between 40 – 60 saturation. The precipitate was
resuspended in a buffer consisting of 20 mM Tris – HCl pH 7.4, 2 mM DTT, 15 glycerol, 4 mM
MnCl
2
, 2 mM MgCl
2
buffer A, supplemented with 1 M NH
4 2
SO
4
and it was loaded onto a Phenyl Sepharose 6 Fast Flow column equili-
brated in the same buffer. The proteins were eluted with a 6 h linear gradient ranging from 1 M
to 0 M NH
4
SO
4
of buffer A at a flow rate of 2 mlmin. Fractions of 12.5 ml were collected. Ac-
tive fractions were pooled and concentrated by ultrafiltration using an Amicon YK-30 filter. This
concentrate was desalted over PD-10 columns.
The concentrated active extract was repeatedly chromatographed over a Mono Q strong anion
exchange column equilibrated with buffer A and the proteins were eluted at a rate of 0.4 mlmin
with the following NaCl gradient: 0 – 0.1 M linear gradient over 12.5 min, isocratic at 0.1 M NaCl
until 25 min and then linear to 0.25 M until 75 min. Fractions of 1 ml were collected.
Active fractions were pooled and concentrated with Microcon filters and they were repeatedly
chromatographed at a flow rate of 0.5 mlmin on a Tosohaas G-3000 SW column equilibrated with
20 mM Hepes pH 7.1, 2 mM DTT, 15 glycerol, 4 mM MnCl
2
and 2 mM MgCl
2
buffer B. The fraction size was 0.5 ml.
Active fractions were pooled, concentrated with Microcon filters and applied on a Mono P weak
anion exchange column. Proteins were eluted at a rate of 0.4 mlmin with a linear gradient of 0 to
0.5 M NaCl in buffer B, in 100 min. 0.3 ml fractions were collected. The Mono P step resulted
in a homogeneous GGPP synthase.
2
.
4
. Molecular mass determination The molecular mass of the purified enzyme as-
suming globular proteins was estimated by gel filtration on a TSK – GEL G3000 SW column
Tosohaas, 0.0075 × 0.6 m, equilibrated with buffer B. It was eluted with the same buffer at 0.75
mlmin. The markers used to calibrate the column were: blue dextran 2000 kDa, aldolase 158
kDa, bovine serum albumine 67 kDa, ovalbu- mine 43 kDa and chymotrypsinogen A 25 kDa.
Molecular weight was also determined by gel electrophoresis on SDS-PAGE, performed with
the PhastSystem Pharmacia: a 12.5 homoge- neous PhastGel with SDS bufferstrips was used.
Protein visualisation was obtained by silver staining.
Table 1 Purification of GGPP synthase from Taxus baccata cell cultures
Total protein Activity
Yield Specific activity nkatg of protein
Purification Purification -fold
mg step
nkat 106
26 –
Cell extract 4080
77 38.8
1980 15
40−60 AS 72
HIC 139
64 460.4
17.7 60
41 7321
5.6 281.5
Mono-Q 38
0.38 TSK-G3000
6.9 18 158
698 6.5
0.01 Mono-P
2.3 230 000
8846 2.1
2
.
5
. Electrophoresis Native and SDS PAGE was performed with
PhastSystem in PhastGel homogeneous medium 12.5. Detection of proteins was with silver
staining.
2
.
6
. Enzyme kinetics Kinetic data were fitted to various models using
the EZ-FIT curve-fitting computer programme. Initial rates were measured in duplicate using the
above described GGPP synthase assay at 10 differ- ent concentrations, under conditions where the
rate of product formation was linear.
The K
m
for FPP was calculated in the range of 0.5 to 45 mM. The IPP concentration was held
constant at 25mM. The K
m
for IPP was measured between 0.05 an 25 mM of IPP, while FPP’s con-
centration was held constant at 45 mM.
3. Results and discussion
Kategori